Bonness, Kathy Marie, Ph.D., University of South Alabama, May 2009. ...... Killilea, S. Derek, Mellgren, Ronald L., Aylward, James H., and Lee, Ernest Y. C..
THE ROLE OF SERINE/THREONINE PROTEIN PHOSPHATASES IN MITOSIS
A Dissertation
Submitted to the Graduate Faculty of the University of South Alabama in partial fulfillment of the requirements for the degree of Doctor of Philosophy Basic Medical Sciences in Department of Biochemistry & Molecular Biology College of Medicine
by Kathy Marie Bonness B.S., Santa Clara University, 1992 May 2009
This dissertation is dedicated to MY FAMILY who supported me with love, confidence, and humor. To my Mom and Frank and The Sisters-Beth, Mary, Ellen and Amy. To my Dad and sister Meg, whose character-defining moments, both in life and death, changed me forever. My deepest gratitude goes to all of my family for the inspiration, strength and motivation to pursue this goal. To my Siberian Huskies-Orion, Saki and Chloe whose unconditional love taught me and sustained me; whose Shenanigans humored and grounded me; and whose mere presence in my life saved me.
“The highest reward for a person's toil is not what they get for it, but what they become by it." - John Ruski
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ACKNOWLEDGMENTS
"Man's mind, once stretched by a new idea, never regains its original dimensions. -Oliver Wendell Holmes Jr.
This research project would not have been possible without the combined effort and input of a wide and varied group of people throughout my interdisciplinary studies. I consider myself very lucky and am truly grateful to all who have crossed my path over these years and left their footprints during my journey. I wish to express my appreciation to Dr. Richard Honkanen and the members of my thesis supervisory committee, Dr. Ron Balczon, Dr. Sailen Barik, Dr. Roger Lane, Dr. Susan LeDoux, and Dr. Jonathan Scammell. The combined knowledge, support and guidance have been abundantly helpful throughout the years, and without, this study would not have been successful. I want to express a special thanks to Dr. Solomon Ofori-Acquah and Dr. Ron Balczon who graciously provided me with laboratory facilities and mentorship both academically and personally. The additional effort and guidance extended to me has been truly appreciated. I am grateful to the faculty/staff/students in the College of Medicine, all of which have, in some manner, assisted me and participated in my interdisciplinary training in the Ph.D. program. Specifically, the departments who opened their doors to me and provided iii
an educational environment and financial support including the Department of Biochemistry & Molecular Biology, the Department of Cell Biology & Neuroscience and The Center for Lung Biology. I would like to extend a special thanks to Cathy Thompson, Betty Farley, Donna Willis, Lanette Flagge, Jeanette Schwartz and Charlene Jordan who have all helped me beyond the call of duty when I have needed it most. I am genuinely thankful and forever indebted to all of my friends who have seen me through the good times and remained at my side during the more challenging times, fondly referred to as my “opportunities for growth”. I have appreciated the academic dialogue, the helping hands after hurricanes, taking care of my huskies, and most importantly, the strength I drew from their presence in my, life regardless of the distance. Lastly, I would like to acknowledge my family for credit much deserved. For my parents who instilled me with all the right stuff including personal responsibility, priorities, creativity and the value of individuality, as well as teamwork. And, to my sisters, who nurtured my curiosity and thankfully never inflicted irreparable damage in response to my endless litany of questions. Finally, I would like to acknowledge Orion, Saki and Chloe for unconditionally putting up with it all and remaining my grounding balance.
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TABLE OF CONTENTS
Page LIST OF TABLES ...................................................................................................... viii LIST OF FIGURES ........................................................................................................ix LIST OF ABBREVIATIONS ......................................................................................... xi ABSTRACT ................................................................................................................. xvi INTRODUCTION ........................................................................................................... 1 BACKGROUND ............................................................................................................. 9 Reversible Phosphorylation........................................................................................ 9 Protein Phosphatases................................................................................................ 11 PPases ................................................................................................................. 12 PP2A-Related Phosphatase ................................................................................. 18 Protein phosphatase 2A (PP2A) ...................................................................... 20 Protein phosphatase 4 (PP4, PPP4, PPX) ......................................................... 23 Protein phosphatase 6 (PP6) ............................................................................ 25 Natural Inhibitors of the Serine/Threonine Protein Phosphatases .............................. 25 Cantharidin ......................................................................................................... 26 Fostriecin ............................................................................................................ 28 Cell Cycle and Mitosis ............................................................................................. 32 Summary and Hypothesis ........................................................................................ 37 MATERIALS & METHODS ........................................................................................ 39 Cell Culture ............................................................................................................. 39 Flow Cytometry ....................................................................................................... 39 Viability and Proliferation Assays ............................................................................ 40 v
Phosphatase Assays ................................................................................................ 41 Indirect Immunofluorescence Microscopy .............................................................. 41 Live Cell Imaging and Time-lapse Video Microscopy............................................. 42 Oligonucleotide Synthesis and Assay for Oligonucleotide Inhibition of PP2AExpression ................................................................................................ 44 Immunoblotting of PP2A ........................................................................................ 45 Oligonucleotide Synthesis and Assay for Oligonucleotide Inhibition of PP4 Expression ......................................................................................................... 46 Immunoblotting of PP4 ........................................................................................... 48
RESULTS ..................................................................................................................... 49 Overall Hypothesis and Aims: ................................................................................. 49 Aim 1 ............................................................................................................... 49 Aim 1 .............................................................................................. 50 Aim 2 .............................................................................................. 52 Aim 3 .............................................................................................. 56 Aim 4 .............................................................................................. 68 Aim 2
................................................................................................ 79 Aim 1 Aim 2 Aim 3
.............................................................................................. 84 .............................................................................................. 87 .............................................................................................. 87
Aim 3 ............................................................................................................... 93 Aim 1 Aim 2 Aim 3
.............................................................................................. 93 .............................................................................................. 96 .............................................................................................. 97
Summary and Conclusion ...................................................................................... 107 DISCUSSION AND FUTURE IMPLICATIONS ....................................................... 113 The Anti-proliferative and Cytotoxic Effects of Cantharidin and Fostriecin are Due to Disruptions in Mitotic Progression Resulting from the Inhibition of Serine/Threonine PPase Activity .................................................................. 114 Cantharidin- and Fostriecin-Mediated Disruptions in Mitotic Progression are Due, in part, to the Inhibition of PP2A Activity ........................................ 117 Cantharidin- and Fostriecin-Mediated Disruptions in Mitotic Progression are Due, in part, to the Inhibition of PP4 Activity ............................................. 119 REFERENCES ............................................................................................................ 131 vi
BIOGRAPHICAL SKETCH ....................................................................................... 146
vii
LIST OF TABLES
Table 1.
Page Summary of Morphological Changes in Mitotic HeLa-H2B-GFP Cells ............ 108
viii
LIST OF FIGURES
Figure
Page
1.
The Phases of Mitosis .......................................................................................... 5
2.
Reversible Phosphorylation and Regulation ....................................................... 10
3.
Homology of the Ser/Thr Protein Phosphatases ................................................. 14
4.
Sequence Alignment of the Catalytic Subunit of PP2A with other Homologous Phosphatases of Human Origin ..................................................... 19
5.
Structure of the PP2A Holo-enzyme .................................................................. 21
6.
Structure of Cantharidin .................................................................................... 27
7.
Structure of Fostriecin (CI-920) ........................................................................ 30
8.
Phosphatase Inhibition Mediated by Cantharidin Correlates with Antiproliferation and Cytotoxicity in A549 Cells ................................................... .51
9.
Cantharidin Treatment Alters Cell Cycle Progression of A549 Cells as Measured by Flow Assisted Cell Sorting (FACS) Analysis of DNA Content ...... 54
10.
Fluorescent Confocal Microscopy Images of Microtubules and DNA Illustrating Metaphase Alignment of a Representative Control A549 Cell .......... 58
11.
Fluorescent Confocal Microscopy Images of Microtubules and DNA Illustrating Representative Aberrations in Metaphase Alignment Resulting from Cantharidin Treatment of A549 Cells (2 mol/L for 24 hours) ................... 61
12.
Time-lapse Live Cell Microscopy Illustrating Typical Cell Cycle Progression of HeLa-H2B-GFP Cells Compared to Cells Treated with Cantharidin ................................................................................................ 66
13.
Semi-Selective Inhibition of PPase Activity by Fostriecin .................................. 69
ix
14.
Time-lapse Live Cell Microscopy Illustrating Representative Abnormalities in Mitotic Progression Resulting from 50 M Fostriecin Treatment of Hela-H2BGFP Cells for 24 hours ....................................................................................... 72
x
15.
Enlarged Images Highlight Abnormalities in Chromosome Alignment during Metaphase Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours....................................................................................................... 74
16.
Enlarged Images Highlight Abnormalities in Chromosome Segregation Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours ............................................................................................................. 76
17.
Enlarged Sequential Images Highlight Cytokinesis Abnormalities Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours ..................... 78
18.
Specific and Potent Inhibition of PP2A by Treatment with Antisense Oligodeoxynucleotides ....................................................................................... 81
19.
Enlarged Images of Representative Cells Suppressed of PP2A via Antisense Oligonucleotide-Mediated Suppression .............................................................. 89
20.
Time-lapse Live Cell Microscopy Illustrating Irregularities in Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP2A Compared to Controls ......................................................................................................... 92
21.
Specific Inhibition of PP4 by Treatment with Antisense Oligodeoxynucleotides ....................................................................................... 94
22.
Confocal Z-sections of Cells Suppressed of PP4 ................................................ 96
23.
Time-lapse Live Cell Microscopy Illustrating Irregularities in Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP4 Compared to Controls ....................................................................................................... 100
24.
Enlarged Images of Representative Cells Suppressed of PP4 via Antisense Oligonucleotide-Mediated Suppression ............................................................ 102
25.
Enlarged Images Highlight Abnormalities in Chromosome Segregation Observed in Hela-H2B-GFP Cells Treated with ISIS 134947 Targeting PP4 .................................................................................................................. 103
26.
Enlarged Sequential Images Highlight Cytokinesis Abnormalities Observed in Hela-H2B-GFP Cells Treated with ISIS 134947 Targeting PP4 .................... 106
27.
Time-lapse Live Cell Microscopy Illustrating Control-like Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP2Aβ, PP1 and PP5 ..... 111
xi
LIST OF ABBREVIATIONS
±SD
standard deviation
2N
DNA content in G2 (46 total chromosomes)
32
radioactive isotope of phosphorus include
P
4N
DNA content in G1 (92 total chromosomes)
AMP
aberrant metaphase phenotype
APC
anaphase-promoting complex
AS
antisense oligonucleotide
ATCC
American Type Culture Collection
ATP
adenosine-5'-triphosphate
CAK
Cdk-activating protein
CaM
Ca2+-calmodulin
CCNSC
Cancer Chemotherapy National Service Center
Cdks
cyclin-dependent kinases
cDNA
complementary Deoxyribonucleic acid
CFTR
cystic fibrosis transmembrane conductance regulator
CI-920
fostriecin
cmm
centrosomes minus microtubules
CO2
carbon dioxide
DIC
differential interference contrast (Numarski) xii
DMEM
Dulbecco’s Modified Eagle’s Medium
MF
dimethylformamide
DNA
deoxyribonucleic acid
DSP-1
diarrheic seafood poisoning
DSPases
dual-specificity phosphatases
DTT
dithiothreitol
ECL
enhanced chemiluminescence
eGFP
enhanced green fluorescent protein
FACS
Flow Activated Cell Sorting
FBS
fetal bovine serum
FCS
fetal calf serum
FLAG
peptide sequence of the FLAG-tag is as follows: N-DYKDDDDK-C
FLT3
FMS-related tyrosine kinase 3
G1
Gap 1
G2
Gap 2
G3PDH
glyceraldehyde 3-phosphate dehydrogenase
GAP
phosphorothioate oligodeoxy residues
GTPases
guanosine triphosphate binding hydrolase enzymes
H
hour
H2B
human histone 2B gene
HEAT
Huntington/elongation/ A-subunit/TOR
HEK293
human embryonic kidney cell line
HR
hour xiii
hSgo1/2
human Shugoshin protein 1 and 2
I-1
Inhibitor-1
I-2
Inhibitor-2
IC50
half the maximal inhibitory concentration
INCENP
Inner Centromere Protein
ISIS
Isis Pharmaceuticals, Inc.
KSP
kinesin spindle protein
LCMT1
leucine carboxyl transferase 1
Min
minute
MKP
MAP kinase phosphatases
MM
mismatch oligonucleotide
MOE
metholxyethyl
M-phase
mitosis
mRNA
messenger ribonucleic acid
MTD
maximum tolerated dosage
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole
NaCl
sodium cloride
NCI
National Cancer Institute
NEB
nuclear envelope breakdown
NIH
National Institute of Health
nM
nanomolar
nMol/L
nanomole per liter
OA
okadaic acid xiv
PBS
phosphate buffered saline
PI
propidium iodide
PKA
protein kinase A
Plk
Polo like kinase
PLK
Polo like kinase
PP1
type I phosphatases
PP2
type II phosphatases
PP2A
Protein phosphatase 2A
PP4
Protein phosphatase 4
PPP4
Protein phosphatase 4
PPX
Protein phosphatase 4
PP6
Protein phosphatase 6
PPases
serine/threonine phosphatases
PTPases
protein tyrosine phosphatases
RNA
ribonucleic acid
RNAi
RNA interference
RNase
ribonuclease
RNase-H
ribonuclease enzyme
ROI
region of interest
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Ser
serine
S-phase
synthesis
SSC
saline-sodium citrate buffer xv
Thr
threonine
TOR
targets of rapamycin
TPR
tetratricopeptide
Tris
tris(hydroxymethyl)aminomethane
Tyr
tyrosine
uM
micromolar
UV
ultraviolet
WD
tryptophan-aspartate
xvi
ABSTRACT
Bonness, Kathy Marie, Ph.D., University of South Alabama, May 2009. The role of serine/threonine protein phosphatases in mitosis.Chair of Committee: Dr. Richard E. Honkanen Previous studies of two naturally-occurring compounds suggested a correlation between protein phosphatase inhibition and anti-cancer qualities; however, the mechanism was unclear. Strong evidence suggested the mechanism involved inhibition of PPP phosphatases based on a common catalytic region, “inhibitor-binding domain”; however, identification of specific phosphatase(s) and associated functions was unclear. Collectively, this dissertation identifies PP2A and PP4 as the target phosphatases with critical roles in mitotic progression, whose selective inhibition is sufficient to induce comparable anti-mitotic effects associated with these compounds. Initial studies characterized the anti-proliferative and cytotoxic effects of cantharidin demonstrating a dose-dependent inhibition of PPP-phosphatases at concentrations consistent with the dose/time-dependent decrease in viability and proliferation and increase in G2/M cell cycle arrest. Microscopic examination revealed disruptions in mitotic spindle formation supported by time-lapse studies revealing disruptions in attaining and maintaining metaphase alignment. Comparative examination of fostriecin at concentrations capable of the complete inhibition of PP2A/PP4 and 50% of PP1/PP5 activity (in vitro) revealed disruptions in metaphase alignment, mitotic exit and cytokinesis. xvii
Subsequent studies identified PP2A and PP4 as the PPases with novel and critical mitotic roles, further implicating PP2A and PP4 as the target PPases responsible for the anti-mitotic effects attributed to cantharidin and fostriecin. Selective PPase inhibition using antisense oligonucleotide-mediated suppression was confirmed by Northern and Western analysis as an effective technique to suppress individual isoforms without altering other related PPases. Time-lapse microscopy revealed selective suppression of PP2A or PP4 resulted in prominent disruptions during mitosis consistent with those exhibited by cantharidin and fostriecin; comparatively, suppression of PP1, PP2A and PP5 exhibited typical mitotic progression. These results demonstrate novel roles for PP2A and PP4 as essential regulators in mitotic progression: PP2A via chromosome alignment and segregation, and PP4 via chromosome alignment/segregation, mitotic checkpoint fidelity and cytokinesis completion. Collectively, this dissertation identifies PP2A and PP4 as the target PPP phosphatases suppressed by cantharidin and fostriecin predominantly responsible for the cytotoxic and anti-proliferative effects. Furthermore, suppression of either PP2A or PP4 is sufficient to alter mitotic progression in a manner that emulates the anti-proliferative effects of cantharidin and fostriecin and thereby warrant further evaluation.
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INTRODUCTION
In America alone, it is estimated that one in three women and one in two men will develop cancer in their lifetime. Cancer refers to a class of diseases with features including uncontrolled growth, invasiveness, and may evolve metastatic traits in time as compared to benign or self-limiting tumors. The American Cancer Society expects 1 of 4 or 565,650 Americans to die of cancer in 2008, exceeded only by heart disease as the second most common cause of death. Furthermore, the National Institute of Health estimated the overall costs of cancer in 2007 to be $219.2 billion: $89 billion for direct medical costs (health expenditure); $18.2 billion for indirect morbidity (loss of productivity); and $112 billion for indirect mortality (death) (1). Thus, there exists an exquisite cost both in human lives and economic burden. At present, patients typically undergo combination therapy including surgery, radiation and chemotherapy. Upon diagnosis, approximately 70% of patients will receive systemic chemotherapy to combat the spread of tumors to other parts of the body. Most patients (90%) will receive classic chemotherapeutic agents such as the taxanes, camptothecins, platinates and anthracyclines. Regrettably, principal limitations continue to plague therapeutic effectiveness due to a combination of complications that often culminate in treatment failure, including related toxicities, lack of target selectivity and resistance. Overcoming today’s limitations require further exploration into novel
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therapeutic targets, a challenge that has been greatly facilitated by the advances in molecularly-targeted therapies and progress on the human genome project. Strategies designed to disrupt mitosis have been successful in abrogating highly proliferative cells. Thus, exploration into novel anti-mitotic targets is warranted which may be targeted alone or in combination with cornerstone chemotherapeutics. Unregulated cellular proliferation is a hallmark of cancer and is facilitated by the dysregulation of cell cycle components, specifically by altering genes that regulate cell growth and differentiation. In healthy tissue, a series of carefully regulated and balanced events allow cells to grow and die to maintain tissue homeostasis. Comparatively, cancer cells demonstrate increased cellular proliferation, reduced cell death or an inauspicious combination often leading to tumor formation. As a result, historical cancer therapies have exploited this characteristic effectively by targeting the cell cycle. Humans have been afflicted by cancer throughout history. One of the earliest writings dating back to 1600 B.C., illustrated eight cases of untreatable ulcers or tumors of the breast that were managed by cauterization utilizing the “fire drill” (2). The Greek physician Hippocrates (460-370 B.C.), considered the Father of Medicine, has been cited for the first use of the Greek terms carcinos and carcinoma to describe tumors, presumably in depiction of the spreading projections radiating from these masses (3). The Renaissance furthered our knowledge regarding the human body and scientific methodology from Galileo to Newton and, in 1761, the pathological findings of Giovanni Morgagni Padua marked the first use of autopsies to correlate illness and disease (4). Later, with the discovery of the modern microscope in the 19th century, the birth of modern scientific oncology emerged with Rudolf Virchow who expanded upon
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postmortem gross visual examination to include microscopic examination of pathological tissues after surgical resection (5). By the early 1900’s, surgery dominated over the classic medieval treatments of arsenic, zinc or alkaline caustic assortments. The next era of chemotherapy would soon follow although the precise origins have been disputed. In 1935, A.P. Dustin published the first report on the anti-mitotic properties of colchicines (6). Still others argue the momentous origin of today’s chemotherapies was spurred from WWII observations that chemicals, nitrogen mustards in this case, could be used to kill cancer cells (7). Nonetheless, there is little dispute that in recent decades the discovery and development of effective chemotherapeutics has been clinically effective, many of which have origins based in natural compounds (8). In the early 1950’s, the Sloan-Kettering Institute initiated the evaluation of natural compounds as potential therapeutics as a direct result of the post-WWII observations with nitrogen mustards. In a concerted effort, the NCI created the Cancer Chemotherapy National Service Center (CCNSC) in 1955 to organize and publicly screen compounds submitted by companies and institutions with well-known chemical structures (9). By 1960, the program expanded their scope to screen natural plant and animal extracts as complex mixtures of structurally-unknown compounds. When both scientific and public demand found its voice in the lobbying efforts of the American Cancer Society, the NCI (National Cancer Institute) expanded its funding from $0.5 million in 1937 to $110 million in 1961, which did not include any additional monetary support from the National Institute of Health (NIH) nor private sources. The initial screening of thousands of plant extracts examined for their anti-
3
proliferative and cytotoxic qualities was a collaborative effort between the USDA and NCI Cancer Chemotherapy National Service Center. Subsequently, it found its way to the newly founded Research Triangle Institute with Dr. Wall heading the Natural Products Laboratory. The discoveries of camptothecin and Taxol® by Dr. Wall, Dr. Wani and colleagues are two of the most momentous in cancer chemotherapy (8). Through the isolation and structure elucidation of the bioactive components found in the natural products Taxol© and camptothecin, their collaborative research not only improved the lives of people afflicted with cancer but, notably, unearthed new strategies for inhibiting cancer cell growth. As a result of this new paradigm, the discoveries of novel bioactive compounds combined with the dawn of molecularly-targeted drug discovery, the recognition of a new generation of therapies that target specific proteins have been made possible. “Undoubtedly, there are other highly active natural products from the plant, marine, and fungal sources as yet unknown which, when discovered, will have therapeutic value. Cancer is not one, but several hundred diseases and will require many different types of agents” (10). To date, one of the most successful cancer chemotherapeutic strategies targets mitosis by disrupting microtubule dynamics for the management of cancer. Microtubule stabilizers (Docetaxel and Paclitaxel) and microtubule destabilizers (Vinblastine, Vincristine and Vinorelbine) are successful anti-mitotic agents that exploit the mitotic requirement of microtubule dynamics (11). Current advances have furthered our knowledge of the distinct morphological changes a cell exhibits throughout mitotic progression which have greatly increased our understanding of the signaling mediators that function at these distinct phases. This in turn has led to the emerging success of
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Figure 1. The Phases of Mitosis. The progression of mitosis through the canonical morphological stages is shown. Specific druggable protein targets that function during mitosis are highlighted. Kinesin spindle protein (KSP) is required to establish mitotic spindle bipolarity through driving centrosome separation. Centromeric protein E (CENPE) is required for accurate chromosome congression at metaphase during mitosis. Aurora A is crucial for centrosome maturation and separation during early prophase. Aurora B is a member of the chromosomal passenger complex (CPC) and is involved in histone H3 phosphorylation, chromosomal condensation, chromosomal alignment on the metaphase plate, bi-polar centromere-microtubule attachments, spindle checkpoint and cytokinesis. During mitosis, Polo-like kinase 1 (PLK1) is involved in centrosome maturation and formation of the mitotic spindle. PLK1 is also required for exit from mitosis and the separation of sister chromatids during anaphase. PLK1 might also have a role in cytokinesis through the phosphorylation of the kinesin-like motor protein MKLP1.
