Transforming Growth Factor fi@

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Robert C. Bast, Jr., and Andrew Berchuck2. Departments of ..... N. F., Parker, L., Lagasse, L. D., and Knapp, R. C. Intraperitoneal immunotherapy of ... Bartlett, J. M. S., Rabiasz, G. J., Scott, W. N., Langdon, S. P., Smyth, J. F., and Miller,.
[CANCERRESEARCH55, 944-948, February15, 1995]

Regulation of Apoptosis in Normal and Malignant Ovarian Epithelial Cells by

Transforming Growth Factor fi@ Laura J. Havrilesky, Jean A. Hurteau, Regina S. Whitaker, Alaa Elbendary, Shu Wu, Gustavo C. Rodriguez, Robert C. Bast, Jr., and Andrew Berchuck2 Departments of Obstetrics and Gynecology/Gynecological Oncology (L J. H., J. A. H., R. S. W., A. E., G. C. R., A. B.] and Medicine (S. W, R. C. B.) and Duke University Comprehensive Cancer Center, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT Previously, we found that transforming growth factor @3(TGF43) in hibits proliferation of normal human ovarian epithelial cells. In addition, although only 1 of S immortalized ovarian cancer cell lines was inhibited, TGF-@3 inhibited proliferation of 19 of 20 primary epithelial ovarian cancers. In this study, we examined whether TGF-fi induces apoptosis in

normal and malignant ovarian epitheial cells. Among 5 immortalized cell lines, only OVCA 420 is markedly growth inhibited by TGF-@3,and this was the only cell line in which

TGF-@3 elicited

DNA fragmentation

char

acteristic of apoptosis. Induction of apoptosis in OVCA 420 was time and concentration dependent and could be partially inhibited by concurrent treatment

with an anti-TGF-@3

mAb.

Although

apoptosis

was not seen in

normal ovarian epithelial cells (n = 7), [3H]thymidine incorporation was inhibited

in all cases [mean

61.2 ±7.2% (SD) of untreated

control;

P < 0.01]. Similarly, TGF43 inhibited [3Hjthymidine incorporation in all 10 primary ovarian cancers(mean 40.4 ±7.1% of control; P < 0.01), but only 3 of 10 (30%) were found to undergo apoptosis when treated with TGF-@. There was no relationship

between p53 status of the ovarian

cancers and the ability of TGF-@3 to elicit apoptosis. In conclusion, TGF-@ inhibits proliferation but does not induce apoptosis in normal human ovarian epithelial cells. In contrast, some ovarian cancers that are growth inhibited by TGF-@also undergo apoptosis. These data are consistent with the hypothesis that malignant cells are more susceptible to apoptosis than their normal nontransformed counterparts.

process of immortalization of ovarian cancer cells in vitro but not in the development of human ovarian cancer. In addition to inhibiting progression through the cell cycle, it has been shown that TGF-@3can elicit programmed cell death (apoptosis) in both normal and malignant cells under certain circumstances.

TGF-f3has been found to induce apoptosis in endometrialcells (7), gastric carcinoma cells (8), normal and transformed hepatocytes (9, 10), acute myelogenous leukemia cell lines (11), and a number of other cell types. It is unclear, however, why TGF-j3 causes reversible growth arrest of some cells while others undergo apoptosis. In the present study, we have examined whether, in addition to inhibiting proliferation, TGF-f3 is able to induce apoptosis in normal and malignant ovarian epithelial cells.

MATERIALS

AND METHODS

Ovarian Cancer Cell Lines. Four epitheial ovarian cancer cell lines previously established in our laboratory (OVCA 420, OVCA 429, OVCA 432, and OVCA 433) (12) and one commercially available cell line (OVCAR 3)

were grown in monolayer culture in DMEM supplemented with 10% FCS, L-glutamine (2 mM), and penicillin/streptomycin

(100 units/mi).

