Stratton. Inc. the cell membrane to diacylglycerol and inositol trisphos- phate.6 ..... Dorset,. UK) at. 2 x 1O mol/L;. (c) TPA. (2 x i0- mol/L) plus A23187. (Sigma) in.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
1987 70: 1536-1542
Synergistic action of calcium ionophore A23187 and phorbol ester TPA on B-chronic lymphocytic leukemia cells HG Drexler, MK Brenner, E Coustan-Smith, RG Wickremasinghe and AV Hoffbrand
Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
Synergistic
Action
of Calcium
tonophore
B-Chronic By Hans
G. Drexler,
Malcolm
Lymphocytic
K. Brenner,
Elaine
blood mononuclear cells from 1 6 patients lymphocytic leukemia (B-CLL. n = 13). leukemia (B-PLL. n = 2) or hairy cell = 1 ) were incubated in the presence of the phorbol ester 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA) and the calcium ionophore A231 87. A synergy between these inducers was found with respect to morphological changes and B cell proliferation and differentiation. A23187 used alone did not activate the cells. B-CLL cells treated with the double stimulus acquired a plasmacytoid morphology. showed significantly higher incorporation of 3H-thymidine and 3H-uridine. and produced significantly higher amounts of monoclonal immunoglobulin compared with the same cells exposed to either of the inducers alone.
HRONIC
LYMPHOCYTIC
in most cells
cases,
in which
differentiation
leading
to a clonal
defect
LEUKEMIA
a monoclonal blockage
excess
(TPA)
CLL
(B-CLL)
elicit
a variety
can
cells
to be
cells.
stimulate
biologic
more
Release
mature
responses.
idiotypically
identical
immunoglobulin
changes
in
surface antigens.3 Recent studies
have
ular
basis
ofsignal
act
tion
is initiated and
linking
provided
leads
Royal
Free
the
the
of
generation
Hematology,
and
ment
London payment.
“advertisement” indicate (c)
I 987
the
cell
2QG,
Royal
These that
the
mobilization
Ca2
from
causes located
Trust
protein activation.
Free
C-mediated
protein
ion flux that
are
an
of intracellular
increase
article
accordance
article must with
therefore
and
/8
U.S.C.
the in
& Stratton,
pH.4’5
or
These
kinase
to alterations events
and
in and
seem
to be
growth.4
inositol trisphosmimicked by
A23 I 87) and A23187 causes
transport
rather than TPA increases
Patient No.
Society in
PhD,
Pond
Depart-
Si,
Hamp-
in part by page hereby §1734
marked solely
to
this fact. by Grune
influx
of
depolarization
from
phorbol esters Ca2 mobilizaintracytoplasmic
the effect of A23 structure, substitutes
by inducing the affinity
187. for
the formation of protein kinase
1
.
Cl inical
D ata on Patients
Studied
WBC(x
published
MD,
be
(calcium
Table
(H.G.D.)
were defrayed
protein
leads
activation
as
by release diacylglycerol
subsequent
in membrane
ionophores (such as TPA), respectively.
parallel mediates
C, a key calcium-dependent
phosphorylation
to cellular
trisphostwo
calcium
whereas
The
manifested
related
diacylglycerol diacylglycerol.
The
American
Hospital,
kinase
inositol
trigger trisphosphate
free
stores,
in cell
(suppl). G. Drexler,
of intracellular
activates
causally
and
messengers Inositol
intracellular
enzyme
1987.
Francisco
to diacylglycerol
antirecep-
Immunology,
ofthe
Hoffbrand
Inc.
two second act in concert.
directly
UK.
costs ofthis in
San
1985
to Hans
The
This
Leukaemia
A. Victor
(MHC)
or
10,
& Stratton.