5
specific drug targets. Today, nonstructural components in mitosis have also demonstrated success including the mitotic kinesins, the Aurora kinases and the Polo-like kinases as illustrated in Figure 1 (12). Therefore, the protein phosphatases may also lead to effective nonstructural anti-mitotic agents and warrant further investigation. The following section is meant to highlight successful strategies and validate the rationale supporting the aforementioned novel targets, although it is not an exhaustive discussion. The first example is directed against a kinesin motor protein, called monastrol, which was the first kinesin spindle protein (KSP) inhibitor (13-15). It was originally identified in a cell-based screen searching for non-structural compounds that induce mitotic arrest. Mitotic arrest induced by KSP inhibitors results in apoptosis, ubiquitously inhibiting cellular proliferation rather than cancerous cells specifically. The associated mitotic arrest phenotype is described as exhibiting mono-orientated chromosomes around centrosomes that do not separate. Other kinesin inhibitors have entered clinical trials and include: SB-715992 or ispinesib which demonstrates 40,000 times more selectivity toward KSP versus other kinesins (16-18); and MK-0731 demonstrating 20,000 fold more selectivity due to allosteric binding to a site not shared with other structurally-related kinesins (19). Other examples are directed against the protein kinases. The first Aurora kinase inhibitor to enter clinical trials was VX-680 (MK-0457) which functions as a competitive inhibitor with nanamolar potency in cell culture against Aurora A-C and FMS-related tyrosine kinase 3 (FLT3) (20-23). Based upon pre-clinical observations, it is expected to induce a delay in mitotic progression, premature mitotic exit and cytokinesis failure, which are common precursors to aneuploidy and subsequent cell death. A selective ATP-
6
competitive inhibitor of Aurora B, AXD1152, is also being explored in phase I clinical trials demonstrating inhibition of cellular proliferation with an IC 50 of 5-35 nM (24-27). Other protein kinase examples include BI2536, which entered phase I development as the first ATP-competitive inhibitor of Polo-like kinase (PLK1) (28-30), and ON 01910, thought to interfere with substrate binding acting as an ATP-non-competitive inhibitor (31). PLK1 inactivation has proven to be an effective tactic to induce pro-metaphase arrest followed by cell death prior to exiting mitosis suggesting mitotic checkpoint activation. The availability of selective inhibitors targeting alternate mitotic components and inducing mitotic arrest has proven clinically effective, in addition to, demonstrating effective strategic design. Thus, the discovery, development and identification of the bioactive components of natural products and their mechanisms of action hold much promise for identifying unique mitotic targets. While this is true, clinical development is still posed with many challenges and limitations. A unique challenge persists in that the precise link coupling mitotic injury with subsequent cell death remains unclear and suggests that the events determining cell death lie downstream of these individual compounds and their unique modes of action. Additionally, these compounds ubiquitously target proliferating cells during mitosis without regard to normal versus cancerous tissues. For consideration of clinical development, these novel candidates may ultimately require verification of tumor inhibition, optimization of dosage and scheduling, and identification of potential determinant sensitivities. The distinct manner in which these new compounds interfere with mitosis has the potential to address current therapeutic limitations yet may introduce new limitations. Still, these new compounds
7
may expand the efficacy of established therapies by creating synergistic combinations. Ultimately, potentially significant strategic advantage may lie in the timing and order of drugs targeting mitosis to maximize efficacy and presumably minimize toxicity. Regardless, many of these compounds may serve as valuable tools to elucidate unique molecular mechanisms, and while doing so may also provide insight into the less understood but nonetheless promising inhibitors. In this regard, a number of independent groups continue to explore two natural compounds, cantharidin and fostriecin, for insights into their anti-proliferative actions. The ability to inhibit the serine/threonine phosphatases at concentrations that correlate with the anti-proliferative and cytotoxic actions sparked initial attention. Studies into the mechanism of action and pre-clinical success attributed to fostriecin (32-35), coupled to the lack of myelosuppression demonstrated by cantharidin (36-38), have made them increasingly attractive to study. Furthermore, this suggests that the selective inhibition of PPases may result in novel anti-tumor actions. Thus, it will be helpful to first discuss the phosphatases themselves, their naturally-occurring inhibitors, and the cell cycle.
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BACKGROUND
Reversible Phosphorylation Most cells require the ability to adapt quickly in response to external cues within dynamic environments. A number of post-translational modifications allow this to occur including methylation, acetylation, glycosylation, ubiquitination, and phosphorylation which was the first described modulation of enzymatic activity. Almost a century after discovering that phosphates are covalently bound to proteins in the early 19 th century, the existence of phosphoproteins was merely considered little more than a consequence of metabolic reactions (39). Phosphoproteins emerged later as key regulators of cellular life after the 1954 observation of phosphorylation. Later, the enzymes responsible would became known as the protein kinases (40). Today, in the human genome, the literature is well-cited with examples of covalently bound phosphates in at least 30% of proteins. In fact, many human diseases are now recognized to have origins in abnormal phosphorylation. This fact was acknowledged in 1992 when Fischer and Krebs received the Nobel Prize in medicine for their pioneering efforts in the discovery of reversible protein phosphorylation as a biological regulatory mechanism (41,42). Today, there is growing evidence of naturally occurring toxins that exert their effects on both protein kinases and protein phosphatases. Originally these compounds were used as valuable
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Figure 2. Reversible Phosphorylation and Regulation. A) Basic Mechanism in Protein Phosphorylation. A protein kinase transfers a phosphate group from ATP to a protein altering its shape and function. A protein phosphatase removes the phosphate returning the protein to its original conformation. B) Diverse Regulation Attributed to Reversible Phosphorylation.
tools to dissect the biological functions of these enzymes, and now they offer potential insight into the development of novel therapeutic intervention. Reversible protein phosphorylation modulates various cellular functions including developmental processes, growth factors, environmental and hormonal stimuli, metabolic control, cell cycle events, growth factor response, hormones, environmental stimuli, metabolic control, and developmental processes (43-48). The conformational changes induced by the interchanging phosphorylation state modifies the function of proteins in a variety of ways: increasing or decreasing bioactivity, inhibiting or facilitating subcellular localization, creating or disrupting protein-protein interactions, and stabilizing or targeting proteins for destruction. Figure 2A and 2B illustrate reversible phosphorylation with examples of regulation, respectively (49,50).
10
The protein kinases all share characteristic structural motifs and related conformational structures which facilitate the transfer of a phosphate from ATP to a protein through covalent attachment. The kinases are classified by the residues they act upon: Ser/Thr residues, Tyr residues, and some can act on both and are termed dualspecificity kinases (51). Protein phosphatases are similarly classified by the residues they act upon; however, they catalyze the reverse mechanism and remove a phosphate from a phosphoprotein (52). A large number of kinases have been well-studied compared to the proportionately fewer protein phosphatases. Comparatively, the phosphatases embody a structurally and evolutionarily diverse group which allows this considerably small number of phosphatases to counteract such a large number of kinases.
Protein Phosphatases Protein phosphatases are classified into three groups based on the residues they act upon: the protein tyrosine phosphatases (PTPases), dual-specificity phosphatases (DSPases), and the serine/threonine phosphatases (PPases). The PTPases dephosphorylate phosphotyrosine residues of proteins, display diverse structural features, and play important roles in the regulation of cell proliferation, differentiation, cell adhesion and motility, and cytoskeletal function (53). The DSPases, also called the MAP kinase phosphatases (MKPs), play a key role in the dephosphorylation of MAP kinases and, as their name suggests, are capable of dephosphorylating all three residues (54). The PPases relevant to this thesis study dephosphorylate phosphoserine and phosphothreonine residues; however recently some have been reported to act upon phosphotyrosine
11
residues as well (55-57). Notably, it is on the serine and threonine residues that >98% of phosphorylation events occur. The PTPases and DSPases comprise a common evolutionary family distinct from the PPases both in origin and mechanism. The former shares a catalytic mechanism based on a conserved cysteine residue within the catalytic core motif. The nucleophilic cysteine displaces the phosphate from the substrate creating a phosphoryl-cysteinyl intermediate. A conserved aspartate also contributes to the phosphate’s removal in this two step reaction (58). Comparatively, the PPases’ catalytic mechanism is facilitated by a number of conserved amino acids within a common region that complex two metal ions (59-62). In this acid-base catalytic mechanism, the nucleophilic attack by the metal-bound water molecule acts to directly displace the phosphate from the substrate residue in one step (63). Therefore, the two groups share a common dephosphorylation function; however, they differ in their greater affinity toward specific residues.
PPases Traditionally, the PPases were classified into four major families: PP1, PP2A, PP2B, and PP2C. The nomenclature established by Ingebritsen and Cohen in the early 1980’s divided the known PPases into two major groups, PP1 and PP2, based on a number of parameters: biochemical properties, substrate specificity and sensitivity to natural inhibitors (64). It should be taken into consideration that, at the time, the ability to specifically identify and measure all PPases was limited. Therefore, it was not until later that technological advances more clearly demonstrated roles for individual PPases rather than an entire group of related enzymes. Nonetheless, early studies found that type
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I phosphatases (PP1) were sensitive to inhibition by two heat-stable proteins known as Inhibitor-1 (I-1) and Inhibitor-2 (I-2), unlike the type II phosphatases (PP2), which were not affected by these two compounds. The former preferentially dephosphorylates the subunit of phosphorylase kinase while the later preferentially dephosphorylates the subunit of phosphorylase kinase and include (PP2A), Ca2+-dependent (PP2B), and Mg2+dependent (PP2C) phosphatases (65). Approximately 50% sequence identity is shared between PP1 and PP2A overall, whereas the structurally distinct PP2C belongs to an entirely different gene family. From another angle, the division into two major gene families transpired based upon similarities in primary amino acid sequence: firstly, the Mg2+-dependent family, termed PPM, includes the PP2C family and pyruvate dehydrogenase phosphatases (66,67); and secondly, relevant to this dissertation, the largest and most well-known PPP family that is Mg2+-independent and includes PP1, PP2A, PP2B (calcineurin), PP4, PP5, PP6 and PP7 (66,68). Along with sequence similarities, the PPP and PPM families share a related structural fold, suggesting a common mechanism of catalysis (69) (Figure 3A). Of special note and relevance to this thesis proposal, one further distinction divides the PPP family into two subgroups based on their sensitivity to a number of naturallyproduced inhibitors including okadaic acid (OA), microcystin, calyculin A, tautomycin, cantharidin and fostriecin (70-72). All members of the toxin-sensitive PPP subgroup are susceptible, albeit to varying degrees, to inhibition by these compounds. This is due to a conserved region termed the “toxin-binding domain” located near the C-terminal end of the catalytic subunit (59-62) (Figure 3B). The two remaining PPP enzymes, PP2B and PP7, both contain inserts within the “toxin-binding domain” rendering them insensitive to
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Figure 3. Homology of the Ser/Thr Protein Phosphatases. A) Phylogenic tree depicting similarity between known PPases based on their primary amino acid sequence. (PP1-PP7) belong to a single gene family (PPP) structurally distinct from the PP2C family (PPM). Phosphatases above the line are highly sensitive to inhibition by cantharidin, fostriecin, okadaic acid, microcystin and calyculin A (toxin sensitive). B) Schematic representation of the PP1-PP7 family of PPases containing a common catalytic core domain, conserved among species. PP1, PP2A, PP4 and PP6 are highly homologous enzymes, differing primarily in their N- and C-terminal domains. PP2B differs in that it contains a Ca2+-calmodulin (CaM) binding site. PP5 contains an extended N-terminal region with three tetratricopeptide (TPR) domains, for proteinprotein interactions. PP7 contains five EF and EF-like hand domains (indicated by black boxes) in the C-terminal calcium binding domain. Both PP2B and PP7 contain an insert in the catalytic core (indicated by open boxes) that alters the toxin-binding sites contained in PP1-PP6.
inhibition by this group of natural products. The cation-dependent PPM enzymes are insensitive to inhibition as well (62). Therefore, for the purpose of this thesis, only the PPP enzymes which are sensitive to inhibition by natural products will be discussed in more detail. Initial studies utilized many of the aforementioned natural inhibitors as tools to provide valuable insights into the function and catalytic mechanism of these highly homologous PPases. To date, the most selective inhibitors include: fostriecin,
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demonstrating 104 more selectivity toward PP2A/PP4 than PP1/PP5; OA, demonstrating PP5>PP1; and tautomycin and tautomycetin demonstrating selectivity toward PP1 versus PP2A/PP4 at approximately 5and 40-fold, respectively (70,71). In humans, the highly homologous PPP family is encoded by 12 genes which are capable of counteracting the actions of a much larger family of genes encoding the kinases. This is due to a number of characteristics. The PPases have unique regions at the catalytic N- and C-terminal domains effectively facilitating functional variation. Additional variation can be attributed to the numerous and variable holoenzyme configurations possible with PP1 and PP2A-related PPases. The existence of an assortment of unrelated regulatory subunits bestows diverse substrate specificity, subcellular localization, regulatory mechanisms and physiological functions (73). Furthermore, the potential variability in function is increased further by the presence of multiple isoforms and splice variants. One of the earliest PPases studied was PP1, a Mn2+-dependent phosphatase composed of a core heterocomplex consisting of a 330 amino acid catalytic subunit that binds to a variety of regulatory subunits which are responsible for the localization and activity of the enzyme. In mammals, PP1 is encoded by three genes and has multiple isoforms: PP1, PP1/, PP11 and PP12. PP1 and PP1/ are encoded by unique genes (74,75); whereas PP11 and PP12 are produced from alternative splice variants of a single gene (76). The four isoforms of PP1 identified in humans are highly homologous and retain a high degree of conservation among species, sharing 80% and 60% identity
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with yeast and plants, respectively (77). PP1-binding proteins in mammals have been identified via biochemical methods and include a wide spectrum of major regulatory proteins including glycogen and myofibrillar targeting subunits, nuclear targeting and inhibiting proteins, cytoskeletal and plasma membrane proteins, and regulators of apoptosis (78,79). Relevant to this study, PP1 had previously been implicated in the end stages of cytokinesis. One study in A549 cells demonstrated that antisense-mediated suppression of PP1 resulted in the prolonged retention of anaphase bridges and another study implicates PP1 in lamin dephosphorylation necessary for nuclear envelop reformation in the final stages of mitosis (80). PP2B (calcineurin) is a Ca2+/calmodulin-dependent protein phosphatase that functions as a heterodimer composed of a 60 kDa catalytic subunit and a 19 kDa regulatory subunit. Although the catalytic subunit of PP2B shares about 40% sequence homology with that of PP1 and PP2A, it contains inserts within the “toxin-binding domain” that renders it insensitive to inhibitor binding. Additionally, PP2B distinctively contains an additional 170-amino acid C-terminal region where the calmodulin-binding domain resides, as well as, additional Ca2+ binding sites on its regulatory subunit (81,82). PP2B activity is also modulated by FK506-binding proteins, cyclophilins, and requires bivalent Mn2+or Mg2+ for optimal activity (83-85). In the brain, PP2B is abundantly expressed in regions susceptible to neurodegenerative diseases, epilepsy and stroke and has been implicated in memory function by modulating long-term potentiation (81). Additional roles for PP2B include inflammation and immunosuppression as inhibitory studies with cyclosporin A and FK506 suppresses IL-2 production and other cytokines in activated T cells (84).