Primary Ovarian Cancer Cells. Ovarian cancer cells were grown in monolayer culture as described previously from the solid tumor or ascites of patients with stage llh/IV epithelial ovarian cancer who underwent surgery at

Duke University Medical Center between 1993 and 1994 (6). In some cases,

INTRODUCTION

cells

TGF-31,3 1@2'and I3@are produced by humans and inhibit prolifer ation of a wide range of cell types including epithelial cells, hepato cytes, and hematopoietic cells (1). Although the signal transduction pathways involved are not yet well understood, it has been shown that the biological activity of TGF-@3is dependent on the presence of type I and II serine/threonine kinase membrane receptors (2, 3). Binding of TGF-@ to these receptors initiates a cascade of molecular events that are thought to culminate in decreased activity of cyclin-dependent kinase 4, which prevents progression from G@into S phase of DNA synthesis (4). Previously, we examined the effect of TGF-J3 on proliferation of normal and malignant human ovarian epithelial cells in monolayer culture. Growth of primary cultures of normal ovarian epithelial cells was found to be inhibited by 40—70%(5). In contrast, similar to many other types of immortalized cancer cell lines, we found that four of five ovarian cancer cells lines were relatively resistant to the growth inhibitory effect of TGF-@ (5). More recently, however, we found that 19 of 20 (95%) primary epithelial ovarian cancer cell cultures ob tamed directly from ascites were inhibited by TGF-@ (6). These data suggest that loss of responsiveness to TGF-@3 is involved in the

I Supported

by

requests

whom

National

Cancer for

Institute

reprints

should

Grant be

at

Duke

University

abbreviation

used

is: TGF-@3,

transforming

growth

factor

ascites

by centrifugation

and stored

over

liquid

medium supplemented with 10% FCS, L-glutamine (2 mM), and penicillin/ streptomycin

(100 units/ml). Purification of ovarian cancer cells was accom

plished using discontinuous gradient centrifugation with Percoll (Pharmacia, Piscataway, NJ) density gradients (1.070—1.023 g/cm3) (6). Normal Ovarian Epithellal Cells. Primary monolayer cultures of ovarian epithelial cells were established from surgical specimens of normal ovaries as described previously (5). Briefly, the surface of the ovary was scraped gently,

and the epithelial cells were then plated in a 1:1 mixture of MCDB 105/M199 medium epidermal

supplemented growth

factor

with

15%

(10 ng/ml).

heat-inactivated

fetal

Cells were cultured

bovine

serum

and

at 37°C in 5% CO2

and 95% humidified air. DNA Fragmentation Assay. TGF-131(R&D Systems, Minneapolis,MN) was dissolved in 4 mM HO-i mg/mI BSA and stored at —20°C. Cells were grown to confluence on 6-well plates, and then treated with TGF-(31 (10.0, 5.0,

1.0, 0.1, or 0 ng/ml) for 72 h. Then cells were harvested, centrifuged at

6000 X g for 10 mm, and the resultantpelletswere resuspendedin 200 ,.d nuclei lysis buffer (5 M guanidine thiocyanate-25 mM sodium citrate, pH 7.0-100 mMf3-mercaptoethanol).DNA was precipitated with an equal volume of isopropanol at —70°C for 1 h. Samples were centrifuged for 30 mm at 12,000 X g at 4°C,and the DNA pellets were washed in 70% ethanol at room temperature. Pellets were resuspended and incubated overnight at 37°Cwith

a Perkin Elmer Cetus Lambda 3B UV/vis spectrophotometer jected to electrophoresis

to determine the

on a horizontal 1.5% agarose gel containing ethidium

bromide and visualized under UV illumination. In cells in which apoptosis was not observed, the culture medium was also examined for the presence of floating cells, but these were not seen in significant numbers.

Medical

Center, Box 3079, Durham, NC 27710. 3 The

from

concentration of DNA. Equal amounts of each DNA sample were then sub

CA55640. addressed,

removed

0.5 mg/ml RNase A (Sigma Chemical Co, St. Louis, MO). Pellets were again resuspended and an absorbance reading at 260 nm wavelength was obtained on

Received 8/18/94; accepted 12/13/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 To

were

nitrogen. For experiments, cells were thawed and washed in MCDB 105/M199

g3.