membrane
phate.6 pathways
London. July
Meeting
1 986, 68:94a,
0006-4971/87/7005-0038$3.00/0
1536
ofMedicine,
Allen
requests
NW3
The publication charge
by Grune
a 1987
by cross-
interaction bisphosphate
accepted
Annual
December
of Haematology,
stead,
School
at the
reprint
antigen
and
and
indicate that phorbol ester and calcium synergistically on B-Cu cells to induce proliferation and differentiation. B-PLL cells responded more vigorously to the signals provided by TPA and A231 87. Previous studies showed that TPA and A231 87 can mimic the two physiological second messengers diacylglycerol and inositol trisphosphate in the transduction of signals leading to cell activation. proliferation. and differentiation in normal B cells. The present findings suggest that the capacity of B-Cu and B-Ph cells to differentiate in response to signals of the second messenger pathway is intact.
tion
activa-
on
an antigen
in B cells
by
17, 1986;
in Blood
Address
between
Wickremasinghe,
calcium stores). EDTA abrogates TPA, having a diacylglycerol-like
products
Cell
TPA
These results ionophore act
(such
activation
complex
receptor4
by the Sharon
the molec-
two
Ester
The intracellular signals triggered by phate and diacylglycerol can be independently
char-
transduction inositol
of
of Haematology and
in part
abstractform
caused
of cell activation.
immunoglobulin
October
Presented
cells
cellular
by interaction
T cell
Hospital
Supported
and
into
histocompatibility
Departments
Submitted
forms’
cell-associated
insights
tor antibodies.5 This receptor-ligand hydrolysis of phosphatidyl inositol
the
B
triggering
to
a major
of receptor
From
and
of
new
messengers in T cells
with
molecule
(Ig)
expression
transduction
as second
associated
B cell
with a more the secretion of
The key event in this signal hydrolysis of membrane-associated that
that
of
Phorbol Cells
R. Gitendra
calcium
and proliferation. pathway is the phospholipids
the
of this studies
TPA-treated
showed altered cell morphology associated differentiated state,2 and TPA alone induced acteristic
B
a basic
differentiation
into
is,
neoplastic
benefits. Numerous ester 1 2-0-tetradecanoylphor-
in vitro
of
of
appears
of these
blockage might have therapeutic have shown that the phorbol bol-13-acetate
(CLL)
expansion
and
Leukemia
Coustan-Smith,
The peripheral with B-chronic B-prolymphocytic leukemia (n
C
A23187
Inc.
Age
Sex
1
B-CLL
56
M
88
2
B-CLL
72
F
187
None
3
B-CLL
69
M
213
None
4
B-CLL
70
F
25
None
5
B-CLL
65
M
164
P,CbI
6
B-CLL
63
M
23
None
7
B-CLL
84
F
40
None
8
B-CLL
64
F
98
None
9
B-CLL
65
F
28
None
10
B-CLL
65
F
46
None
11
B-CLL
6 1
M
17
None
12
B-CLL
56
F
32
None
13
B-CLL
72
M
28
None
14
B-PLL
77
M
32
None
15
B-PLL
82
M
67
None
16
HCL
62
F
28
Abbreviations: CbI,
10’/L) of Study
Diagnosis
C, cyclophosphamide;
at Time
0,
Oncovin;
Treatment COP
None
P. prednisolone;
chlorambucil.
Blood,
Vol 70,
No 5 (November),
1987:
pp 1536-1542
of C
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
SYNERGISTIC
OF
ACTION
A23187
AND
Table
2.
TPA
Surfac
ON
1537
B-CLL
e Mark er Ph enoty
pes of Patie nts Stu died
(Before Patient
Reagent
Reactivity
CD
1
3
83
95
RFDR2
HLA-DR(classllantigen)
RFAL3
Common
RFB4
Pan-B
CD22
RFB6
C3dreceptor
CD21
90
RBF7
Pan-B
CD2O
90
84
SmIgM/G
Ig heavy
-
86
Smlgsc/X
Ig light chain
RFT1
Pan-T
CD5
E rosette receptor
CD2
RFT12
Pan-T
CD6
FMC7I
Mature
Tac
IL-2
RFT
11
91t
2
-
ALL antigen
CD1O
chain
0 85
-
6
7
8
9
10
11
12
13
14
15
97
74
84
87
80
90
69
64
90
83
88
0
0
0
0
0
0
0
0
0
0
0
0
0
94
0
78
0
2
70
74
49
0
44
81
6
92
5
1
95
70
79
94
83
96
72
80
82
ndt
80
96
70
K
ndt
K
ndl
92
94
8 95
89
K
2
0
96
91 7
98 2
of positive
for
of B-PLL
calcium8
but
0
0
0
0 77
70
75
74
49
89
43
80
78
76
78
52
90
88
81
78
ndt
80
ndt
70
77
50
90
73
98
63
nd*
K
nd*
K
K
K
K
K
85
95
93
95
85
71
77
94
11
98
9
17
22
10
18
14
19
25
10
10
10
95
86
94
15
85
93
10
95
10
2
0
6
0
61
82
83
84
56
0
nt
nt
91
94
interleukin
2.