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PP2C is a 42 kDa Mg2+/Mn2+-dependent monomeric enzyme that displays some overlapping substrate specificity with PP1 and PP2A despite the lack of substantial homology with the other evolutionarily-related PPases. Multiple isoforms of PP2C have been recognized including PP2C, PP2C, and PP2C in humans and multiple homologues in yeast and mice (86,87). PP2C is rich in heart and skeletal muscle and widely expressed in mammalian tissues. Studies have associated PP2C with negative regulation of the cell cycle via inactivation of the cyclin-dependent kinases (Cdks), human Cdk2 in HeLa cells and Cdc28p in budding yeast (88,89). PP2C reverses the activating phosphorylation catalyzed by Cdk-activating protein (CAK). Notably, it has been implicated in cystic fibrosis via inactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) (90-95). A single gene encodes PP5 which was identified much later as an ubiquitously expressed member of the “toxin-sensitive” PPases sharing only ~43% identity with PP1 and PP2A. This enzyme also contains three tetratricopeptide repeat (TPR) domains which facilitate protein-protein interactions (96-102). PP5 exhibits 97% identity across human, mouse and rat. While this high degree of conservation across species suggests a fundamental biological role, only the knockout of PP5 in Drosophila melanogaster has effectively demonstrated embryonic lethality (103). Studies have been undertaken on the yeast homologue, PPT1, yet no readily apparent phenotype has been shown to date (99). In PP5-deficient mice, viability and survival throughout postnatal life has been demonstrated by a number of groups (unpublished data). Recently, studies in PP5deficient MEF cells indicate a defect in the DNA damage checkpoint in response to ionizing radiation in G2/M (104,105). PP5 has been implicated in diverse signaling
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cascades in response to cellular stress, growth factors and hormones through an assortment of interactions facilitated by the TPR domains including: radiation-induced DNA damage, glucocorticoid receptor signaling, p53-mediated growth arrest and oxidative-stress mediated apoptosis (106-109). Interestingly, implications regarding an opposing function to PP2A suggest the TPR domain of PP5 has been shown to competitively associate with regulatory subunits of PP2A. Specifically, the PR65 scaffolding subunit has been shown to interact with PP5 by yeast two-hybrid screen and the B” (PR72) subunit of PP2A has been shown to co-immunoprecipitate with PP5 (110). The two genes encoding PP7 (also called PPEF1 and PPEF2) share less than 35% identity with PP1 and PP2A and contain the conserved toxin-binding domain in the catalytic subunit, however inserts within this region render PP7 insensitive to inhibition by the inhibitors (111). A variation within the structure of the enzyme is that it includes EF hand motifs located in the C-terminus that function to activate PP7 by facilitating Ca2+ binding. Finally, expression of PP7 is limited to the developing fetal brain, primary sensory neurons and the retina (111). PP2A-Related Phosphatases The PP2A family includes PP2A, PP4 and PP6 which are structurally-related serine/threonine protein phosphatases sharing a high degree of amino acid identity. The catalytic subunits of PP4 (originally PPX) and PP6 are ~65% and 57% identical to PP2A, respectively (112), yet are encoded by distinct genes and classified as distinct families (Figure 4) (113). The PP2A family members also share a wide variety of functional diversity due to the numerous heterotrimeric configurations possible. Whereas studies examining
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Figure 4. Sequence Alignment of the Catalytic Subunit of PP2A with other Homologous Phosphatases of Human Origin. Both and isoforms of PP2A are shown. Secondary structural elements of the catalytic subunit of PP2A are indicated above the sequences. Conserved residues are highlighted in yellow. Residues that make hydrogen bonds to the scaffolding subunit via their sidechain and main-chain atoms are identified by red and green circles, respectively. Residues that contribute to van der Waals interactions with the scaffolding subunit are denoted by blue squares. Residues that specifically recognize the toxins through hydrogen bonds and van der Waals contacts are shown in red and yellow triangles, respectively.
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PP4 and PP6 have only recently emerged, PP2A has been extensively studied and implicated in the regulation of a number of remarkably diverse cell signaling cascades. This pleiotropic characteristic is largely due to differential regulation, restricted substrate specificity, and tissue- and cell-specific distribution attributed to the individual heterotrimeric configurations. Protein phosphatase 2A (PP2A) represents a family of holoenzyme complexes formed by three distinct subunits, a 36 kDa catalytic subunit (PP2Ac) which forms the core dimer when bound to the scaffolding regulatory subunit, 65 kDa (PR65). This dimer associates with one of many variable regulatory subunits that confer extensive diversity to the complex (Figure 5) (114). Human PP2A has two conserved isoforms of the catalytic subunit which share >97% identity to each other, PP2A and PP2A, and share ~80% identity with yeast PP2A homologues (73,115,116). The alpha subunit is expressed ~10fold more than the beta subunit due to a weak promoter. The catalytic subunit of PP2A forms a bi-metallic active site for phosphor-ester hydrolysis and is regulated by phosphorylation at the C-terminus Tyr307 by the tyrosine kinases p60 v-src, p56 lck, and by EGFR-kinase, which reduces enzyme activity by over 90% and can be restored with dephosphorylation (73). Methylation on Leu309 by S-adenosylmethionine-dependent leucine carboxyl transferase 1 (LCMT1) is required for the binding of family members within the PR55B subunits (117,118). Due to tight regulation at both the translational and post-translational levels, ectopic expression of the catalytic subunit of PP2A has remained elusive (112,119). Nonetheless, the importance of this phosphatase is underscored by the resulting lethality in yeast and mice from loss of PP2A (120).
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Figure 5. Structure of the PP2A Holo-enzyme. Schematic overview of PP2A holoenzyme composition showing interaction of the PP2A catalytic subunit to the PR65 scaffold protein at five C-terminal Huntington/elongation/ A-subunit/TOR (HEAT) repeats. The variable B subunit interacts with the N-terminal HEAT domains of the PR65 scaffold subunit or directly to the PP2A catalytic subunit. The B family is encoded by four related genes (///); the B’ family is encoded by five related genes (////) some of which give rise to alternative splice variants; the B’’ family contains three related genes encoding PR48/70 and G5PR with splice variants PR72 and PR130; and the B’’’ family consists of PR93 and PR110. The boxes highlight the cancer relevant mutations described to date in the two isoforms of PR65.
The catalytic subunit of PP2A binds to either of the regulatory subunit isoforms, PR65 or PR65. Studies suggest that the formation of the core enzyme changes the conformation of the scaffolding subunit into a horse shoe-like or bent structure that facilitates unimpeded binding of the mutually exclusive B regulatory subunits to the core dimer (55). PR65 has demonstrated the ability to interact with each of the B families of
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regulatory subunits; however PR65 has been shown to preferentially bind to PR72. Although the isoforms share 87% sequence identity, loss of PR65 isoform, which is expressed 10-fold higher, cannot be rescued by substitution of the isoform due to unique biochemical properties which alter the ability to bind to the regulatory subunits (55,73). Cancer producing mutations have been identified in both isoforms including: PR65 mutations E64D, E64G, R418W, and 171-589; and PR65 mutations G8R, P65S, G90D, L101P, K343E, D504G, V545A, V448A AND 344-E388 (121). Fifteen human genes have been identified that encode at least 26 alternate transcripts and splice variants of the B subunits. Tissue specific expression also participates in the substrate specificity ascribed to the individual PP2A holoenzymes. Most regulatory PP2A subunits are ubiquitously expressed; however, PR55 and PR55 are mainly expressed in neuronal tissue whereas PR72 is found in muscle tissue. The regulatory B families are further divided into five groups due to the lack of sequence similarity with the exception of the conserved amino acids necessary for interaction with the N-terminal HEAT domains of the scaffolding subunit. The regulatory B family of subunits currently include the following families, each will be discussed briefly: the B/PR55 (,,, and ), B’/PR61/PR56 (,,,, and ), B’’/PR72 (PR130, PR48/70, and G5PR), B’’’PR110/PR93 family, and the PTPA /PR53 family (73,116,122-124). There are at least six B/PR55 members transcribed from four different genes exhibiting developmental regulation and distinct spatial distribution, directly or indirectly via binding partners. Substrate specificity for this family is dependent upon five WD repeats that contain a conserved sequence of 40 amino acids ending in tryptophan-
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aspartate (WD) which mediates direct protein-protein interactions. Additional regulation lies in the methylation of Leu309 and dephosphorylation of Thr 304 which is required for binding to the B/PR55 subunit family members (125,126). The B’/PR61/PR56 family shows diverse tissue distribution within the five different genes and shares little similarity with the other B families. This family is distinguished by the presence of -helices akin to the PR65 scaffolding subunit of the core enzyme. It is thought that the highly acidic side of B’-PR61/PR56 is primed for recruiting basic substrate proteins into its concave side (115,123). The PR72 and PR130 subunits were the first B’’/PR72 family members discovered and found to have different promoters yet are transcribed from the same gene. Differing tissue specificities result from a functional replacement of amino acids in the N-terminus. PR72, which binds pRB, contains calcium binding EFX hand domains while newer members such as G5PR contain EFX hand domains that have been found to associate with PP5 and PP2A scaffolding subunit PR65 (127). Other members containing EFX hand domains include splice variants PR70 and PR48, the later interacting with cdc6 (128). The last B regulatory family is classified more accurately as a chaperone due to its direct role in regulating PP2A’s enzymatic activity and is referred to as the PTP/PR53 family with 8 splice variants (121). Protein phosphatase 4 (PP4, PPP4, PPX) is a highly conserved ubiquitous serine/threonine phosphatase, now identified as a regulator of various cellular pathways independent of the highly homologous and structurally-related PP2A. The PP4 heterotrimer is composed of a catalytic subunit (PP4c) that interacts with two regulatory
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subunits which control and regulate the activity of PP4: a 105 kDa subunit, R1; and a 55 kDa subunit, R2 (112). The 35kDa catalytic subunit shares ~65% identity with PP2Ac (129,130). R2 appears to act as a core regulatory subunit for other subunits and a prerequisite for binding of either the - or - isoform of the 95 kDa mammalian subunit R3 (131,132). The catalytic PP4 subunit has been shown to localize in the pericentriolar region around the centrosomes, found in the nucleus and to a lesser degree cytoplasmically in both human and Drosophila cells. Early studies have also shown that R2 also is present at the centrosome in human cells (131,133). Disrupting the PP4 catalytic subunit in D. melanogaster generated the centrosomes minus microtubules (cmm) semi-lethal phenotype described as having centrosomes displaying no radiating microtubules with spindles missing or disconnected from the centrosomes (133). Furthermore, RNAi of PP4 in C. elegans resulted in a comparable phenotype with abnormal spindle formation both in mitosis and sperm meiosis (134). Finally, studies of PP4 in D. melanogaster (133) and C. elegans (134) demonstrate that deficiencies in this enzyme block the formation of the mitotic spindle and indicate that PP4 is required for the maturation of the centrosome. Although PP4 is required for centrosome function in invertebrates, its function in mammals is less understood. PP4 has also been implicated in other cellular processes including components of the motor neuron complex and spliceosomal assembly (129,131). PP4 has been shown to have a role in diverse signaling pathways, thus a minimum level of PP4 activity is likely to be necessary for cell survival (9-12). Evidence of diverse PP4c regulation has been
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shown in NFB, JNK, and rapamycin (TOR) pathways, TNF-dependent insulin receptor signaling and histone deacetylase activity (135-138). Protein phosphatase 6 (PP6) There is an abundance of well-documented literature on PP2A and recently more has been learned regarding PP4; comparatively, the function and regulation of PP6 is substantially less understood. Recent studies suggest that PP6 functions like PP2A and PP4 as a heterotrimer. What is known currently is that PP6 functions as a heterotrimer composed of a catalytic subunit bound to a scaffolding subunit that is conserved in eukaryotes (112,139,140).
Natural Inhibitors of the Serine/Threonine Protein Phosphatases Many PPase inhibitors were identified as eukaryotic cell toxins derived from biologically diverse natural products produced by marine sponges (okadaic acid (OA) and calyculin A), cyanobacteria (nodularin and microcystin variants), Streptomyces (fostriecin and tautomycin) and blister beetles (cantharidin) (70). Original studies into the function of the serine/threonine phosphatases were developed from the discovery and isolation of okadaic acid (OA) produced from a common black sponge, Halichondria okadaii, which was later identified as the causative agent in diarrheic seafood poisoning (DSP-1) (141-143). The landmark studies into the molecular mechanism of OA were compiled by Bialojan and Takai demonstrating potent inhibition of PP1 and PP2A (144). Later, OA was determined to be a relatively potent inhibitor of PP1, PP2A, PP4 and PP5, and a weak inhibitor of PP2B. PP2C and PP7 are not sensitive to OA (70). There is a group of PPase inhibitors (calyculin A, microcystin-LR and nodularin) that are highly potent, relatively non-selective and highly toxic; however, a number of
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less toxic inhibitors have been studied that demonstrate higher selectivity within the PPP family (70,72). Notably, some of these demonstrate promising anti-proliferative and cytotoxic effects, both in vitro and in vivo disease models, as well as clinical settings. Because of this, further inquiry into the mechanisms of action are warranted for two of the most promising compounds, cantharidin and fostriecin, which have been studied in humans. While seemingly unrelated and derived from different sources, both natural compounds were initially found to exhibit anti-proliferative and cytotoxic activity. It was not determined until later that the potent inhibition of PPases was substantial and may offer insights into the mechanism of action surrounding the anti-proliferative and cytotoxic effects. However, identification of the specific phosphatase(s) and associated functions critical for proliferation had not been identified.
Cantharidin Ancient writings in the form of tablets dating back to ~180 AD from the Han Dynasty describe the use of Mylabris, a concoction produced from the Chinese blister beetle, for the treatment of abdominal mass (145). Thus, Mylabris represents one of the oldest treatments for cancer in recorded history. In 1810, a French pharmacist (Robiquet) developed a procedure to produce an active constituent of Mylabris, cantharidin (exo-, exo-2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride) which was followed by a period in which cantharidin was employed as an antitumor drug in Europe (Figure 6). By the early 1900’s, however, cantharidin was considered by most physicians as too toxic for internal use and fell out of favor as a treatment for human cancer (146). Today, Mylabris is still employed in traditional Chinese medicine, and
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Figure 6. Structure of Cantharidin. (exo, exo-2,3-dimethyl-7-oxabicyclo [2.2.1]heptane-2,3-dicarboxylic acid anhydride) Cantharidin is a chemical compound secreted by many species of male blister beetles, most notably by the Spanish Fly (Lytta vesicatoria) and given to the female during mating to protect her eggs from predators. Cantharidin was first isolated by Pierre Robiquet in 1810 and is currently used today only in a diluted form to remove warts, tattoos and Molluscum contagiosum due to potentially fatal doses of 10 mg/kg when consumed by humans. Symptoms of cantharidin poisoning include haematuria and abdominal pains. Cantharidin is mistaken as an aphrodisiac due to renal irritation presenting as genital swelling or priapism. However, it can easily cause death which has deemed it illegal to use or sell for this purpose.
recently, interest in the clinical use of cantharidin as an anti-cancer agent has re-emerged following reports that cantharidin and synthetic derivatives produce cytotoxic effects in a number of human tumor cell lines. Still, the clinical usefulness of cantharidin is limited by renal and mucous membrane toxicity (147,148). Case studies of human poisonings indicate that at high concentration, cantharidin acts as an acute toxin, producing nausea, severe abdominal pain, shock, convulsions, coma, and death (146). In cultured cells, when applied at concentrations >10 mol/L, cantharidin also kills animal cells acutely (50% decrease in tumor colony forming units in 42% breast, 33% ovarian and 38% nonsmall cell lung cancer sample (35). Additionally, solid tumor models in mice demonstrated that fostriecin generated rapid necrosis in advanced implanted murine colon tumors (161). The lack of reduced-folate carrier in other solid tumors correlated with
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insensitivity to methotrexate as well as fostriecin due to carrier-mediated uptake of fostriecin. Toxicity studies were conducted primarily in rat and mouse models with limited toxicity data; however, the major toxic effects in mice resulted from 17.5 mg/kg fostriecin dosages and included bone marrow hypocellularity, leukopenia, neutropenia, thrombocytopenia, and diffuse necrosis in lymphoid tissues, all of which were reversible (32,158). Renal toxicities were also reversible including increases in serum BUN, creatinine and 24-hour glucose excretion, and renal lesions (162,163). Clinical data are limited due to stability and supply challenges of fostriecin. As a result, phase I clinical trials were prematurely halted prior to attaining the maximum tolerated dosage (MTD) (32). Nonetheless, encouraging in vitro and in vivo evidence has demonstrated the potential therapeutic effectiveness of fostriecin and, therefore, has sparked the interest of a number of groups to identify promising and stable synthetic analogues that retain the advantageous qualities demonstrated by fostriecin thus far. Collectively, fostriecin treatment demonstrated marked anti-proliferative and cytotoxic qualities at equivalent concentrations reported to demonstrate inhibition of PPase activity. Growth arrest in the G2/M phase of the cell cycle has been shown and is associated with abnormalities in mitotic structures upon microscopic examination of microtubules, centrosomes and chromosomes within cell cultures. Nonetheless, the biological effects of cantharidin and fostriecin, specifically in altering cell cycle progression through mitosis, are poorly understood and thus merit further examination. More detailed studies may offer additional insight into the individual PPases whose inhibition may be responsible for the alterations in cell cycle progression, specifically in
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G2/M. This is especially significant as greater attention has been focused on G2abrogators as anti-cancer therapeutics. This is due to the discovery that G1-checkpoint defects are common in a large number of cancer cells which render them dependent upon the G2-checkpoint. The limitations in targeting replicating cells irrespective of their cancerous state could be addressed, in part, by exploiting this susceptibility of cancerous cells. Therefore, valuable insight may be gleaned through the identification of cell cycle disruptions induced by cantharidin and fostriecin, the identification of the individual PPases they inhibit, as well as their respective functions during mitosis. First, it will be helpful to highlight relevant aspects of the cell cycle with particular attention paid to the dynamic nature of mitosis.
Cell Cycle and Mitosis Great strides have been made in understanding the cell cycle since Robert Hooke, "the father of microscopy", first identified the basic unit of life as a cell in the 17th century (164). Expanding upon this, Rudolf Virchow published his theory Omnis cellula e cellula or "every cell originates from another existing cell like it" in 1858, which is a theory still accepted today (5). Thus, for the continuation of life, division is accomplished in nature through an orderly sequence known as the cell cycle. With the advent of technological advances, the ability to visualize the cell and its components has provided valuable information including the morphological changes that occur throughout cell cycle progression resulting in successful creation of its progeny. Today, morphological changes may be used to identify the distinct phases of the cell cycle, to visualize abnormal alterations found in disease states, and to distinguish effective
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temporal and spatial events that may provide insight into therapeutic intervention. This is especially true for the effective strategies used today by impeding highly proliferative cancer cells by targeting mitosis and cell cycle progression. To do so, an understanding of the distinct morphological changes as a normal cell progresses through the cell cycle will be helpful. The objective of the cell cycle is to generate two genetically equal cells from one. This is done through highly regulated and complex processes. Although variations are present between species, accurately copying and passing on of genetic material to the next generation remains universal. To provide both daughter cells with complete and accurate genetic copies of the genome, two critical events are indispensable: the accurate replication of DNA in Sphase, and the subsequent segregation of chromosomes and division into two cells during M-phase. In approximately one hour, mammalian cells characteristically complete mitosis. This is a relatively short time period compared to non-dividing phases of the cell cycle. The cell cycle progresses through four distinct and successive phases: Gap 1 (G1), Synthesis (S-phase), Gap 2 (G2)-, and Mitosis (M-phase) (165). During interphase, two “gap” phases flank DNA synthesis in the S-phase including G1 prior to S-phase, and G2 prior to M-phase. These decision-making intervals, or gaps, provide the cell with checkpoints to prepare for the next phase and only initiate entrance into the next phase when a threshold of requirements is met. Mitosis (M-phase) is separated into a number of successive phases that must be completed prior to initiating the next stage: Prophase, Pro-metaphase, Metaphase, Anaphase, Telophase and Cytokinesis. Each phase has distinguished functions and morphological characteristics which will be explained in
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further detail. Ultimately, two major events occur, chromosome division during Anaphase and cytoplasmic division during Cytokinesis. The following section will highlight the major events that must be completed to progress to the next stage. While this is by no means exhaustive, it will emphasize relevant matters to this thesis, experimental design and interpretations of the data. Interphase is the longest phase of the cell cycle in which the cells grow by producing more protein and cytoplasmic components throughout G1, S-phase, and G2 (166). At the end of the previous M-phase, G1 begins with a higher rate in the biosynthetic activities required in S-phase such as those necessary for DNA replication. Duration of G1 is highly variable, even among different cells of the same species, and ends with the start of S-phase. The replication of DNA only occurs during the S-phase. G2 follows S-phase preparing the cell for the onset of mitosis. Throughout interphase, chromatin is visualized as loosely bundled coils of genetic material that will double in size during DNA replication. During this time, the cell does not exhibit any gross morphological changes compared to the dynamic alterations visualized throughout the individual phases of mitosis as seen under the microscope. The onset of prophase is first apparent with chromosome condensation, at which time the replicated sister chromatids become bound together at the centromere by the cohesion complex (167). These chromosomes are seen as distinct coils, and the plasma membrane takes on the classically round shape, which are both indicative characteristics of mitosis. Although not as easily visualized, the two centrosomes, for which replication occurred in S-phase separate to opposing sides of the cell and function as coordinating centers for microtubules in animal cells.