944

APOPMSIS 1N OVARIAN CANCER

e- [ horporatioa Assay. Cells w m plated (2 X 10" cells! well) in sin replicate wells in %-well culture pletes. fills were then allowed to gmw to W id u e a c e . Humaa mmmb'ylsnt TljF-8, (10.0,S.O 1.0,O.l. or 0 nglml) was added, and cells wue incubated at 370C for 48 48. Duriqg the last 6 h, 1 pCi of [3Hlthymid~ne (6.7 CJmmbVlitcr)was edded to eacb well. The culture d u r n was then runcwed, aad cells wen wsshed 3 times and h a d Incorporated t a d i d v i t y was mwsured in a Packad liquid scintillation muatcr. Immanobistochemistry. F'rimsry ovarian cancer and normal ovarian epitheIial cells of identical passage to those wed for DNA Eragmentatioa and thymidine incorporation experiments were used to prepare cytospins for immunohistochemical staining. Staining was pcrformd using the Vcctastain ABC kit (Vector Laboratories. Burlingame, CA). A panel of five mAbs [260F9,317G5.741F8 (from Pcrkin Umer Cents), BT424.1, and BT8FF1.5 ( d e v e l m in tbip Iaboratory)] shown lo be r d v e with surface markers w fwe epithelial ovarian cancer cell lims, but rwt with masothelial and inflammatory ails from the benign peritoneal fluid of eight patients (6).was used to WHCSS the purity of the ovarian cancer cells & a i d from &tea and scraped tumor. mAb 1801 (Oncogene Scimct, Manbassttt, NY) (5 p g M ) was used as the primary antibodyfo~imrnuno~tainin~ of p53 as -bed previously (13).

Purified mause IgG, specific for nonhuman tissue (Coulter Immunology. Hialeah, FL),was used as a negative control. Anticytokeratin antibody AElIAE3 (Boehringer Mannheim Biochemicals, Indianapolis, M) was umd as a positive control. The slides were developed for 4 min with the enzyme substrate diaminobenzidine (0.5%diaminobenzidinein 0.05% Tris buffer-0.6% hydrogen peroxide). Slides were then rinsed in water, wunterstained with methyl green, dehydrated, and mounted. StaMcs. The Student's t test was used to analyze experiments in which mean values were compand.

W e examined whether TGF-f3 (10.0 ndd) induced apoptosis in immortalized epithelial ovatian cancer cell ha.Among the five cell lines studied, a DNA ladder consistent with apoptosis was seen on agamie gel electrophoresis only in the OVCA 420 cell Iine (Fig. 1). This result was reproducible under different experimental conditions in which the d i density (coduent versus iubcoduent), s e m content (10% vvrrsus serum-free), or incubation time (0, 6, 12, 24, 48, o r 96 h) were varied. Thc induction of apoptosis in OVCA 420 cells by TGF-/3 was found to be time and concentration dependent (Fig. 2, A and B). Maximum DNA fragmentation was seen at 48-96 b using 5.0-10.0 ng/ml of TGF-f3. Induction of apoptosis by TGF-0 was partially reversed by concurrent treatment of OVCA 420 cells with anti-TGF-8 mAb 1D11.16 (10 gglml; Fig. 2C). Finally, the presence of apoptosis in TGFP-treated OVCA 420 cells was confirmed by electron microscopy (data not shown). As described previously, we were able to obtain relatively pure populations of watian caucer cells directly from patients with

96 12 2434 48 72 96

Time (Hours)

B

Fig. 2. Induction of apoptosis in OVCA 420 ceUs by transforminggrowth factor 6.(A) Dose-response (0,O.l. 1.0,5.0,and 10.0 nglml). (B) Time course (12,24,34,48,72, and % h). (C) Partial revenal of transforming growth factor pinduced apoptosis using anti-transforming growth factor antibody.

Pig. 1. Effacf of mforming growth fnctor B (10.0 @ml) on apqpt-8 in imm0rt.lEad marian c a m cell l h DNA laddm consistentwith &i s seen in OVCA 420 cclt tine, but nm ia OVCA 429. OVCA 432. OVCA 433. m OVCAR 3.

advanced stage disease as evidenced by immunostaining with the panel of antibodies reactive with ovarian cancers (6). Greater than 90% of cells in cultures established from 10 patients were reactive with the panel of antibodies that recognize ovarian cancer-associated antigens (data not shown). TGF-p (10.0 ng/ml) induced DNA fragmentation characteristic of apoptosis in 3 of 10 (30%) primary ovarian cancers (Fig. 3A). In contrast, [3H]thymidine incorporation of all 10 primary ovarian cancers was significantly inhibited by TGF-/3

APOFFO5I5IN ov@ai@n CANCER

TGF@@ -

+

-

untreated control; P < 0.05; Fig. 4B). Significant inhibition of [3HJthymidineincorporationalso was seen when cells were treated with 1.0 ng/ml TGF-j3 (mean inhibition = 57.7 ± 11.0% of control) or 0.1 ng/ml TGF-f3 (mean inhibition = 77.6 ± 7.2% of control; P < 0.05). We also examined the relationshipbetween p53 status and the ability of TGF-@3to elicit apoptosisin ovarian cancers(Table 1). Previously, we had found that OVCA 432 has a missensemutation in codon 277 (cysteine to phenylalanine), while the other 4 immortalized cell lines have normal p53 genes. Although the one cell line with a mutant p53 gene (OVCA 432) did not undergo apoptosis, only 1 of 4 lines with normal p53 genes underwent apoptosisin responseto TGF-@. Among the 10 primary ovarian cancers, in 7 casescells were