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Pro-metaphase initiates open mitosis in most multi-cellular organisms in which the nuclear envelope disassembles allowing the growing microtubules to invade the nuclear space and capture the newly replicated chromosomes (168). The proteinacious trilaminar plates, terrmed kinetochores, form and provide a central dock for microtubules to connect to chromosomes. The kinetochores are composed of a number of proteins, many of which have an integral function in maintaining the integrity of the cell through checkpoints (169). The growing and shrinking of the microtubules, termed “rescue and catastrophe” respectively, provide the dynamic instability necessary for the oscillating movement of chromosomes, termed “congression” (170). During this time, chromosomes can be visualized as moving back and forth throughout the entirety of the cell until the centromeres convene at the equatorial plate and achieve proper chromosome alignment at the metaphase plate. The alignment upon the equatorial plate, or metaphase plate, is indicative of metaphase and is visualized by the central alignment of sister chromosomes equidistant from opposing spindle poles (171). The normal cell is equipped with safety mechanisms called checkpoints that prevent premature progression to the next phase. The specific transition from metaphase to anaphase is held in check by the mitotic checkpoint that generates “wait” signals due to the presence of unattached kinetochores, unsuitable tension or a combination of the two. The latter is more likely considering the intertwined nature of the signaling cascades and the number of proteins involved (172). Currently it is understood that while the actual number of microtubule-binding regions may vary by species, there exists a critical threshold of both microtubule binding and resulting tension that must be met to silence the checkpoint and thus progress to anaphase (172). The
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details surrounding the proteins that participate in these intertwined signaling cascades is beyond the scope of this thesis; however, it should be noted that the inappropriate exit from mitosis typically leads to cell death as genomic fidelity is not maintained (173). Furthermore, it suggests a role for improper regulation of the mitotic spindle checkpoint and may offer insight into the nature of the failure. Once the critical threshold of tension is reached and attached kinetochores maintain metaphase alignment equidistant from the spindle poles, anaphase is initiated and two main events occur. Sometimes anaphase is separated into two phases in which the first is called early anaphase and the later is called late anaphase. Early anaphase is initiated by the activation of the anaphase-promoting complex (APC) which is composed of several proteins and functions as an E3 ubiquitin ligase that targets multiple proteins for degradation by the 26S proteasome. This gives the cell cycle progression directionality due to the irreversibility of the proteolysis (174, 175). Briefly, the APC indirectly triggers the degradation of cohesin resulting in the segregation of sister chromosomes by targeting securin for degradation. Once securin is degraded, separase is no longer inhibited resulting in the cleavage of cohesin and thus the onset of anaphase (174). Visually, this event is observed as the initial separation of the sister chromosomes and anaphase proceeds as chromosomes move to opposing ends of the cell by the actions of depolymerizing microtubules. It is considered the end of anaphase when the cell has succeeded in separating two identical copies of DNA into two distinct populations which can be easily visualized under the microscope. The next two phases begin simultaneously but effectively involve two distinct processes. Telophase is a period in which the alterations undergone in the initial phases
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are reversed such as elongating the cell, attaching chromosomes to opposing ends of the cell, nuclear envelope reformation, and the formation and unfolding of condensed chromosomes into the diffuse array typical in interphase. While mitosis is considered complete at this point, the physical division of the cell has not come to completion. Cytokinesis, while often thought a part of telophase, is a distinct process beginning at the same time. A cleavage furrow forms which is composed of a contractile ring that pinches off two visibly distinct daughter cells (173). The end of cytokinesis marks the end of mitosis and results in two daughter cells containing a complete genomic copy from the parent cell. Figure 1 illustrates the cell cycle depicting the morphological changes that occur at sequential and distinct phases. This figure also highlights critical events for cell cycle progression, highlighting phases of mitosis where the previously discussed targets function, respectively.
Summary and Hypothesis Previous studies revealed a correlation between the anti-proliferative qualities of two naturally-occurring compounds, cantharidin and fostriecin, and the inhibition of the serine/threonine phosphatases; however, the mechanism was unclear. Strong evidence had suggested the mechanism involved the ability to broadly inhibit the PPP phosphatases based on a common region of the catalytic domain, termed the “toxinbinding domain”; however, identification of the specific phosphatase(s) and associated functions critical for proliferations were unclear. Thus, the goal of these studies was to further understand the bio-molecular mechanisms associated with anti-proliferation and cytotoxicity resulting from cantharidin and fostriecin treatment as it relates to the
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reduction in serine/threonine protein phosphatase activity. The conclusions drawn from these studies may provide further evidence into the identification and novel roles of the individual PPases responsible for these effects; and, in doing so, may also provide further insight into the design and development of alternative mitotic targets in the treatment of human cancers. The overall hypothesis of this dissertation is that cantharidin and fostriecin inhibit proliferation and decrease cell survival by inhibiting the serine/threonine protein phosphatases necessary for proper mitotic spindle formation and normal cell cycle progression. The following specific aims were designed to test this hypothesis: Aim (1) To test whether the anti-proliferative and cytotoxic effects attributed to the serine/threonine phosphatase inhibitors, cantharidin and fostriecin, result from disruptions of mitotic progression ensuing from the inhibition of toxin-sensitive PPase activity; Aim (2) To investigate whether the aberrant mitotic effects leading to cell death resulting from cantharidin and fostriecin treatment are, in part, due to inhibition of PP2A and/or PP2β activity; and Aim (3) To investigate whether the aberrant mitotic effects leading to cell death resulting from cantharidin and fostriecin treatment are, in part, due to inhibition of PP4 activity.
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MATERIALS & METHODS
Cell Culture A549 cells were obtained from ATCC. Hela-H2B-GFP cells, a generous gift, were developed by Dr. Kevin Sullivan, et al (The Scripps Research Institute, La Jolla, CA) (176). Both cell lines were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% Fetal Bovine Serum and L-Glutamine (4.0 mM) at 37° C in 5% CO2. The HeLa-H2B-GFP stable cell line was created by fusing the human histone 2B gene (H2B) to the C-terminus of the gene encoding an enhanced green fluorescent protein (eGFP). This chimeric protein was then sub-cloned into a mammalian expression vector driven by a strong mammalian promoter, EF-1. It was then transfected into human HeLa-cells to generate a stable line constitutively expressing H2B-GFP (176).
Flow Cytometry Propidium iodide (PI) staining was conducted for cell cycle analysis by examination of DNA content by flow cytometry. At the times indicated, media and non-adherent cells were removed from culture dishes (and saved if they are to be analyzed). The cells were treated briefly with trypsin to detach the cells and collected by centrifugation (5 minutes at 1,000 rpm). Supernatant was discarded, pellet was resuspended in 1 ml (PBS + PI (50µg/ml)) buffer, and cell count was determined using a hemacytometer. The cells were
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re-suspended at a concentration of 1 x 106 cells/ml in (PBS + PI (50 µg/ml)). 1 ml of cell suspension was transferred to a 15-ml conical centrifuge tube containing 1 ml of Vindelov’s PI and incubated for at least 1 hr at 4C in the dark or overnight (Vindelov’s PI: 1.21g Tris base (0.01M), 584 mg NaCl (10mM), 10mg Rnase (700 U), 50.1 mg propidium iodide (7.5x10-5M), 1.0 Igepal (equiv to NP-40), pH 8.0, 0.2µ filtered, protected from light and stored at 4C in the dark). The cells were passed through a 20gauge syringe needle separate the cells and filtered through a 30 µm mesh. DNA content was then analyzed using routine methods with a Becton Dickinson fluorescence-activated cell sorter (FACS), gating to exclude cell debris.
Viability and Proliferation Assays The ability of cantharidin to inhibit the growth of cultured cells was determined using a tetrazolium based proliferation assay (MTT) and by counting the number of cells daily using a Coulter counter. Cell death was visualized by trypan blue exclusion and DNA-staining using Propidium Iodide. MTT assays were performed using Cell Titer™ (Promega, Madison, WI) using 96-well plates seeded with 5,000 cells/well in 80 l of medium according to the methods supplied by the manufacturer. Cantharidin (0.5-10 M) was added from concentrated stock solutions in N,N-dimethylformamide (DMF). Cells were seeded in 12-well tissue culture plates at a density of 5.0 X 104 cell/dish. On the next day, the cells were treated with specific antisense oligonucleotide or the scrambled mismatch control at a final concentration of 300 nM as described above. On each of the next five days, the cell cultures were treated briefly with trypsin to detach the cells from the dish (three wells from each test group). The number of cells was then
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determined by counting using a hemacytometer. Cell viability was determined with trypan blue staining. The percentage of viable cells was calculated by dividing the number of cells excluding trypan blue by the total number of cells, and the results are reported as the mean ±SD of data collected from three independent experiments.
Phosphatase Assay The measurement of phosphatase activity was conducted by a typical phosphohistone-based phosphatase assay. Phosphohistone was prepared by phosphorylation of histone 2AS (Sigma) with cAMP-dependent protein kinase A (PKA) in the presence of [32P] ATP. Protein phosphatase activity against phosphohistone was determined by the liberation of 32P using either the purified catalytic subunits of the indicated protein phosphatases or a dilute whole cell homogenate of A549 cells. The dephosphorylation of substrate was 10 mol/L), cantharidin rapidly inhibits proliferation and >95% of the cells were killed in 2 to 5 mol/L cantharidin die (Figure 8C). For A549 cells, the LC50 (concentration killing 50% of the cells) for 48 hours of continuous exposure was ~2.0 mol/L. This correlates with the near complete inhibition of divalent cation-independent PPase activity (i.e., PP1PP6). Aim (2) was designed to examine alterations in cell cycle progression resulting from cantharidin treatment at concentrations that inhibit the PPP family. Previously, cantharidin has been shown to kill animal cells acutely in less than 24 hours when applied at concentrations greater than 10 mol/L. In cultured cells, however, lower concentrations (~2-5 M) of cantharidin can elicit a strong apoptotic response in many types of tumor cells (149-151), characterized by membrane blebbing, caspase activation, DNA fragmentation (151), phosphorylation/activation of p53, and increased levels of
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Figure 9. Cantharidin Treatment Alters Cell Cycle Progression of A549 Cells as Measured by Flow Assisted Cell Sorting (FACS) Analysis of DNA Content. A) Cantharidin dose-response. Logarithmically growing A549 cells were treated with cantharidin at the concentrations indicated for 24 hours. Controls were treated with solvent alone. At the time points indicated, the cells were harvested, and DNA content flow cytometry analysis was conducted. Histograms of DNA content shown (x-axis) as fluorescence, and populations of cells containing 2N and 4N DNA are shaded in red (2N and 4N populations, arrowheads), S-phase cells are shaded with diagonal blue lines. The sub-G1 populations (dead and apoptotic) are shaded in blue. In B) and C), double thymidine treatments were used to arrest A549 cultures in late G1 phase and then treated with solvent B) or cantharidin C) after release from growth suppression. At the times indicated, the cells were fixed and processed for DNA content FACS analysis as described above. D) Cantharidin was added to the culture at the times indicated after release from G1 block. The cells were fixed 16 hours after they were released from double thymidine arrest. Data shown are representative of at least three independent experiments each.
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Figure 9.
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both p21waf1/cip1 (150) and Bax (149). To further characterize the growth arrest and cytotoxicity attributed to cantharidin, A549 cells were analyzed using low concentrations of cantharidin to analyze cell cycle progression in the presence of cantharidin compared to controls. A549 cells were treated with the indicated amount of cantharidin (0-5 mol/L) and then subjected to FACS analysis. FACS analysis of Propidium Iodidestained cells indicated that the number of cells containing twice the G1 content of DNA (4N) increased in a dose-dependent manner 24 hours after treatment with 2-5 mol/L cantharidin (Figure 9A). To further test the effects of cantharidin on cell cycle progression, A549 cell cultures that were synchronized by double thymidine treatment were then treated with cantharidin immediately after release from thymidine-induced G1growth arrest. As seen in Figure 9B, following release from the second thymidine block, control cells rapidly progressed into S-phase, and cells with 4N DNA were observed after ~6-7 hours. At approximately 12 hours after release, a G1 population of cells is again observed suggesting that the cells divided. When double thymidine arrested cells were treated with cantharidin immediately after release from growth suppression, the progression of cells into S-phase was not delayed (compare Figure 9B and 9C; 4 HR). However, following DNA replication, the majority of the cantharidin treated cells arrested, most still containing 4N DNA 16 hours after treatment (compare Figure 9B and C; 12 and 16 HR). These studies suggest that cantharidin-induced growth arrest occurred after DNA replication is complete but before cell division (i.e., G2/M-phase). When cantharidin was added to double thymidine-arrested cell cultures at timed intervals (2, 4, 6, and 7 HRS) after release from G1-growth arrest, FACS analysis showed that all treatment groups contained 4N DNA at 16 hours after release (Figure 9D). This
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study reveals that the cantharidin-induced growth arrest occurring after DNA replication but prior to cell division can be elicited when cantharidin is added at time points well into G2/M. This suggested that the critical mechanism may occur later in G2/M. Aim (3) of aim one was designed to examine abnormalities in mitotic progression resulting from cantharidin treatment by indirect immunofluorescence and time-lapse microscopy of cultured cells at concentrations that correlate with the inhibition of the PPases. To investigate mechanisms associated with cantharidin-induced G2-M phase growth arrest, we examined the effects of cantharidin on microtubules which are integral to the dynamic needs of a dividing cell. Immunostaining with anti--tubulin antibodies revealed marked differences between the control and cantharidin-treated cells. At mitosis, >98% of the control cells contain a typical array of anti-parallel microtubules characteristic of a normal bipolar mitotic spindle and metaphase aligned chromosomes (Figure 10B and 10 C, respectively, merged in Figure 10A and z-sections in Figure 10D). Comparatively, the cantharidin-treated cells exhibited an aberrant mitotic spindle apparatus composed of aggregates or bundles or microtubules and condensed chromosomes that were not aligned at the metaphase plate (Figure 11B and 11C, respectively, merged in Figure 11A). The percentage of cells containing aberrant spindles increased in a dose-dependent manner (Figure 11F). Z-sections of confocal images acquired by fluorescence microscopy of fixed mitotic structures suggest that cantharidin does not prevent the polymerization of microtubule structures in general. Rather, cantharidin disrupts the organization of mitotic spindle microtubules (Figure 11D) and the metaphase alignment of chromosomes (Figure 11E). In non-dividing cells, there was no apparent disparity in the microtubule networks in control compared to
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Figure 10. Fluorescent Confocal Microscopy Images of Microtubules and DNA Illustrating Metaphase Alignment of a Representative Control A549 Cell. A549 cell demonstrating mitotic alignment of chromosomes at the metaphase plate between microtubules arranged in a typical bipolar array. A549 cells were fixed after 24 hours on coverslips and examined by confocal microscopy. A) Representative control cell (n=300) with overlay images of B) microtubules shown in green (anti--tubulin primary antibody and Alexa Fluor 488 secondary antibody) and C) DNA shown in red (Propidium Iodide staining). D) Overlay images of same representative control cell in serial z-sections.
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Figure 10.
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Figure 10, Continued.
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Figure 11. Fluorescent Confocal Microscopy Images of Microtubules and DNA Illustrating Representative Aberrations in Metaphase Alignment Resulting from Cantharidin Treatment of A549 Cells (2 mol/L for 24 hours). Cantharidin treated A549 cell demonstrating mitotic aberrations in chromosome alignment and lacking in bipolar arrangement of mitotic spindles. Representative A549 cells treated with 2 mol/L cantharidin, fixed on coverslips 24 h later and examined by confocal microscopy. A) Representative overlay images of B) microtubules shown in green (anti--tubulin primary antibody and Alexa Fluor 488 secondary antibody) and C) DNA shown in red (Propidium Iodide staining). D) Serial z-sections of microtubules. E) Serial z-sections of DNA. F) Metaphase cells (300 for each concentration of cantharidin) were scored as containing normal or aberrant mitotic spindles and chromosomes. Percentage containing normal bipolar spindles (white columns) or aberrant spindles spindles/chromosomes (filled columns).
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Figure 11.
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Figure 11, Continued.
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Figure 11, Continued.
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cantharidin-treated cultures. Bearing in mind the dynamic nature of mitotic progression and the limitations in making interpretations based on fixed images, further insight required a more thorough investigation into cell cycle progression made possible by employing time-lapse live cell microscopy. To further characterize the cellular effects of cantharidin on cell cycle progression, live cell imaging in combination with time-lapse video microscopy was employed using a HeLa cell line that stably expresses histone-2B (H2B) fused to enhanced green fluorescent protein (eGFP) and named (HeLa-H2B-GFP). This cell line constitutively expresses H2B-GFP and has been shown to allow high-resolution imaging of chromosomes without compromising nuclear and chromosomal structures (176). Simultaneously, differential interference contrast (DIC) images allowed visualization of nuclear-cytoplasmic morphology. This allowed closer analysis of the dynamic nature of chromosome congression to the metaphase plate and progression through the cell cycle in addition to basic plasma membrane alterations characteristic of mitotic cells, as well as, dying cells. Figure 12A is representative of the typical mitotic progression observed in control HeLa-H2B-GFP cells (n=62). Selected images depict morphologies observed throughout cell cycle progression from prophase through the completion of cytokinesis in control cells. In 96.8% of control cells, the average metaphase duration was 1.3 hours. This was calculated by the duration of time between the onset of prophase, determined by the initial visualization of chromosome condensation, and anaphase onset, determined by the visualization of aligned chromosomes physically segregating from the metaphase plate. Subsequently, the formation of a cleavage furrow in the plasma membrane was apparent
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Figure 12. Time-lapse Live Cell Microscopy Illustrating Typical Cell Cycle Progression of HeLa-H2B-GFP Cells Compared to Cells Treated with Cantharidin. Images were selected from time-lapse microscopy experiments in which HeLa-H2B-GFP cells were analyzed over 24 hours. Representative microscopy images of fluorescent (GFP) and corresponding differential interference contrast (DIC) HeLa cells expressing H2B-GFP. Phases of the cell cycle (Interphase, Prophase, etc.) are indicated below sequential images of the same cell from interphase through mitosis. Time shown indicates “time-elapsed” from initial onset of prophase in (min) or (h) for each pair of images composed of: (top panel) indicating chromosome movement with H2B-GFP tagged HeLa cells; and, (bottom panel) indicating plasma membrane alterations by DIC. (A) Cells treated with solvent alone illustrating typical chromosome condensation and alignment on the metaphase plate in ~97% of cells scored (n=62). Representative mitotic cells were selected from time-lapse studies to illustrate mitotic progression through the cell cycle highlighting morphology and timing. Control cells illustrate typical mitotic progression through interphase (0 min), prophase (2 min), pro-metaphase (20 min), metaphase (70 min), early anaphase (80 min), late anaphase (86 min), and cytokinesis completion (132 min). Metaphase duration from prophase (chromosome condensation) to anaphase (chromosome segregation) was observed within 1.3 hours in the presence of chromosome alignment and segregation. Cleavage furrow scission is complete by 2.1 hours resulting in two separate daughter cells. Comparatively, in B) and C), representative mitotic cells were selected from time-lapse studies to highlight abnormalities in morphology and timing resulting from 4 M cantharidin treatment compared to controls. B) Representative images illustrate abnormal metaphase alignment of chromosomes and delayed mitotic progression for ~12 to 15 hours resulting from treatment with 4 uM cantharidin in 100% of cells scored (n=68). C) In cells that had metaphase aligned chromosomes at the time of treatment with 4 M cantharidin, chromosomes fail to separate but maintain alignment for ~ 3 hours until they appear to move away from the metaphase plate in an unorganized and random nature similar to the morphology shown in B).
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Figure 12.