+

available for p53 immunostaining.Previously, our group and others have shown that approximately 90% of cancers in which p53 immu nostaining

A TGF-@3

TOV6TOV7 -

+

-

is seen have point mutations

in the gene (14, 15). Similar

to prior studies (13), we observed p53 immunostaining in 3 of 7(43%) primary ovarian cancers. As in the immortalized cell lines, there was no relationship between p53 status and the ability of TGF-j3 to elicit apoptosis. Apoptosis occurred in 2 of 4 cases in which p53 immu nostaining was not seen and in 1 of 3 casesin which immunostaining was present.

+

100 .@

0

80

0@ CC

6

@2o 40

fl

E

I— I

C')

B @

A “—1

OvarianCancer

.:@ —

0

Fig. 3. Effect oftransforming growthfactor on programmedcell deathin normaland malignant ovarian epithelial cells. (A) Apoptosis was seen in primary ovarian cancer

-4.' I.

TOV7butnotin TOV6.(B) Apoptosis wasnotseenin normalovarianepitheialcell culturesOSE3 or OSE4.

@.

@.80

02 C) C c 60 @0

@

(10.0 ng/ml; mean = 40.4 ±7.1% of untreated control; P < 0.05; Fig. 4A). Significant inhibition of [3H]thymidine incorporation

was also seen when cells were treated with 1.0 ng/ml TGF-@ (mean = 46.2 ± 7.8% of control) or 0.1 ng/ml TGF-j3 (mean = 65.3 ±7.3% of control; P < 0.05). Cells cultured from 7 normal human ovaries demonstrated immu nostaining with anti-cytokeratin antibody AE1/AE3 consistent with their epithelial origin (data not shown). None of the normal ovarian epithelial cell cultures underwent DNA fragmentation associatedwith apoptosis following treatment with TGF-f3 (10.0 ng/ml; Fig. 3B). In contrast, TGF-f3 (10.0 ng/ml) inhibited [3H]thymidine incorporation

E ___ I—

I

B

NormalOvarianEplthelium

Fig.4. Transforminggrowthfactor @3 (10.0ng/ml)inhibits[3H)thymidine incorpora

tion in normaland malignantovarianepitheial cells. (A) Primaryovariancancers

in all 7 of these normal ovarian cell cultures (mean = 61.2 ±7.8% of

(P < 0.05).(B) Normalovarianepitheialcells(P < 0.05). 946

APOPTOSISIN

Table1 Relationshipbetweenp53 StatUsandthe ability of transforminggrowth factor

p to inhibitproliferation apoptosisGrowthp53a andelicit ApoptosisCell

@

—

+

-0VCA432

-

-

-0VCA433 -OVCAR3

+ -

-

-Primary cancers

-

-

TOV1 —TOV2

—

+

-TOV3

-

+

-TOV4

+ +

+ +

-TOV6 TOV5 -TOV7 +TOV8 -TOV9 +TOV1O -a

@

+

@b ND + ND

withdrawal of trophic hormones in the postpartum uterus (23), in

atretic ovarian follicles (24), and in the postcastration prostate (25).

inhibition

linesOVCA42O +0VCA429

@@AR1AN CANCER

+ + + + +

status of cell lines reflects presence of mutation in the gene, while + p53 StatUs

of @rimary cancersreflectsthepresence of immunostaining for p53protein. ND, not done.

DISCUSSION TGF-@3inhibits proliferation of many types of normal cells in primary culture, while most immortalized cancer cell lines usually are resistantto the growth-inhibitoryeffect of TGF-@ (1). Resistancemay be manifestas a requirementfor a higher concentrationof TGF-@3to elicit growth inhibition, a lesserdegreeof inhibition relative to that seen in nontransformed cells, or complete lack of responsiveness to TGF-f3. Studies of the effect of TGF-j3 on proliferation of normal ovarian epithelial cells and ovarian cancer cell lines are consistent with thesestudiesin other cell types. We found that TGF-@3consis