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at regions equidistant from opposing ends of the cell within 90 minutes after prophase. This was followed by scission into two physically distinct daughter cells 140 minutes after prophase indicating cytokinesis completion. To address the dynamic nature of time-lapse live cell microscopy while maintaining consistency and accuracy in quantitatively documenting observations made, the identification of abnormal metaphase cells versus typical metaphase cells is based on two distinguishing parameters. First, abnormal metaphase cells included those cells observed with metaphase durations greater than two hours. This was due to results indicating that control cells spent 1.3 hours on average in metaphase with greater than 99.9% of controls completing metaphase within two hours. Thus, the failure to complete metaphase within two hours is consistent with a metaphase arrest. Second, to be scored as an abnormal metaphase cell, the mitotic cells must also exhibit irregular chromosome congression visualized as the repeated failure of multiple chromosomes to attain alignment at the metaphase plate. In untreated HeLa-H2B-GFP cells, the typical progression from prophase to cytokinesis completion required ~2-2.5 hours. Metaphase alignment was observed ~20 minutes after prophase and the onset of anaphase ~50 minutes thereafter (Figure 12A). In comparison, when HeLa-H2B-GFP cells were treated with cantharidin, although no apparent differences from controls were observed prior to prophase, the chromosomes condensed yet failed to align at the metaphase plate delaying cell cycle progression for ~12-15 hours (Figure 12B). During this period, the cells remained rounded in appearance, and the chromosomes remain condensed. After ~12 to 15 hours, the abnormal metaphase cells showed extensive membrane blebbing and perturbations
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characteristic of apoptosis although definitive conclusion of such would require more directed studies. Additional observations were made in mitotic cells that were aligned at the time of cantharidin treatment (Figure 12C). Although alignment was maintained for ~3 hours, the chromosomes appeared to lose alignment and move away from the metaphase plate. However, unlike controls in which sister chromatids moved towards the opposite poles of the cell in a well-orchestrated and smooth manner, the cantharidin-treated cells displayed unorganized movements that appeared random in nature. Because the concentration of cantharidin used in these studies was sufficient to induce marked inhibition of the activity of all the isoforms of PP1-PP5, it was still not clear which cantharidin-sensitive PPase(s) were required for mitotic progression. Thus, the next aim, designed to examine the effects of fostriecin on mitotic progression using time-lapse microscopy, provided a more detailed analysis with a better characterization of the induced mitotic alterations induced at concentrations consistent with the complete inhibition of PP2A/PP4 and ~50% inhibition of PP1/PP5 activity. These studies provided supportive evidence that the critical PPases may be PP2A and/or PP4 considering the concentrations of fostriecin used although conducting selective inhibition in the later aims would be required to suggest proper conclusions. Aim (4) of aim one was designed to examine mitotic progression by indirect immunofluorescence and time-lapse microscopy after 50 M of fostriecin was added to cultured cells. Fostriecin is the most selective PPase inhibitor to date, exhibiting 104 times greater affinity toward PP2A and PP4 versus PP1/PP5 (IC50 values of ~1µM compared to 50µM) (71). Figure 13 illustrates a typical dose-response inhibition curve obtained by using
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Figure 13. Semi-Selective Inhibition of PPase Activity by Fostriecin. The graph illustrates a typical dose-response inhibition curve obtained by using purified PP2A (PP4 has comparable sensitivities based on extreme conservation of the amino acids within the toxin-binding domain), PP1 and PP5.
purified PP2A, PP4, PP1 and PP5 (71). As illustrated, micromolar concentrations of fostriecin added to crude cell homogenates results in near complete inhibition of PP2A/PP4 with little, if any, effect upon the activities of PP1/PP5. It should be noted that the exact uptake of hydophobic inhibitors by cells is difficult to accurately determine due to variability resulting from (1) partitioning into the aqueous media, (2) passive diffusion through the aqueous media, (3) partitioning into and out of the cell membrane, (4) and finally partitioning through the cytoplasm to bind with high affinity to the
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sensitive phosphatases (71). Thus, for the purpose of this dissertation work, it was reasonable to consider that if mitotic abnormalities are observed at concentrations that completely inhibit the activities of both PP2A/PP4 while only minimally inhibiting the activities of PP1/PP5 (50 M), then it would narrow the potential candidates or key PPases which may be accountable for the fostriecin-mediated anti-mitotic effects. That is, this would suggest a role for isoforms of PP2A/PP4 rather than PP1/PP5; nonetheless, selective inhibition would be required to make conclusions. If true, this would be consistent with previous investigations into the cellular effects of fostriecin. FACS analysis revealed concentration-dependent effects: (25-500 M) induced an apoptotic response, 15 M fostriecin resulted in a large population of cells arrested in S-phase, and, low concentrations of fostriecin (1.5-5 M) resulted in a greater population of cells in the G2/M phase. Morphological analysis of mitotic cells treated at lower concentrations provided further support as evidenced by condensed chromosomes, separated spindle poles surrounded by a rounded plasma membrane (159). Lastly, supportive evidence is based on the correlation between the abnormal mitotic features observed during these earlier studies and concentrations that led to the complete inhibition of PP2A and not PP1 (72). To examine the mitotic alterations in more detail, time-lapse images of HeLa-H2BGFP cells were acquired immediately after adding 50 M fostriecin to a sub-confluent monolayer which was consistent with the complete inhibition of PP2A/PP4 and ~50% inhibition of PP1/PP5 in vitro although exact concentrations per cell are difficult if not impossible to determine. Nonetheless, it was reasonable to consider that considerably less actually partitioned through the cell to bind and inhibit the sensitive PPases.
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Figure 14. Time-lapse Live Cell Microscopy Illustrating Representative Abnormalities in Mitotic Progression Resulting from 50 M Fostriecin Treatment of Hela-H2B-GFP Cells for 24 hours. Images of representative HeLa-H2B-GFP cells were selected from time-lapse microscopy experiments conducted to compare cell cycle progression of 24 hours in controls as compared to 50 M fostriecin treatment. Phases of the cell cycle (Interphase, Prophase, etc.) are indicated below sequential images of the representative cell from interphase through mitosis. Time shown indicates “time-elapsed” from initial onset of prophase in (min) or (h) for each pair of images composed: (top panel) indicating chromosome movement visualized by H2B-GFP tagged HeLa cells; and, (bottom panel) indicating plasma membrane alterations by differential interference contrast (DIC). A) Cells treated with solvent alone illustrate typical chromosome condensation and alignment on the metaphase plate in ~97% of cells scored (n=62). B) Representative cell illustrates abnormal metaphase alignment of chromosomes resulting from treatment with 50 uM fostriecin in 100% of cells scored (n=57). The presence of lagging chromosomes (indicated with asterisks in) are present in two distinct metaphase phenotypes which can be distinguished based on variations in chromosome alignment indicated with asterisks (*) in B): Lagging chromosomes surround metaphase aligned chromosomes in a straight single line (indicated with a white line) at the metaphase plate; and lagging chromosomes aligned in an irregular Y shaped (indicated with white lines) metaphase plate in HeLa-H2B-GFP cells treated with 50 µM fostriecin during a prolonged metaphase arrest >10 hours (delayed metaphase ranged from 2-12.5 hours). Subsequently, chromosomes segregated into >2 unequal chromosome clusters in 89.5% of the aberrant metaphase aligned cells and included irregularities in the plasma membrane. Cleavage furrow formation of the plasma membrane was observed in early in anaphase; however, the cleavage furrow regressed over time resulting in one multinucleated cell. White lines surround the perimeter of the cell of interest for easier viewing of the bottom panel. A small portion of aberrant metaphase cells, 4 of the 5 or 80%, divided into 2 clusters of chromosomes similar to controls.
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Figure 14.
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Acquisition of time-lapse microscopy images was conducted at intervals of five minutes for 24 hours, as previously described. Compared to controls, 100% of cells exposed to 50 M fostriecin exhibited distinct abnormalities while progressing from prophase through cytokinesis. Three abnormal morphologies will be discussed in more detail and described as sequential and phase-specific occurrences. Figure 14A illustrates a representative control cell as it progresses from interphase through cytokinesis during a typical mitotic cycle. Briefly, the newly condensed chromosomes oscillate between spindle poles and metaphase plate until the complete metaphase alignment is attained, shown here within 45 minutes. Subsequent chromosome segregation occurs in less than 1.5 hours from onset of prophase into two equivalent chromosome clusters. Finally, the successful completion of cytokinesis is shown ~ 2 hours later, resulting in two distinct daughter cells. An overview of the altered mitotic progression will be discussed in brief as shown in the time-lapse progression of a representative cell treated with 50 M fostriecin in Figure 14B. Initially, the cell enters prophase similar to controls with chromosome condensation and a rounded plasma membrane characteristic of mitotic cells. However, abnormalities soon become apparent during a prolonged metaphase (asterisks) associated with lagging chromosomes in 100% of the cells entering mitosis after treatment with fostriecin. Subsequently, chromosomes segregated after 5 hours into >2 unequal chromosome clusters in 89.5% of the misaligned cells which also included irregularities in the plasma membrane. Shortly thereafter, cleavage furrow formation of the plasma membrane was observed in early anaphase; however most notable, the cleavage furrow
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Figure 15. Enlarged Images Highlight Abnormalities in Chromosome Alignment during Metaphase Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours. Representative images were selected from time-lapse microscopy experiments as described earlier and analyzed for irregularities in metaphase alignment. A) Cells treated with solvent alone illustrate the typical localization of condensed chromosomes aligned equatorially on the metaphase plate in ~97% of mitotic cells (n=62). 100% of cells (n=57) treated with 50 µM fostriecin demonstrate two distinct and abnormal metaphase phenotypes based on the localization and alignment of chromosomes. Representative images illustrate the presence of lagging chromosomes seen in both metaphase phenotypes (indicated with asterisks); and, two alignment variations shown in B) with chromosomes aligned in a straight single line at the metaphase plate (highlighted with white lines); compared to C) with chromosomes aligned in an irregular Y-shaped metaphase plate and highlighted with white lines.
regressed over time resulting in one multinucleated cell in ~90% of cells dividing into >2 daughters (n= 51). The same time-lapse experiments were analyzed for each sequential and phasespecific abnormality resulting from fostriecin treatment in greater detail. Enlarged images in Figure 15 compare the typical metaphase alignment of mitotic cells to two distinct abnormalities observed during chromosome congression after fostriecin treatment. Both fostriecin treated cells contain lagging chromosomes positioned between the equatorial plane and outer edges of the cell (indicated by asterisks). However, these
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two morphologies were distinguished from each other by the different metaphase alignment shown in the majority of chromosomes (indicated with white lines); that is, metaphase aligned chromosomes in a straight single line at the metaphase plate (Figure 15B); or chromosomes aligned in an irregular Y-shaped metaphase plate (Figure 15C). Metaphase arrest varied between 2 and ~12 hours (5.3 hours shown in Figure 14B) and subsequently appeared to segregate chromosomes regardless of attaining full alignment of chromosomes on the metaphase plate, which may suggest a nonfunctional checkpoint. The next specific disruption in mitotic progression occurred during segregation of chromosomes upon exit from mitosis which is highlighted with enlarged images in Figure 16. Cells treated with solvent alone are represented in Figure 16A exhibiting the typical segregation of chromosomes into two distinct and equivalent chromosome clusters in ~97% of mitotic cells (n=62). Also noted is the appearance of one continuous plasma membrane that is relatively smooth and symmetrical as shown extending in opposing directions which is typical of anaphase cells. Comparatively, 50 µM fostriecin exhibited abnormalities in chromosome segregation shown in Figure 16B. In the majority of fostriecin treated cells, segregation into >2 distinct clusters of chromosomes occurred in 89.5% of mitotic cells. As evidenced by viewing the time-lapse sequentially, clusters of chromosomes appeared to pull apart or segregate during this time; however the presence of two equal and distinct clusters, indicative of typical chromosome segregation was rarely observed. Nonetheless, these cells appeared to initiate cytokinesis, albeit in the presence of lagging chromosomes and significant irregularities in the plasma membrane. It should also be noted that ~9% of mitotic cells divided into two daughter cells, however
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Figure 16. Enlarged Images Highlight Abnormalities in Chromosome Segregation Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours. Representative images were selected from time-lapse microscopy experiments as described earlier and analyzed in more detail for irregularities present upon chromosome segregation. A) Cells treated with solvent alone illustrate the typical segregation of chromosomes into two distinct and equivalent chromosome clusters in ~97% of mitotic cells (n=62). Additionally, a single plasma membrane is shown extending in opposing directions typical of anaphase. Comparatively, abnormal segregation of chromosomes results from treatment with 50 µM fostriecin (n=57) in 89.5% of mitotic cells observed with the Y-shaped aberrant mitotic phenotype as represented in B). White outlines surround the perimeter of the cell to highlight representative cell. Additionally, ~9% of fostriecin treated cells appeared to exit mitosis with two distinct populations of segregated chromosomes similar to controls (representative image not shown).
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some did so in the presence of lagging chromosomes with visible irregularities in the plasma membrane. The third distinct abnormality during mitotic progression occurred during anaphase and cytokinesis involving cleavage furrow regression and subsequent failure to divide into two equal daughter cells. As shown in Figure 17A, typical cleavage furrow formation became apparent shortly after chromosome segregation and was observed as lateral ingressions of the cell margins. In fostriecin treated cells, the formation of a cleavage furrow was also apparent shortly after chromosome segregation. However, in comparison, the initial evidence of cleavage furrow formation, as seen by multiple lateral ingressions of the cell margins, was followed by subsequent regressions and resulted in a single plasma membrane surrounding an irregularly shaped multinucleated cell over a variable but prolonged period of time. Of the aberrant mitotic cells dividing into >2 clusters of chromosomes, 90.2% exhibited cleavage furrow regression leading to multinucleate cells. Interestingly, 80% of the cells dividing into two distinct chromosome clusters, as if dividing into two daughter cells, also exhibited this cleavage furrow regression morphology. Although imaging constraints prevented analysis of subsequent cell death in all of these cells, at least 19 of the 57 (33.3%) mitotic cells displayed features consistent with cell death including chromosome compaction and plasma membrane blebbing; however, this is also consistent with disruption of the actin cytoskeleton and micronuclei formation and cannot be discounted. It should be noted that 50 µM fostriecin inhibits a number of PPases, thus the PPase(s) responsible for the abnormalities in mitotic progression during chromosome congression, chromosome segregation and cytokinesis completion are not known.
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Figure 17. Enlarged Sequential Images Highlight Cytokinesis Abnormalities Observed in Hela-H2B-GFP Cells Treated with 50 M Fostriecin for 24 hours. HeLa-H2B-GFP cells were treated with A) solvent alone or with B) 50 M Fostriecin as previously described. Representative images were selected from time-lapse microscopy experiments and analyzed in more detail for irregularities observed in late anaphase through cytokinesis. A) Cells treated with solvent alone illustrate the typical progression from anaphase through cytokinesis completion. 50 M Fostriecin resulted in two types of cytokinesis abnormalities in which cleavage furrow regression results in multinucleated cells. B) highlights 80% of abnormal mitotic cells that divided into 2 sets of chromosomes and C) highlights 90% of abnormal mitotic cells that divided into >2 sets of chromosomes upon exit from mitosis.
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Nonetheless, because fostriecin inhibits PP2A/PP4 at concentrations ~5,000-fold lower than PP1 or PP5, we hypothesized PP2A and/or PP4 were involved. Therefore, the next aims were designed to examine HeLa-H2B-GFP cells suppressed of the individual PPase isoforms of PP2A (Aim 2) and PP4 (Aim 3) to identify and examine in more detail which PPases play key roles in the anti-mitotic and anti-proliferative molecular actions attributed to cantharidin and fostriecin. Aim 2 PP2A has been a relatively well-studied PPase compared to other structurallyrelated members. Because 1) cantharidin is a documented inhibitor of PP1 and PP2A (70); 2) purification studies reveal the high affinity cantharidin binding protein to be PP2A (183); and 3) other known PPase inhibitors, including OA and fostriecin, show higher affinity for the PP2A family at concentrations that stop proliferation and correlate with antitumor characteristics (148), it was reasonable to begin analysis of the individual suppression of PPases with the isoforms of PP2A. Therefore, Aim 2 was designed to test the hypothesis that the aberrant mitotic effects leading to cell death mediated by cantharidin and fostriecin treatment are, in part, due to inhibition of PP2A activity through the following specific aims: Aim (1) Synthesize and confirm antisense oligonucleotides specificity in targeting unique sequences contained in the mRNA of PP2A and PP2A by phosphatase assay, Northern and Western blot analysis; Aim (2) Examine alterations in components of the mitotic phenotype by indirect immunofluorescence microscopy of cultured cells due to antisense-mediated suppression of PP2A and PP2A; and Aim (3) Determine uncharacteristic variations in the mitotic progression of Hela-H2B-GFP cells suppressed of PP2A activity by measuring mitotic
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Figure 18. Specific and Potent Inhibition of PP2A by Treatment with Antisense Oligodeoxynucleotides. A) Relative positioning of the predicted hybridization sites within the human PP2A or PP2A mRNA of 43 antisense oligonucleotides that were evaluated for inhibition of PP2A expression in cultured A549 cells. Antisense oligodeoxynucleotides that inhibit the expression of PP2A or PP2A mRNA were identified by treating A549 cells with the indicated oligodeoxynucleotides at a concentration of 300 nmol/L. mRNA was prepared 24 hours later and analyzed for PP2A, PP2A and gyceraldehyde-3phosphate dehydrogenase (G3PDH) mRNA levels by Northern blot analysis. B) Inhibition of PP2A levels by ISIS 110179, ISIS 110181, ISIS 110186, or ISIS 101089. C) Inhibition of PP2A by ISIS 110159. IC50 values were estimated from plots produced by quantification of PP2A mRNA levels after normalization to G3PDH mRNA in A549 cells following treatment with increasing concentrations of oligodeoxynucleotides illustrated for ISIS 110159. D) Specificity of oligonucleotides targeting PP2A (ISIS 110159) and PP2A (110181). A549 cells were treated with 300 nmol/L) of the indicated antisense oligodeoxynucleotides or corresponding mismatched control analogues (MM) that contain 13 base changes (mismatches) within the sequence of the indicated target. Total mRNA was prepared 24 hours later and analyzed for PP2A, PP2A and G3PDH mRNA levels by Northern blot analysis. E) Western blots of PP2A protein levels in A549 cells. Cells were treated with the mismatch control oligodeoxynucleotides, ISIS 110159 targeting PP2A, or ISIS 110181 targeting PP2A. Protein extracts were prepared 48 or 76 hours later. Each lane contained 40 g protein. F) Target-specific inhibition of ISIS 110159 and ISIS 110181. A549 cells were treated with 200 or 400 nmol/L antisense oligodeoxynucleotides targeting the indicated isoform of PP2A. Total protein was prepared 48-72 hours later and analyzed for PP4 and PP6 by Western blot.
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Figure 18.
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Figure 18, Continued.
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Figure 18, Continued.