tently inhibits proliferationof normalhumanovarianepithelialcellsin monolayer culture by 40—70%(5). In contrast, only one of five immortalized ovarian cancer cell lines that we have examined is markedly growth inhibited by TGF-(3 (5). Likewise, studies by other groupshave shown that proliferationof only 5 of 10 ovarian cancer cell lines were inhibited by greaterthan 25% by TGF-f3 (16—19). Because of the difficulty in obtaining and culturing material di rectly from patients, few studies have examined the effect of TGF-(3

on primary human cancer cells. Recently, we reported that in 19 of 20 cases (95%), proliferation of ovarian cancer cells purified from the ascites of patients with advanced stage disease was significantly inhibited by TGF-f3 (6). As in the present study, there was no differ ence in the magnitudeof growth inhibition, or the concentrationof TGF-j3 required to achieve maximal growth inhibition between nor mal ovarian epithelial cells and primary ovarian cancers.Similarly,

Danielset a!. (20) foundthat TGF-@3 inhibitedcolonyformationof 7 of 9 fresh ovarian cancersin soft agar. Studiesthat have examined proliferation of leukemia cells obtained directly from patientshave shown these cells also are growth inhibited by TGF-@3(21, 22). These

studiesin ovarian cancerand leukemiaare amongthe few that have examined the effect of TGF-(3 on proliferation of early passagecancer cells from patients.They suggestthat while lossof responsiveness to

Although TGF-@1markedly inhibited proliferation of normal ovarian epithelial cells, we were unable to demonstrate that these cells un dergo apoptosisin responseto TGF-f31. It is possiblethat apoptosis could have been induced in a small fraction of cells, but that this was below the threshold of detection of the DNA fragmentation assay. Among the five immortalized ovarian cancer cell lines, the only line that is profoundly growth inhibited by TGF-(3 (OVCA 420) also underwent apoptosis. Apoptosis in OVCA 420 cells was dose and time dependent, and can be partially reversed by concurrent treatment with anti-TGF-fJ mAb. We also examined 10 primary ovarian cancers from patientswith advancedstagedisease.In all 10 cases,prolifera tion was inhibited by TGF-f3, but apoptosis occurred in only 3 cases. There was no relationshipbetween the magnitude of inhibition of proliferation and whether apoptosis occurred. It some cell lines, loss of p53 function due to mutations and/or deletions of this gene during the process of malignant transformation has been associatedwith the development of resistance to the growth inhibitory effect of TGF-j3 (26). Previously, however, we had found that some ovarian cancers with mutant p53 protein were responsive to

TGF-f3, suggesting that the growth-inhibitory effect was not depend ent on the presence of functional p53 (6). It also has been shown, in some cell types, that DNA damage due to radiation can lead to apoptosis and that this process is dependent on the presenceof normal p53 protein (27). It is thought that this serves as a surveillance mechanismfor elimination of cells with genetic damage, which are more likely to undergo malignant transformation. Similarly, transfec tion of normal p53 genes into cancer cells that lack normal p53 induces apoptosisin some cases (28, 29). Since p53 is altered in

approximately one-half of epithelial ovarian cancers (13) and normal p53 appears to play a role in inducing apoptosis under certain cir cumstances, we examined whether there was a relationship between the status of p53 and the ability of TGF-@ to induce apoptosis in ovarian cancers. The absence of such a relationship in these ovarian

cancers suggests that TGF-f3 induced apoptosis occurs via molecular pathways that are independent of p53. Similarly, another group has shown that transfection of a mutant p53 gene into a hepatoma cell line did not negate the ability of TGF-@3to induce apoptosis of these cells (30). Presently, it is unclear why only a fraction of ovarian cancers undergo apoptosisin responseto TGF-@, whereas proliferation of almost all of these cancers is arrested by this growth inhibitor. Although

it has been postulated

that cancer cells may be more sus

ceptible to apoptosisthan their normal counterpartsdue to the pres enceof geneticdamage,mostadvancedovariancancersare aneuploid (31). Thus, if a significant load of genetic damage is the primary factor involved in an increasedpropensityto undergo apoptosis,a larger fraction of ovarian cancers would have been expected to undergo apotosis in responseto TGF-(3. Further studies that elucidate the specific molecular pathways involved are needed to determine why only a minority of ovarian cancers that are growth inhibited by

TGF-j3undergo apoptosis.

TGF-@3often occurs in vitro during the process of immortalization, it may not commonly occur during the development of human cancers. Although immortalized cell lines are useful models for cancer re search,mosthumancancercells are not immortal, and the alterations

in growth regulatory pathways that occur during the process of

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APOPTOSIS

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