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duration, dynamic chromosome alignment and cell death utilizing time-lapse video microscopy. Aim (1) was designed to allow the identification of oligonucleotides that would suppress PP2A production. For these studies, ~45 different oligonucleotides targeting unique sequences contained in the mRNA of the respective PPases were synthesized and tested. Each antisense oligonucleotide tested was 20 bases in length and designed to hybridize to a unique region of human PP2A or PP2B. Twenty-three were designed to target PP2A and twenty targeted PP2B (Figure 18A). All of the oligonucleotides tested in the primary screen were second generation chimeric oligonucleotides, containing 10 central phosphorothioate oligodeoxy residues (gap) flanked by five to six 2’metholxyethyl-modified residues on the 3’ and 5’ ends. These modifications have been shown previously to enhance the potency of antisense oligonucleotides targeting mRNAs encoding other proteins (178,179). Because phosphorothioate oligonucleotides have been shown to commonly act through RNase H-dependent mRNA cleavage mechanism in cells (184), the ability of each oligodeoxynucleotide to specifically inhibit the expression of PP2A was initially determined by Northern blot analysis probing for levels of either PP2A- or PP2A- mRNA. A comparison of PP2A mRNA levels in A549 human lung carcinoma cells treated with (300 nmol/L) PP2Ac-specific antisense oligonucleotides in the presence of cationic lipids is shown in Figure 18B. The antisense oligonucleotides with the most potent activity against the PP2A mRNA identified in this series were ISIS 110159 and ISIS 110163 and the PP2A mRNA identified in this series were ISIS 110179, ISIS 110181 and ISIS 110186 (arrows in Figure 18A). Increasing concentrations (0-300 nmol/L) of
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PP2Ac-specific antisense oligonucleotides are shown in Figure 18B and demonstrate a concentration-dependent reduction in PP2A mRNA levels. In Figure 18C, increasing concentrations (0-300 nmol/L) of PP2A-specific antisense oligonucleotide (ISIS 110159 shown) demonstrate a concentration-dependent reduction in PP2A mRNA levels. The effective oligonucleotides and several of the oligonucleotides with moderate or no effect were also tested against other human cell lines for effects on the expression of PP2A mRNA levels with essentially identical results. To examine the specificity of oligonucleotides targeting PP2A (ISIS 110159 and ISIS 110163) and PP2A (ISIS 110179, ISIS 110181, ISIS 110186, and ISIS 110189), the effects of each oligonucleotide and appropriate mismatch control analogues (designated ISIS 110159 MM1, etc) were tested. The mismatch control analogues contain the same base composition as the corresponding antisense oligonucleotide but with sequences non-complementary to the target mRNA. As indicated in Figure 18D, treatment of A549 cells resulted in the reduction of the appropriate PP2A mRNA levels with the most potent antisense oligonucleotides having IC50s 20 times the IC 50 (17.8 nmol/L) for PP2A (Figure 18F). Because the sequence targeted by ISIS 110159 is not contained in PP4 or PP6, as expected the ISIS 110159 did not inhibit the expression of these proteins. In the same manner, the sequence targeted by ISIS 110181 was also not expected to inhibit the expression of PP4 nor PP6; however 200 nM ISIS 110181 targeting PP2Aβ appears to decrease the amount of PP4 protein levels at 48 hours. This may be due to inappropriate loading of less protein considering protein levels by 76 hours do not appear decreased.
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Aim (2) was designed to determine if aberrant alterations in mitotic Hela-H2B-GFP cells suppressed of PP2A activity were present. Immuno-staining with anti--tubulin antibodies revealed differences in the appearance of the mitotic spindles only in the ISIS 110159 (targeting PP2A)-treated cells (compare Figures 10A-D and 19A-E); whereas the ISIS 110181 (targeting PP2Aβ)-treated cells were similar to controls with a bipolar array of microtubules. As observed following treatment with cantharidin and fostriecin, cells treated with ISIS 110159 but not ISIS 110181 contained condensed yet scattered and misaligned chromosomes (Figure 19C). In addition, the mitotic spindles in ISIS 110159-treated cells were not well organized as compared to controls and ISIS 110181treated cells. However, most mitotic cells still produced a somewhat bipolar spindle apparatus (Figure 19A) and the microtubules of non-dividing cells with suppressed PP2A expression were similar in appearance to untreated, mismatched controls and ISIS 110181-treated cells. To test for centrosome abnormalities, immuno-staining with antibodies targeting pericentrin revealed the normal complement of two centrosomes in ISIS 110159-treated A549 cells (Figure 19B), as well as, in ISIS 110181-treated A549 cells which previously were shown with control-like mitotic spindles. An overlaid image of a representative ISIS 110159-treated cell in Figure 19D is also shown as overlaid z-sections in Figure 19E to demonstrate alterations in mitotic spindle formation. Aim (3) was designed to test the hypothesis that the reduction of PP2A expression with ISIS 110159 leads to abnormal metaphase progression. Thus, to further characterize
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Figure 19. Enlarged Images of Representative Cells Suppressed of PP2A via Antisense Oligonucleotide-Mediated Suppression. Representative confocal microscopic images of A549 cells treated with ISIS 110159 obtained from five or more independent experiments. A549 cells were treated with 300 nmol/L ISIS 110159, fixed on coverslips 48 hours later and examined by confocal microscopy. A) Microtubules are shown in green (anti--tubulin primary antibody and Alexa Fluor 488 secondary antibody). B) Arrows point to two centrosomes shown in red (anti-pericentrin primary and Alexa Fluor 594 antibodies). C) DNA is shown in blue (staining with Draq5). D) Computer-assisted merge of images A-C. E) Serial zsections of same representative mitotic cell treated with ISIS 1101059 suppressing PP2A.
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Figure 19.
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Figure 19, Continued.
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the mitotic alterations attributed to ISIS 110159, we acquired images of HeLa-H2B-GFP cells treated with ISIS 110159 beginning 24 hours post transfection through 48 hours as described earlier. Cell division occurred normally prior to 30 hours. After 30 hours, time-lapse fluorescence microscopy revealed a majority of chromosomes condensed and eventually maintained alignment at the metaphase plate. However, as compared to controls shown in Figure 20A, anaphase onset was delayed for >15 hours in the presence of lagging chromosomes that exhibited a continuous oscillatory congression of chromosomes yet never achieved nor maintained a full alignment of chromosomes at the metaphase plate. This was true in 89% of Hela-H2B-GFP cells suppressed of PP2A and entering mitosis 30 hours post treatment (n=45) (Figure 20B as compared to 20A). Comparatively, 95% of Hela-H2B-GFP cells suppressed of PP2Aβ (n=29) demonstrated progression through mitosis comparable to controls (see Figure 27A). This mitotic morphology was strikingly similar to the earlier metaphase phenotype observed in studies examining mitotic HeLa-H2B-GFP cells treated with 50 µM fostriecin in which lagging chromosomes were observed surrounding a single straight metaphase plate (Compare Figures 20B with 14B and enlarged image in Figure 15B). Considering that 50 µM fostriecin is consistent with >99% inhibition of PP2A, observations of similar alterations during mitotic progression are not surprising. Comparatively, the additional morphology observed later in the fostriecin treated cells distinguished by lagging chromosomes surrounding a Y-shaped metaphase plate, (Figure 15B) was not observed in the studies with PP2A suggesting a functional checkpoint with no anaphase onset. These results suggest that the inhibition of another PPase may be responsible and, thus, the focus of Aim 3.
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Figure 20. Time-lapse Live Cell Microscopy Illustrating Irregularities in Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP2A Compared to Controls. Time-lapse microscopy experiments were conducted as described earlier in which HeLaH2B-GFP cells were treated with ISIS 110159 targeting PP2A for 30 hours prior to acquisition of time-lapse images. Representative microscopy images of fluorescent (GFP) and corresponding differential interference contrast (DIC) HeLa cells expressing H2B-GFP. Phases of the cell cycle (Interphase, Prophase, etc.) are indicated below sequential images of the same cell from interphase through mitosis. Time shown indicates “time-elapsed” from initial onset of prophase in (min) or (h) for each pair of images composed of: (top panel) indicating chromosome movement with H2B-GFP tagged HeLa cells; and, (bottom panel) indicating plasma membrane alterations by DIC. A) As shown earlier, cells treated with solvent alone illustrating typical chromosome condensation and alignment on the metaphase plate in ~97% of cells scored (n=62) and are shown again for comparison. B) Representative mitotic cells were selected from time-lapse studies of HeLa-H2B-GFP cells treated with SIS 110159 (targeting PP2A) to compare with untreated cells. Representative images illustrate a majority of chromosomes attain alignment at the metaphase plate, however, a small portion of chromosomes are shown lagging during a delayed metaphase. Mitotic arrest is prolonged for > 15 hours without attaining full alignment in 89% of mitotic cells scored (n=45).
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Aim 3 A growing body of evidence suggests that PP4 also plays important roles in mitosis, suggesting that fostriecin-mediated suppression of PP4 contributes in the fostriecinmediated mitotic phenotype. PP4 was determined to be essential in Drosophila embryos and was required for microtubule organization at the centromere (133). PP4 has also been determined to be necessary in C. elegans for centrosome maturation in mitosis and sperm meiosis (134). Therefore, Aim 3 was designed to test the hypothesis that the aberrant mitotic effects leading to cell death mediated by cantharidin and fostriecin treatment are, in part, due to inhibition of PP4 activity through the following specific subaims: Aim (1) Synthesize and confirm oligonucleotides specificity in targeting unique sequences contained in the mRNA of PP4 by phosphatase assay, Northern and Western blot analysis; Aim (2) Examine alterations in components of the mitotic phenotype by indirect immune-fluorescence microscopy of cultured cells due to antisense-mediated suppression of PP4; and Aim (3) Determine uncharacteristic variations in the mitotic progression of Hela-H2B-GFP cells suppressed of PP4 activity by measuring mitotic duration, dynamic chromosome alignment and cell death utilizing time-lapse video microscopy. Aim (1) was designed to allow synthesis and confirmation of oligonucleotides specificity in targeting unique sequences contained in the mRNA of PP4 by Northern and Western blot analysis. To develop antisense oligonucleotides that potently suppress the expression of the PP4, ~20 different oligonucleotides (20 bases in length) were designed and synthesized to hybridize to unique sequences contained in the mRNA of PP4 (186).
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Figure 21. Specific Inhibition of PP4 by Treatment with Antisense Oligodeoxynucleotides. A) Northern blots of PP4 mRNA levels in A549 cells. Antisense oligodeoxynucleotidemediated inhibition of the expression of PP4 mRNA was identified by treating A549 cells with increasing concentrations (0-500 nmol/L) of the oligodeoxynucleotides, ISIS 134947 (targeting PP4) or ISIS MM (mismatch) which contains 13 base changes within the sequence of the indicated target. mRNA was prepared 24 hours later and analyzed for PP4 and gyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA levels by Northern blot analysis. B) Western blots of PP4 protein levels in A549 cells. Cells were treated with the mismatch control oligodeoxynucleotides or ISIS 134947 targeting PP4 at indicated concentrations. Total protein was prepared at indicated times, each lane contained 40 g protein and was analyzed for PP2A, PP5 and PP6 by Western blot.
All of the oligonucleotides were second generation chimeric oligonucleotides, and the characteristics of second generation oligonucleotides were described in Aim 2 (134). Specific inhibition of PP4 expression was determined by Northern blot analysis. PP4 mRNA was detected using PP4-specific cDNA probes forming a hybrid with a single transcript of the target PP4 mRNA. In mammalian cells, levels of PP4 mRNA
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were compared after treatment with Lipofectin (DOTMA.DOPE; Lipofectin) to facilitate uptake and 20-500 nmol/L of ISIS 134947, the PP4-specific antisense oligonucleotide with the most potent activity against PP4. Results demonstrate that PP4 mRNA expression was reduced in a dose dependent manner exhibiting pronounced effects at both 300 and 500 nmol/L (Figure 21A). Blots were stripped and re-probed with GADPH cDNA probe to demonstrate equal RNA loading (Figure 21A). To confirm the specificity of ISIS 134947 in targeting PP4, mismatch control analogues were tested. The mismatch analogues were designed to contain the same base composition as ISIS 134947 but with non-complementary sequences to the target PP4 mRNA. Treatment with increasing concentrations (25-500 nM) of mismatch control analogues resulted in no observable effect on protein levels as compared to increasing concentrations of (25-500 nM) ISIS 134947 antisense oligonucleotides tested by Western analysis (Figure 21B). To further test the specificity of the PP4-specific antisense oligonucleotides, expression levels of three structurally related PPases, PP2A, PP6, and PP5 were tested by Western analysis after transfection with ISIS 134947. The individual antibodies were generated against peptides unique to PP4, PP2A, PP6 and PP5. ISIS 134947 showed no observable effect on the protein expression of PP2A, PP6 nor PP5 even at concentrations that significantly reduced the protein expression of PP4 (Figure 21B). Inhibition of these structurally related PPases using RNaseH-mediated suppression was not expected as the unique sequence targeted is not present in PP2A, PP6 or PP5. Additionally, as expected, there were no observable effects on cells treated with mismatch controls when measured for the specific protein levels of PP2A, PP6 and PP5. Therefore, transfection of HeLa-
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Figure 22. Confocal Z-sections of Cells Suppressed of PP4. Representative confocal microscopic images of HeLa-H2B-GFP cells treated with ISIS 134947 obtained from five or more independent experiments. Cells were treated with 500 nmol/L ISIS 134947, fixed on coverslips 48 hours later and examined by confocal microscopy. Serial z-sections of representative mitotic cell treated with ISIS 134947 suppressing PP4. A) Chromosomes shown in green (H2B-GFP) and centrosomes shown in red and pointed to with red arrows (anti-pericentrin primary and Alexa Fluor 594 antibodies). B) Chromosomes shown in green (H2B-GFP) and microtubules are shown in red (anti--tubulin primary antibody and Alexa Fluor 594 secondary antibody).
H2B-GFP cells with ISIS 134947 is capable of significant reduction in the levels of PP4 protein without altering the protein levels of the other structurally-related PPases. Aim (2) was designed to examine alterations in components of the mitotic phenotype by indirect immunofluorescence microscopy of cultured cells due to antisense-mediated suppression of PP4. I immunostaining revealed disruptions although they were not as uniform as those observed after PP2A suppression. Figure 22 highlights two representative cells exhibiting abnormal mitotic features. In Figure 22A, the mitotic cell suppressed of PP4 represents a population of cells that shared similarities to the PP2A
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suppressed cells. For easier viewing, four serial z-sections are shown and contain condensed chromosomes (green) and immunostaining with antibodies targeting pericentrin revealed the presence of two centrosomes (red). Microtubules appeared similar to those seen in cells suppressed of PP2A. However, there was also a population of PP4 suppressed cells that had accentuated disruptions. Figure 22B highlights these abnormalities as four serial z-sections as well. In this figure, chromosomes (green) are condensed and immunostaining with antibodies targeting tubulin revealed the presence of microtubule aggregates. However, consistent centrosome staining with pericentrin was difficult to achieve in mitotic cells with accentuated disruptions. Because of the limitations of fixed microscopy and the availability of live cell analysis, a more detailed analysis with time-lapse was conducted to gain better insight. Nonetheless, as observed with cantharidin, fostriecin and PP2Aspecific antisense oligonucleotides, the microtubules in non-dividing cells with suppressed PP4 expression were similar in appearance to untreated or mismatched controls. Aim (3) was designed to test the hypothesis that the reduction of PP4 expression with ISIS 134947 leads to abnormal mitotic progression. To examine the dynamic effects of PP4 suppression on chromosome morphology and cell cycle progression, we once again utilized fluorescent microscopy to analyze asynchronous log phase HeLa-H2B-GFP cells treated with antisense oligonucleotide ISIS 134947, specifically targeting PP4c. Acquisition of time-lapse was initiated 24 hours post transfection and continued for another 48 thereafter at five minute intervals. Quantitative analysis of cells was then
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limited to cells entering mitosis 40 hours post transfection when PP4 levels were sufficiently suppressed. Compared to controls, 67% of mitotic cells exposed to ISIS 500 nM 13947 exhibited distinct abnormalities in mitotic progression. Three abnormal morphologies, in particular, will be discussed in more detail and described as sequential and phase-specific occurrences. Figure 23A is shown to illustrate the mitotic progression of a control cell as described previously as compared to Figure 23B illustrating a representative HeLa-H2BGFP cell treated with 500 nM ISIS 13947. A general overview of the altered mitotic progression will be discussed in brief and followed by a more detailed analysis. Initially, PP4 suppressed cells entered prophase similar to controls with chromosome condensation and rounded plasma membrane characteristic of mitotic cells. However abnormalities soon become apparent during a prolonged metaphase (asterisks) associated with lagging chromosomes in 67% (n=82) of the cells entering mitosis beyond 40 hours post treatment with ISIS 13947. Subsequently, chromosomes segregated into either 2 or >2 clusters of chromosomes, 47% and 31% (n=55), respectively. Shortly thereafter, cleavage furrow formation of the plasma membrane was observed in early anaphase; however most notably, the cleavage furrow regressed over time resulting in one multinucleated cell in 38% of those cells that segregated into 2 clusters (n=26) and 88% of those cells that segregated into >2 clusters (n=17). These results suggested that an active checkpoint was not maintained in the presence of abnormal mitotic spindles. Furthermore, this aberrant progression resulted in cytokinesis abnormalities although whether this was a direct or indirect result was not clear.
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Figure 23. Time-lapse Live Cell Microscopy Illustrating Irregularities in Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP4 Compared to Controls. Images were selected from time-lapse microscopy experiments in which HeLa-H2B-GFP cells were analyzed from 24 hours through 72 hours post transfection. Representative images where chosen to illustrate irregularities in cell cycle progression resulting from PP4 suppression compared to controls. Phases of the cell cycle (Interphase, Prophase, etc.) are indicated below sequential images of the same cell from interphase through mitosis. Time shown indicates “time-elapsed” from initial onset of prophase in (min) or (h) for each pair of images composed of: (top panel) indicating chromosome movement with H2B-GFP tagged HeLa cells; and, (bottom panel) indicating plasma membrane alterations by differential interference contrast). A) Cells treated with solvent alone illustrating typical chromosome condensation and alignment on the metaphase plate in ~97% of cells scored (n=62). Representative images illustrate abnormal metaphase alignment of chromosomes resulting from treatment with ISIS 134947 targeting PP4 in 67% of cells scored (n=82). The presence of lagging chromosomes (indicated with asterisks in) is present in both mitotic phenotypes. Two distinct mitotic phenotypes are present based on variations in chromosome alignment (asterisks) in B): Lagging chromosomes surround metaphase aligned chromosomes in a straight single line at the metaphase plate (white lines); and lagging chromosomes aligned in an irregular Y-shaped metaphase plate (white lines) in HeLa-H2B-GFP cells treated with (400 nM) PP4 ISIS 134947 during a prolonged metaphase arrest ~15 hours (delayed metaphase ranged from 2-20.5 hours). Additionally, chromosome congression aberrations resulted in chromosomes segregating into either 2 or >2 unequal chromosome clusters (47% and 31%, respectively, of aberrant mitotic cells n=55) with the later demonstrating irregular plasma membrane alterations. The formations of cleavage furrows in the plasma membrane were observed in anaphase initially; however, this regressed over time resulting in one multinucleated cell. White lines surround the perimeter of the cell of interest for easier viewing. 10 of the 26 aberrant mitotic cells divided into 2 (38.5%) and 15 of 17 aberrant mitotic cells divided into >2 (88.2%).
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Figure 23.
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The same time-lapse experiments were analyzed for each sequential and phasespecific abnormality resulting from antisense-mediated PP4 suppression in greater detail. Enlarged images in Figure 24 compare the typical metaphase alignment of mitotic control cells (Figure 24A) to two distinct abnormalities (Figure 24B-C) observed during chromosome congression in 67% of metaphase cells suppressed of PP4. Both aberrant morphologies resulting from PP4 suppression contain chromosomes positioned between the equatorial plane and outer edges of the cell (indicated by asterisks). However, these two morphologies were distinguished from each other by the different metaphase alignment shown in the majority of chromosomes (indicated with white lines); that is, metaphase aligned chromosomes in a straight single line at the metaphase plate; or chromosomes aligned in an irregular Y-shaped metaphase plate. Metaphase arrest varied between 2 and ~20 hours (15 hours shown in Figure 14B) and subsequently chromosome segregation occurred regardless of attaining full alignment of chromosomes on the metaphase plate, which may suggest a nonfunctional checkpoint. Both abnormal mitotic alignment morphologies were reminiscent of those resulting from 50 M fostriecin. Additionally, as in the fostriecin treated cells, a morphology reminiscent of that observed in the PP2A suppressed cells was also observed (compare with Figure 20B). The next specific disruption in mitotic progression occurred during segregation of chromosomes upon exit from mitosis. Comparisons are highlighted in enlarged images in Figure 25. Cells treated with solvent alone are represented in Figure 25A exhibiting the typical segregation of chromosomes into two distinct and equivalent chromosome clusters in 97% of mitotic cells (n=62). Also noted is the appearance of one continuous plasma membrane that is relatively smooth and symmetrical as shown extending in
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Figure 24. Enlarged Images of Representative Cells Suppressed of PP4 via Antisense Oligonucleotide-Mediated Suppression. Representative images were selected from time-lapse microscopy experiments as described earlier and analyzed for irregularities in metaphase alignment. (A) Cells treated with solvent alone illustrating the typical localization of condensed chromosomes aligned equatorially on the metaphase plate in ~97% of mitotic cells (n=62). ~67% of cells (n=55) treated with ISIS 134947 targeting PP4 demonstrate two distinct and abnormal metaphase phenotypes based on the localization and alignment of chromosomes. Representative images illustrate the presence of lagging chromosomes seen in both metaphase phenotypes (indicated with asterisks); and, two alignment variations shown in (B) with chromosomes aligned in a straight single line at the metaphase plate and highlighted with a white line; compared to (C) with chromosomes aligned in an irregular Y-shaped metaphase plate and highlighted with white lines.
opposing directions which is typical of anaphase cells. Comparatively, cells treated with ISIS 13947 suppressing PP4 exhibited abnormalities in chromosome segregation. In the majority of PP4 suppressed cells exhibiting an abnormal mitotic morphology, segregation followed yielding 2 (image not shown) and >2 distinct clusters (Figure 25B) of chromosomes in 47% and 31% (n=55), respectively. As evidence by viewing time-lapse images sequentially, clusters of chromosomes appear to pull apart and segregate during this time; however, the presence of two equivalent clusters indicative of normal chromosome segregation is rarely observed. Nonetheless, these cells appear to initiate
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Figure 25. Enlarged Images Highlight Abnormalities in Chromosome Segregation Observed in Hela-H2B-GFP Cells Treated with ISIS 134947 Targeting PP4. Representative images were selected from time-lapse microscopy experiments as described earlier and analyzed in more detail for irregularities present upon chromosome segregation. A) Cells treated with solvent alone illustrate the typical segregation of chromosomes into two distinct and equivalent chromosome clusters in ~97% of mitotic cells (n=62). Additionally, a single plasma membrane is shown extending in opposing directions typical of anaphase. Comparatively, abnormal segregation of chromosomes results from treatment with 500 nM ISIS 134947 in 31% (n=55) of mitotic cells observed with the Y-shaped aberrant mitotic phenotype as represented in B). White outlines surround the perimeter of the cell to highlight representative cell. Additionally, 47% of 500 nM ISIS 134947 (n=55) treated cells appeared to exit mitosis with two distinct populations of segregated chromosomes similar to controls (representative image not shown).
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cytokinesis, albeit in the presence of lagging chromosomes and significant irregularities in the plasma membrane. The fostriecin treatment group, based on the greater percentage of cells segregating into >2 clusters compared to the PP4 suppressed treatment group, 89.5% compared to 31% respectively, suggests that the fostriecin-mediated morphologies are, in large part, due to the suppression of PP4 but also may require the suppression of PP2A as well. However, due to the time constraints used to acquire images by timelapse, the PP4 suppressed cells may have been observed when PP4 levels were not as comparably suppressed and may mimic the fostriecin-mediated morphology more so at later times when PP4 is comparably suppressed. This would not be surprising considering that analysis of the raw data demonstrated increasingly more aberrant morphologies as time passed which would correlate with decreasing levels of PP4 in cells entering mitosis later in the experiment. The third distinct abnormality in mitotic progression occurs during anaphase and cytokinesis involving cleavage furrow regression and subsequent failure to divide into two equal daughter cells. As shown in Figure 26A, typical cleavage furrow formation becomes apparent shortly after chromosome segregation and is observed as lateral ingressions of the cell margins. In PP4 suppressed cells, the formation of the cleavage furrow was also apparent shortly after chromosome segregation. However, in comparison, the initial evidence of cleavage furrow formation was followed by subsequent regression and resulted in a single plasma membrane surrounding an irregularly shaped multinucleated cell over a variable but prolonged period of time. Of the aberrant mitotic cells resulting from ISIS 13947 treatment that segregated into 2 or >2 clusters of chromosomes, ~39% (n=26) and 88% (n=17) exhibited cleavage furrow
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Figure 26. Enlarged Sequential Images Highlight Cytokinesis Abnormalities Observed in Hela-H2B-GFP Cells Treated with ISIS 134947 Targeting PP4. HeLa-H2B-GFP cells were treated with A) solvent alone or with B) ISIS 134947 as previously described. Representative images were selected from time-lapse microscopy experiments and analyzed in more detail for irregularities observed in late anaphase and telophase. A) Cells treated with solvent alone illustrate the typical progression from anaphase through cytokinesis completion. Treatment of cells with ISIS 134947 resulted in two types of cytokinesis abnormalities in which cleavage furrow regression results in multinucleated cells. B) Highlights 38.5% of abnormal mitotic cells that divided into 2 sets of chromosomes and C) Highlights 88.2% of abnormal mitotic cells that divided into >2 sets of chromosomes upon exit from mitosis.
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Figure 26.
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regression leading to multinucleate cells. Enlarged images compare cleavage furrow formation and regression beginning in anaphase highlighting the defect in cytokinesis regardless of mitotic phenotype or the number of chromosome clusters segregated at anaphase onset as shown in images Figures 26B and 26C, respectively. Although imaging constraints prevented accurate analysis of the subsequent cell death resulting in the formation of multinucleated cells, at least 24 of the 55 (~45%) mitotic cells displayed features consistent with cell death including chromosome compaction and plasma membrane blebbing; however, this is also consistent with disruption of the actin cytoskeleton and micronuclei formation.
Summary and Conclusions Previous studies revealed a correlation between the anti-proliferative qualities of two naturally-occurring compounds, cantharidin and fostriecin, and the inhibition of the serine/threonine phosphatases; however, the mechanism was unclear. Strong evidence had suggested the mechanism involved the ability to broadly inhibit the PPP phosphatases based on a common catalytic domain, referred to as the “toxin-binding domain”; however, identification of the specific phosphatase(s) and associated functions critical for proliferations were unclear. Collectively, this dissertation identifies disruptions in mitotic progression induced by cantharidin and fostriecin that contribute to the anti-proliferative and cytotoxic qualities of these natural products. Moreover, this dissertation identifies both PP2A and PP4 as the two key PPP phosphatases whose inhibition by these natural inhibitors contribute to the mitotic disruptions that were identified as critical to the anti-proliferative and cytotoxic mechanism of action.
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Table I. Summary of Morphological Changes in Mitotic HeLa-H2B-GFP Cells. Analysis of all mitotic cells in which asynchronous HeLa-H2B-GFP cells were treated with indicated treatment as described previously. All mitotic HeLa-H2B-GFP cells were analyzed by time-lapse microscopy from at least three separate experiments and pooled to represent “total cell number” for individual treatments. Table numbers indicate number of cells and (#) and (%) reflects the percentage. The aberrant mitotic phenotype or “AMP” includes all mitotic cells within the total mitotic cells counted that are defined as exhibiting a mitotic arrest longer than 2hours in the presence of lagging chromosomes. Chromosome Segregation of AMP* is the number of cells from the AMP pool that segregate into 2 or >2 groups, indicating the percentage of AMP cells respectively. Cleavage Furrow Regression** is the number of AMP cells segregating into the two subgroups (2 or >2) that also exhibit regression of the cleavage furrow, indicating the percentage respectively.
Quantitative analysis is summarized in Table I from all five treatment groups with respect to the number of cells demonstrating aberrant metaphase morphologies, segregation into 2 or >2 chromosome clusters, and regression of the cleavage furrow.
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Aim one characterizes the cellular effects of cantharidin and fostriecin. Results indicate cantharidin inhibits the PPases in a dose- and time-dependent manner and this correlates with decreased cell viability and proliferation, increased accumulation of cells in G2/M-phase and increased number of cells exhibiting aberrant mitotic microtubules and scattered chromosomes. The nature of the mitotic arrest resulting from cantharidin was revealed using time-lapse microscopy which demonstrated a metaphase arrest due to disruptions in attaining and maintaining chromosome alignment. At concentrations inhibiting nearly all PP2A/PP4 activity and 50% of PP1/PP5 activity, similar time-lapse microscopy examination with fostriecin revealed multiple disruptions throughout mitotic progression from pro-metaphase through cytokinesis. Initially, cells arrest in metaphase due to disruptions in chromosome alignment. However, metaphase exit follows regardless of proper metaphase alignment. This aberrant progression through the metaphase-anaphase transition was followed by cytokinesis defects, due to cleavage furrow regression, and resulted in multinucleated cells. Aims two and three identified PP2A and PP4, respectively, as the key PPases whose inhibition is chiefly responsible for the mechanism of inducing mitotic aberrations attributed to fostriecin and cantharidin. Individual PPase inhibition was achieved utilizing antisense oligonucleotide-mediated suppression and was confirmed by Northern and Western analysis. Time-lapse microscopy revealed that the selective suppression of PP2A or PP4 resulted in prominent alterations during mitosis consistent with the mitotic disruptions exhibited by cantharidin and fostriecin; whereas, the antisense oligonucleotide-mediated suppression of PP1 (187), PP2A, and PP5 (188) exhibit mitotic progression similar to mismatch controls and untreated cells (Figure 27).
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Figure 27. Time-lapse Live Cell Microscopy Illustrating Control-like Mitotic Progression of HeLa-H2B-GFP Cells Suppressed of PP2Aβ, PP1 and PP5. Time-lapse microscopy experiments were conducted as described earlier in which HeLaH2B-GFP cells were treated with ISIS antisense oligonucleotides as indicated for 30 hours prior to acquisition of time-lapse images. Representative microscopy images of fluorescent (GFP) and corresponding differential interference contrast (DIC) HeLa cells expressing H2B-GFP. Time shown indicates “time-elapsed” from initial onset of prophase in (min) or (h) for each pair of images composed of: (top panel) indicating chromosome movement with H2B-GFP tagged HeLa cells; and, (bottom panel) indicating plasma membrane alterations by DIC. A) Cells treated with ISIS 11018 (antiPP2Aβ) illustrated typical chromosome condensation and alignment on the metaphase plate in ~95% of cells scored (n=29) represented here. B) Cells treated with ISIS 14435 (anti-PP1) illustrated typical chromosome condensation and alignment on the metaphase plate in ~95% of cells scored (n=24) represented here. C) Cells treated with ISIS 15534 (anti-PP5) illustrated typical chromosome condensation and alignment on the metaphase plate in ~96% of cells scored (n=41) represented here.
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Figure 27.
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Specifically, these results demonstrate both overlapping and distinct roles for PP2A and PP4 as essential regulators in mitotic progression: PP2A via chromosome alignment and segregation, and PP4 via chromosome alignment and segregation, mitotic checkpoint fidelity and cytokinesis completion. Thus, this work identifies PP2A and PP4 as the key phosphatases inhibited by cantharidin and fostriecin responsible for the cytotoxic and anti-proliferative effects; and, furthermore, that the suppression of either PP2A or PP4 is sufficient to alter mitotic progression unlike the suppression of PP2Aβ, PP1 or PP5. These results offer potential insight into the development of synthetic analogues to cantharidin and fostriecin that selectively target only PP2A or PP4 to minimize toxicity while retaining therapeutic value for the management of human cancer.
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DISCUSSION AND FUTURE IMPLICATIONS
The experiments in this dissertation demonstrate that: 1) the cantharidin-mediated suppression of PPase activity in cell culture results in metaphase arrest induced by failures in chromosome alignment; 2) the fostriecin-mediated suppression of nearly 100% of PP2A/PP4 activity and ~50% of PP1/PP5 activity in cell culture results in progressive disruptions from pro-metaphase through cytokinesis (metaphase arrest induced by incomplete and improper alignment of chromosomes, anaphase onset in the presence of improperly aligned chromosomes, and subsequent regression of the cleavage furrow resulting in multi-nucleated cells); 3) the antisense-mediated selective suppression of PP2A activity in cell culture results in metaphase arrest induced by the incomplete alignment of a portion of chromosomes; and 4) the antisense-mediated selective suppression of PP4 activity in cell culture results in progressive disruptions from prometaphase through cytokinesis (metaphase arrest induced by incomplete and improper alignment of chromosomes, anaphase onset in the presence of improperly aligned chromosomes, and subsequent regression of the cleavage furrow resulting in multinucleated cells). Collectively, our results support the conclusion that the antiproliferative mechanism of action attributed to both cantharidin and fostriecin is largely due to disruptions in chromosome regulation throughout mitotic progression resulting from the inhibition of PP2A and PP4 activity.
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The Anti-proliferative and Cytotoxic Effects of Cantharidin and Fostriecin are Due to Disruptions in Mitotic Progression Resulting from the Inhibition of Serine/Threonine PPase Activity Aim one of this dissertation further characterized the anti-proliferative and cytotoxic effects of cantharidin and fostriecin, identifying specific disruptions throughout mitosis. Our results indicate that cantharidin inhibits the toxin-sensitive PPP phosphatases in a dose-dependent manner which correlates with decreased viability and proliferation, accumulation of cells within G2/M-phase, and an increased number of cells demonstrating aberrant mitotic morphologies in a dose- and time-dependent manner. The nature of the mitotic arrest resulting from cantharidin was revealed in our studies using time-lapse microscopy which consistently showed a prolonged metaphase arrest induced by disruptions in attaining and maintaining chromosome alignment. This is consistent with previously documented cytotoxic effects in a number of human tumor cell lines and primary tumor cells (147,148). Our studies are also consistent with previous findings of aberrant mitotic spindles in CHO cells, notably at concentrations that correlate with the potent inhibition of three families of structurally-related PPases (PP1/PP2A/PP5) (154). Collectively, this supports the conclusion that the mechanism of action of cantharidinmediated anti-proliferative and cytotoxic effects is due to the disruptions in metaphase that arrest mitotic progression resulting from the inhibition of toxin-sensitive PPase activity. Nonetheless, identification of the key PPase(s) involved was still unclear. To address this, similar studies were conducted employing the semi-selective capability of fostriecin, using concentrations capable of inhibiting ~100% of PP2A/PP4 activity and ~50% of PP1/PP5 activity (71). Fostriecin has been well-examined in cell cultures, tumor models and Phase I clinical trials where it was entered as a potential
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antitumor therapeutic; however little was known regarding the nature of the cellular arrest preceding cell death (70,72,158). The live cell time-lapse microscopy experiments conducted in this thesis work exposed multiple disruptions from prophase through cytokinesis when HeLa-H2B-GFP cells were treated with 50M fostriecin; and thus offered additional insight into the potential mechanism of fostriecin-induced antiproliferation. Initially, two fostriecin-mediated metaphase morphologies were identified: one showed a small portion of lagging chromosomes surrounding the metaphase plate, and the other showed a small portion of lagging chromosomes surrounding a Y-shaped metaphase plate. The next abnormality shown in mitotic progression occurred in both of the aforementioned metaphase morphologies described. They both exhibited improper metaphase alignments and lacked complete alignment of metaphase chromosomes yet exited metaphase regardless. This improper progression through the metaphase-anaphase transition resulted in the unequal segregation of chromosomes which was subsequently followed by disruptions in cytokinesis. Although these studies demonstrated initial formation of the cleavage furrow, subsequent regression of the cleavage furrow resulted in multinucleated cells and failure to complete cytokinesis. Our studies are consistent with previous reports demonstrating that fostriecin inhibited tumor cell proliferation and induced cell cycle arrest with an accumulation of cells in the G2/M-phase at concentrations that inhibit the activity of the toxin-sensitive PPases in a semi-selective manner (159). Furthermore, the morphologies observed in the human tumor cell lines used in these studies are consistent with the aberrant mitotic structures reported in previous studies examining the molecular mechanism of fostriecin-
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mediated anti-proliferative and cytotoxic effects in CHO cells (159). However, the studies presented here are the first to demonstrate the succession of mitotic disruptions in human cells as they progress through mitosis. Taken together, these studies support the conclusion that the common antiproliferative and cytotoxic mechanism of action attributed to cantharidin and fostriecin is due to the multiple disruptions throughout mitotic progression resulting from the semiselective inhibition of the PPases. While the specific PPases responsible were far from confirmed, our results implicated PP2A and PP4 as potentially significant contributing PPases responsible. The semi-selectivity of fostriecin demonstrated at concentrations that inhibit ~100% of PP2A/PP4 activity while only inhibiting ~50% of PP1/PP5 activity are consistent with a larger role for PP2A and/or PP4, although not conclusive (71). The concentrations of cantharidin used effectively inhibited all of the toxin-sensitive PPases, however the higher affinity of cantharidin toward the PP2A-like PPases and the resulting metaphase arrest sharing phenotypic similarities is therefore not inconsistent (154). It seemed reasonable then that cantharidin treatment resulted in a more prominent and accentuated abnormal mitotic phenotype due to broad inhibition of the PPases as compared to the phenotypes resulting from semi-selective inhibition resulting from fostriecin treatment. Nonetheless, specific identification of the individual PPases responsible remained unknown and required selective inhibition to make further conclusions.
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Cantharidin- and Fostriecin-Mediated Disruptions in Mitotic Progression are Due, in part, to the Inhibition of PP2A Activity Aim two of this dissertation identified a role for PP2A in cell survival due to regulation of mitotic progression. Our results indicate that the antisense-mediated selective inhibition of PP2A results in mitotic arrest resulting from the failure to properly align all chromosomes at the metaphase plate. The nature of the mitotic arrest resulting from PP2A suppression was revealed by time-lapse microscopy which consistently showed a prolonged metaphase arrest induced by disruptions in attaining chromosome alignment of a small portion of chromosomes. Furthermore, it should be noted that the prolonged arrest in the presence of lagging chromosomes suggests a functional mitotic spindle checkpoint. These results are consistent with previous reports regarding cantharidin that demonstrate cantharidin-mediated inhibition of PP2A (183). The effects observed in mitosis resulting from suppression of PP2A are also supported by reports demonstrating the higher selectivity of fostriecin toward the structurally-related PP2A PPases at concentrations that decrease proliferation and decrease cell survival. Recently, three papers from independent laboratories described a role for PP2A in regulating Shugoshin (Sgo), a family of centromere protector proteins localized to the inner centromere during prophase to protect centromere cohesion (189-191). Subsequent studies identified two Shugoshin homologues in humans, hSgo1 and hSgo2, and reported their role in centromeric protection of cohesion. Specifically, it was found that PP2A localization to the centromere was dependent upon hSgo2 recruitment; however hSgo1 localization was dependent upon PP2A localization at the centromere. PP2A-dependent
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dephosphorylation of hSgo1 protects hSgo1 and thereby prevents premature disassociation from cohesion until later at the onset of anaphase when dissociation is necessary for chromosome segregation. At that time, Plk1-dependent phosphorylation facilitates the dissociation to allow for proper chromosome segregation. The results from our time-lapse microscopy, which demonstrated misaligned chromosomes, are consistent and supported by these reports. The three groups that reported PP2A regulatory subunits bound to hSgo1, specifically identifying the B’/PR61/PR56 family as responsible for targeting PP2A to hSgo1, through co-immunoprecipitation or tandem affinity purification studies (189-191). It should be noted that the conclusions drawn regarding B families were premature as the indicated peptide sequence is shared in all B families. Nonetheless, antibody staining supports the B’ family as they demonstrated co-localization of hSgo with the -isoform of PP2A-B’/PR61/PR56 within the inner centromeres detected during pro-metaphase and not in anaphase. This is consistent with the proper dissociation of cohesin required to segregate chromosomes and exit mitosis. Later this interaction was shown to be mediated by the N-terminus of Sgo1 in Drosophila mei-S332 (192). Finally, there is additional evidence supporting this relationship coming from an unrelated set of PP2A structural studies. It was shown that the B’/PR61/PR56 family contains -helices akin to the PR65 scaffolding subunit of the core enzyme, which differ from the other B families, and is suggested by the authors to be primed for recruiting basic substrate proteins into its concave side. They cite examples including Shugoshin, notably due to the highly acidic side of PR61 (113).
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Collectively, our data identifies PP2A as one of the critical PPases with a role in mitosis, specifically playing a regulatory role in the proper alignment and segregation of chromosomes, which was later identified by others to protect Shugoshin. Furthermore, the inhibition of PP2A resulting from cantharidin and fostriecin treatment plays a significant role in the molecular mechanism associated with the anti-proliferative and cytotoxic effects. However, the additional abnormalities exhibited later in mitosis following fostriecin treatment could not be attributed to PP2A. Thus, these results also suggested the involvement of another toxin-sensitive PPase and therefore the pursuit of the last aim.
Cantharidin- and Fostriecin-Mediated Disruptions in Mitotic Progression are Due, in part, to the Inhibition of PP4 Activity To identify other PPases that may contribute to the anti-proliferative and cytotoxic effects shown with cantharidin and fostriecin treatment, similar examination of cells suppressed of PP4 activity via antisense oligonucleotide methods were conducted. Our time-lapse microscopy studies revealed the nature of multiple disruptions during mitotic progression. Notably PP4 suppression shared remarkable similarities to the number of mitotic disruptions documented in cells treated with fostriecin. Initially disturbances were apparent after mitotic onset and two metaphase morphologies were identified: one showed a small portion of lagging chromosomes surrounding the metaphase plate, and the other showed a small portion of lagging chromosomes surrounding a Y-shaped metaphase plate. In spite of the inability to align chromosomes properly, many cells exited mitosis regardless. This aberrant progression through the metaphase-anaphase
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transition resulted in the unequal segregation of chromosomes which was then followed by cytokinesis disruptions. Specifically, our studies showed for the first time, regression of the cleavage furrow and the formation of multinucleated cells in cells lacking functional PP4. These results were strikingly similar, morphologically, to our studies with fostriecin; however there were differences in the portion of cells demonstrating the different mitotic morphologies. That is, 47% of PP4 suppressed cells versus ~9% of fostriecin treated cells were shown with the first described metaphase morphology described as containing a small portion of lagging chromosomes surrounding a single metaphase plate. Although chromosomes segregated into two groups, the distribution was not always equivalent. Interestingly, the first mitotic phenotype was also shared by ~89% of cells suppressed of PP2A. Comparatively, these cells maintained a prolonged mitotic arrest which suggests a functional mitotic checkpoint because the cells never transitioned from metaphase to anaphase in the presence of improperly aligned chromosomes. Comparatively, 31% of PP4 suppressed cells and ~90% of fostriecin treated cells exhibited the second aberrant metaphase morphology. That is, a portion of lagging chromosomes exhibited alignment difficulties along an improper Y-shaped metaphase plate. In these cells, chromosomes segregated into two or more unequal portions of chromosomes. Notably, the PP2A suppressed cells did not demonstrate the second mitotic morphology shared in both the PP4 suppressed and fostriecin treated cells which suggested additional roles for PP4 in a direct or indirect capacity.
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Fostriecin treated cells demonstrated an aberrant mitotic morphology in 100% of mitotic cells observed during these studies. Approximately 9% and 90% of these aberrant mitotic cells segregated into 2 or >2 groups of chromosomes, respectively, and subsequently demonstrated cleavage furrow regression in 80% of those aberrant mitotic cells segregating into 2 groups and 90% of those aberrant mitotic cells segregating into >2 groups of chromosomes. Thus, ~88% of all aberrant mitotic cells were observed with regression of the cleavage furrow resulting from fostriecin treatment, which is conservative due to time-lapse acquisition constraints. This trend was also seen in the PP4 suppressed cells although the percentages in each group varied in comparison. Specifically, the PP4 suppressed cells demonstrated an aberrant mitotic morphology in 67% of all cells entering mitosis after 40 hours post transfection when PP4 was suppressed. Approximately 47% and 31% of these aberrant mitotic cells segregated into 2 and >2 groups of chromosomes, respectively, and further demonstrated cleavage furrow regression in ~39% of those segregating into 2 and 88% segregating into >2 groups of chromosomes. Therefore, ~44% of all aberrant mitotic cells were observed with regression of the cleavage furrow resulting from suppression of PP4, which is a conservative percentage due to time constraints with time-lapse acquisition. Thus, these studies support the conclusion that fostriecin’s anti-proliferative and cytotoxic qualities result from disruptions in mitotic progression due to the inhibition of PP2A and PP4. Furthermore, the selective inhibition of PP4 is sufficient to induce remarkably similar disruptions in mitosis as those resulting from fostriecin treatment. The fact that a greater percentage of cells exhibited cleavage furrow regression in the fostriecin treated cells versus the cells suppressed of PP4, may suggest the additional role played by PP2A
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suppression. Nonetheless, it is not clear whether the multiple disruptions due to PP4 suppression or fostriecin treatment are generated from multiple independent roles attributed to PP4 in mitosis or if there is a consequential progression of disruptions generated from disruptions in earlier mitotic events. Because of the high degree of homology within the toxin-sensitive PPases, comparable live cell time-lapse experiments were also conducted in HeLa-H2B-GFP to determine if the individual suppression of these PPases resulted in disruptions throughout mitosis cells by utilizing antisense-mediated suppression of PP1 (ISIS 14435), PP2A (ISIS110181) and PP5 (ISIS15534). In our studies, suppression of PP1, PP2A and PP5 did not elicit morphologically apparent disruptions in mitotic progression as shown earlier (Figure 27A-C). Specifically, there was no evidence of premature exit from mitosis in the presence of improperly aligned chromosomes and cleavage furrow formation resulted in the formation of two separate daughter cells. These observations were comparable to untreated cells and mismatched controls. This is not surprising as previously reports demonstrated a role for PP1 in final scission of the anaphase bridge between two newly formed daughter cells (80) and recent evidence that PP5-deficient mice are viable and survive through postnatal life (104,105). However, it is interesting that the PP2A/PR65 scaffolding subunit has been identified as a PP5 interacting protein in yeast two-hybrid screening (110), in addition to, two subunits of the anaphase promoting complex (APC), CDC16 and CDC27 (193). In preliminary (unpublished) experiments, the dual suppression of PP2A and PP5 with 250 nM ISIS 110159 and 115534, respectively, resulted in a comparable metaphase arrest associated with abnormal mitotic spindle formation however was followed by subsequent premature exit from
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metaphase. These preliminary results may be consistent with a regulatory role in proper functioning of the anaphase promoting complex to ensure metaphase arrest until proper alignment is attained and thus may offer additional insight into the development of selective PPase inhibitors for the potential development of anti-cancer therapeutics. Nonetheless, these are only preliminary results and require further testing. From the studies presented here, the critical PPases significantly contributing to the fostriecin-mediated anti-proliferative and cytotoxic effects have been identified as PP2A and PP4. Considering PP2A has been identified as protecting Shugoshin and preventing premature chromosome segregation, this can explain the aberrant mitotic features demonstrated in our time-lapse experiments (191). This is also supported by recent studies that have reported a heterotrimeric PP2A-separase-securin complex identifying PP2A as a new interaction partner with human separase (Holland 2007), including PP2A-specific phosphorylation sites have been mapped on securin. However, relatively less is known about PP4 and it is still not clear from our studies how PP4 specifically regulates mitotic progression. Nonetheless, previous studies in D. melanogaster and C. elegans showed evidence of abnormal mitotic spindle phenotypes in the presence of suppressed or non-functional PP4, which is supportive of our findings (133,134). Our studies support the conclusion that PP4 is the other key PPase, in addition to PP2A, contributing to the fostriecin-induced disruptions throughout mitosis. Suppression of PP4 has been identified as playing a regulatory role in: 1) metaphase alignment and segregation of chromosomes; 2) the metaphase-anaphase transition suggesting participation with the mitotic spindle checkpoint; and 3) cytokinesis
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completion. The specific downstream targets of PP4 have yet to be identified however; the literature suggests a number of potential candidates, some of which will be discussed briefly here. In the past, much attention has been given to anti-mitotic agents that effectively disrupt the cell cycle and target highly proliferative cells in human cancers. Comparatively, little attention has been focused on targeting cytokinesis as an alternative or supplementary mechanism to disrupt the cell cycle. In our studies, both fostriecin treated and PP4 suppressed cells exhibited accentuated disruptions later in mitotic progression during cytokinesis. Furthermore, there was evidence of this from cells in both aberrant mitotic cell groups, regardless of segregation into 2 or >2 clusters of chromosomes. In both, cells appeared to initiate cleavage furrow formation. However, subsequent regression resulted in a single multinucleated cell. It is not clear if the multiple disruptions throughout mitosis are generated from independent roles in mitosis or if there is a sequential progression of disruptions generated from unregulated mitotic progression. Nonetheless, as discussed earlier, induction of mitotic delays, followed by premature exit from mitosis and cytokinesis failures are common precursors to aneuploidy and subsequent cell death (12). In animal cells, the cleavage furrow forms a contractile ring composed of actin, myosin and other proteins including the motor proteins. Subsequent ingression creates a barrier between the daughter cells separating cytoplasmic components. Perhaps PP4 plays a role regulating one of these components. For instance, one of the major motor proteins involved include the kinesins, specifically CHO/MKLP families (194,195). In cells lacking the C. elegans CHO/MKLP homologue, ZEN-4, metaphase progression is
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followed by typical cleavage furrow ingression however regression of the furrow results in multinucleated embryos and cytokinesis failure (196,197). Still, there are many other motor proteins essential to the establishment of a proper bipolar mitotic spindle including NuMA, HSET and dynein/dynactin which are transported by dynamic microtubules to localize and establish normal morphology of the spidle (198-201). The Aurora kinases are also proposed to have dual roles in mitosis and cytokinesis and form a complex with INCENP, inner centromere protein, and ZEN-4 (202,203). Lack of aurora kinase also induces cleavage furrow regression thought to be due to failure of directing ZEN-4 properly to the spindle (204). Another similar description of defective cytokinesis is reported in Drosophila polo mutants. Polo (Plk) is a kinase that localizes with CHO/MKLP at the midbody during late cytokinesis (205). Another proposed target may then be the mitotic kinase Plk1 with pleiotropic functions in mitosis that, notably, share many similarities with our findings. Plk1 has been implicated in chromosome condensation in prophase, chromosome-spindle attachment in pro-metaphase, chromosome alignment at the metaphase plate, chromosome segregation at anaphase onset and chromosome decondensation in telophase (206). The possibility seems reasonable coupled with previous studies demonstrated a PP4-dependent recruitment of Plk1 and -tubulin to the centrosomes during mitosis (207). In both human and Drosophila, centrosomal localization of the regulatory R2 subunit of PP4 had also been shown (131,133,208). Recently, in a 2008 report, the centrosomal localization of the endogenous R3 subunit was also shown in human mitotic cells. This suggested that the trimeric complex of PP4c-R2-R3 played a functional role at the centrosomes in mitotic cells (132). Additional studies demonstrated that Plk1
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localization to centrosomes is markedly decreased in HEK293 cells depleted of PP4c, via lentiviral expression of PP4c-sh RNAi constructs, although overall cellular levels and midbody localization remained normal (209). This may supports Plk as a potential downstream target of PP4. Additional support from these studies was shown in their model using HEK293 cells depleted of PP4. The cells presented with abnormalities in mitosis specifically exhibiting multiple spindles, aberrant chromosome distribution, and evidence of multiple centrosomes, which our studies could not consistently replicate. Although evidence of the normal complement of two centrosomes has been observed in our PP4 suppressed mitotic cells presenting with an aberrant mitotic morphology, consistent observations were only evidenced in those aberrant mitotic cells that exhibited lagging chromosomes surrounding a single straight metaphase plate. It should be noted that difficulties in attaining prominent pericentrin staining or the lack of typically strong centrosomal staining may have been perceived as artifacts and not scored as such. Of potential relevance, while examining the time-lapse movies constructed from the sequentially acquired images of PP4 suppressed cells over time, an observation made in some of the cells was noted but was not a parameter thoroughly analyzed. That is, the abnormal mitotic phenotypes typically presented with the straight metaphase plate alignment earlier in the movies progression. The accentuated abnormalities observed with Y-shaped alignment typically presented later as levels of PP4 presumably became increasingly suppressed. Additional observations include some cells that originally presented with a single metaphase plate and appeared to change formation into the Yshaped metaphase plate as chromosomes continuously failed to attain or maintain alignment. This may explain our observations of a normal complement of centrosomes in
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one mitotic phenotype and suggest secondary consequences rather than aberrant centrosome duplication. Comparatively, another study examining cellular migration and regulation of Rho GTPases (207), noted the presence of multiple centrosomes in PP4 suppressed HEK293 cells and Drosophila; however, the study did not thoroughly examine mitotic progression. They did however report that in adult mammalian cells, the other highly homologous PPases, including PP2A, were unable to functionally replace PP4. This supports the mitotic abnormalities observed in our studies resulting in cells suppressed of PP2A or PP4, suggesting that in HeLa-H2B-GFP cells these two enzymes cannot functionally replace each other as well. Nonetheless, our studies are the first to demonstrate a distinct disruption in cytokinesis suggesting an additional role for PP4 in cytokinesis. Perhaps these studies, coupled with recent reports examining the Plk1, may support the following proposed model of PP4 regulation that contributes to the multiple mitotic disruptions demonstrated in our time-lapse experiments. A recent report specifically demonstrates the presence of two noticeably different mitotic phenotypes resulting from mutational analysis of Plk1 on specific phosphorylated sites (210). Mutation of Plk1 at amino acid 210 (T210D) resulted in a delay in mitotic progression lasting from 50 minutes to 10 hours, compared to wild-type which executed metaphase within 50 minutes on average. The authors suggest that Plk1 requires phosphorylation on Thr-210 for proper metaphase progression. Mutation of a second phosphorylation site within Plk1 (S137D) resulted in a mitotic catastrophe or cytokinesis failure described as “normal formation of a cleavage furrow, which subsequently regresssed...ultimately giving rise to only one (multinucleated) daughter cell” (210).
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These findings offers insight from two angles: 1) the aberrant morphologies shown throughout mitotic progression demonstrating striking similarities of mutations of Plk1 at different sites to those shown in our studies with both PP2A and PP4 suggesting a regulatory role either directly or indirectly; and 2) the emerging new concept of dual target potential which has recently received more attention. A potential explanation that is consistent with our data is that PP4 may potentially have dual target potential if PP4 regulates PP2A, regulates Plk1 specifically at S137, and indirectly regulates Plk1 at T210 via the direct regulatory role of PP2A at this site. The morphologies described are phosphorylation-dependent resemble morphologies described for PP2A and PP4, respectively. Studies have reported evidence of interdependent regulation of Plk1 and PP2A in mitosis (211,212); however, little has been studied regarding PP4 which leads to the proposed model that PP4 may potentially have dual target potential if PP4 regulates PP2A, regulates PLK1 specifically at S137, and indirectly regulates PLK1 at T210 via the direct regulatory role of PP2A at this site. This proposed model is supported by the striking similarities in mitotic morphologies and disruptions in mitotic progression. The phenotype described for the site-directed mutation of Plk1, S137, shares a delay in metaphase followed by improper anaphase onset and subsequent regression of the cleavage furrow; therefore it may suggest a role for PP4 in the regulation of Plk1, S137. Comparatively, the phenotype described for the sitedirected mutational analysis of Plk1, T210, may suggest a role for PP2A in the regulation of Plk1, T210, due to the similarities in a prolonged mitotic arrest. Interestingly, they suggested that the mutation in S137D may be dominant over the T210D mutation
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demonstrating that consecutive phosphorylation events are critical to maintain timed executed mitotic events and successful completion of mitosis (210). Perhaps, in a similar manner, this may be true for PP4 in regard to PP2A as well. The regulation of one PPase in regard to another highly homologous PPase has not been examined thoroughly; however it is not without precedent. Early studies were unsuccessful in establishing an association of PP2Ac with PP4c (213); however recent studies using Tap tags demonstrated that low amount of PP2A A also co-purified with FLAGPP4c (214). Despite the common HEAT repeats, earlier studies of the PP4 (PPX) catalytic subunit reported no association with the PP2A/PR65 scaffolding subunit (208). In more recent reports, association of both PP4R1 and PP4R4specifically with PP4c and both PP2A A and PP2A β with PP2Ac were reported, as well as the lack of association between PP4c and PP2A/PR65 (214). A proposed role for PP4 in the regulation of PP2A, albeit indirectly, may be suggested. That is, the initial metaphase abnormalities may be due to disruption of PP2A as it relates to the protection of Shugoshin at cetromeric cohesion from premature dissociation. Perhaps PP4 may potentially contribute to the regulation of Plk1 on two levels. One possibility may include that the initial metaphase abnormalities, sufficient to activate the mitotic checkpoint, may be transient and subsequently followed by disruptions in maintaining checkpoint activation by uncoupling APC activation from spindle checkpoint components. Another level of regulation may involve Plk1-dependent functions in cytokinesis which may or may not be related to earlier metaphase functions. Nonetheless, it is not clear if the resulting disruptions in cytokinesis may result from the direct inactivation of the checkpoint or from rendering the APC non-responsive to
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inhibitory signals. Alternatively, it may be a secondary consequence of improper metaphase events that occurred earlier or associated with an unrelated contractile ring function which could also explain the blebbing observed. For example, previous reports indicate that the metazoan regulatory subunit alpha4 uniquely binds the catalytic subunits of PP2A, PP4 and PP6. The mammalian alpha4 in co-immunoprecipitations, glutathione S-transferase pulldown assays and yeast 2-hybrid assays binds to PP2A, PP4 and PP6 (22-25). Results indicated that alpha4 produced modifications in PP2A and PP6 upon interaction with their catalytic subunits. It is the homologue of the yeast protein Tap42 which has accumulated contradictory findings. In conclusion, our results coupled with this emerging new evidence supports the conclusion that PP4 may indeed be a suitable molecular target in the management of human cancers. Furthermore, development of a synthetic analogue selectively targeting PP4 warrants further evaluation. Still, it is not clear if the observed cytokinesis defects are an indirect consequence of improper chromosome segregation or improper execution of cytokinesis. Additionally, it should be noted that while the precocious mitotic exit resulting in polyploidy predominates as the phenotype attributed to the emerging dual target inhibitors, it does raise concerns regarding the facilitation of transformation by the accumulation of polyploid cells which may result in secondary tumors. Nonetheless, as past successful therapies have taught us, the discovery, development and effective clinical use of pharmacotherapeutics is a process riddled with challenges that may be overcome with collaborative teamwork, diligence and perseverance.
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BIOGRAPHICAL SKETCH
Name of Author:
Kathy Marie Bonness
Place of Birth:
Milwaukee, Wisconsin
Date of Birth:
September 2, 1970
Graduate and Undergraduate Schools Attended: Santa Clara University, Santa Clara, CA University of South Alabama, Mobile, Alabama Degrees Awarded: Bachelor of Arts in Political Science, 1992, Santa Clara, CA Doctor of Philosophy in Basic Medical Sciences, 2009, Mobile, AL Publications: Bonness KM, Dean N, Honkanen RE 2009 The Selective Inhibition of Protein Phosphatase 4 Reveals a Regulatory Function in Mitotic Progression and Suggests a Key Role in the Fostriecin-Induced Anti-Mitotic Effects. (Manuscript in preparation). Bonness KM, Aragon IV, Rutland B, Ofori-Acquah S, Dean ND, and Honkanen RE 2006 Cantharidin-Induced Mitotic Arrest is Associated with the Formation of Aberrant Mitotic Spindles and Lagging Chromosomes Resulting, in part, from the Suppression of PP2A. Mol Cancer Ther 5, 2727-2736. Buck SB, Hardouin C, Ichikawa S, Soenen DR, Gauss C-M, Hwang I, Swingle M, Bonness KM, Honkanen RE and Boger DL 2003 Fundamental Role of the Fostriecin Unsaturated Lactone and Implications for Selective Protein Phosphatase Inhibition. J. Am. Chem. Soc., 125 (51), 15694-15695. Awards and Honors: Institutional pre-doctoral trainee in Cell Signaling and Lung Pathobiology NIH Training Grant T32 HL076125 (Center for Lung Biology) University of South Alabama, Mobile, AL (2004-2007). Hypothesis-drive microscopy images exhibited by PERCIPIOTM: Mobile Museum of Art; The Gulf Coast Emporium Science Center; USA Library; and PERCIPIOTM promotional video (2003-2006).
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