93
6
97
84
4
3
0
0
0
2
CD25
nt
0
81
2
0
1
nt
0
0
surface
53
91
membrane
nd
5
nt
immunoglobulin;
2
IL-2,
1
A
cells.
tSurface Ig expression not detectable; monoclonality could be shown in the immunoglobulin patient no. 9, IgMA; patient no. 12, IgMK). § Patient nos. 4 and 16 were monoclonal for IgG heavy chain (all other cases monoclonal
I “Marker
0
16 82
80
Abbreviations: nt, not tested; nd, not detectable; ALL. acute lymphocytic leukemia; SmIg. #{149}Clusterof differentiation group according to First and Second International Workshop. Percentage
Nos.
5
-
B cells receptor
96
4
T Cel I Depletion)
and
does
HCL
cells.
not
provoke
a significant
increase
ELISA for
assays
(patient
no.
6, IgMK;
patient
no.
7, IgMA;
1gM).
MATERIALS
in
AND
METHODS
Ca2.9 TPA mouse
and and
known
of
possibility ied
A23
1 87
human
B
their
effects
of
the
synergistically ‘
inducing
synergism
various
act
on
B-CLL
differentiation
between
aspects
However,
TPA
of B cell
measured
by
the
measured
by
3I-I-thymidine
phological
changes
and
of
little
cells.
and
differentiation
To
A23
and which to
explore cells,
1 87
including
3H-uridine uptake,
with
respect
to
synthesis
DNA
synthesis
accompany
1
.
or
B-CLL cells treated with of TPA plus A231 87
morcells.
patients
Peripheral with
(B-PLL), nosis
stud-
RNA
Ig-producing
Patients.
is the
we
TPA. (MayGr#{252}nwald-Giemsa stained; original magnification x 1.000; current magnification x 440). (A) Cells at day 0 (typical morphology of small. round B-CLL cells with scanty cytoplasm). (B) Cells after 72 hours of treatment with 2 x lO mol/L TPA (enlarged. irregularly shaped cells with clear cytoplasm). (C) Cells after 72 hours of treatment with 2 x 10-a mol/L TPA plus 0.5 mol/L A23187 (enlarged. round to oval cells with basophilic cytoplasm. clear perinuclear zone. and eccentrically located nucleus). (D) Cells after 72 hours of treatment with 0.5 Mmol/L A23187 (cells are enlarged but remained round with scanty. clear cytoplasm). Fig
A231 87,
Morphology of a combination
normal
in CLL
activation,
uptake
in activating
comparatively
was
blood
B-CLL,
two
and one patient based
on typical
samples
patients
with
were
with
hairy
clinical,
Phenotyping centrifugation
of
cells.
(Lymphoprep, and
analyzed
Mononuclear Nyegaard, by
routine
from
13
leukemia
cell leukemia
morphological,
(l-ICL).
Diag-
cytochemical,
immunologic features. Clinical characteristics are outlined in Table I . Mononuclear cells from of four healthy donors were also investigated. Ficoll-Hypaque
obtained
prolymphocytic
of the
cells
the
cases
peripheral
were
Oslo) leukemia
and studied blood
isolated
density
gradient
phenotyping.
by
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
1538
DREXLER
Direct
and indirect
immunofluorescence
microtiter
plate
system
expression
was
determined
fluorescein sera
(Southern
Biotechnology,
was
monoclonal
antibodies, goat the
rhodamine
antihuman
Ig-specific
reagents.
conjugated against
tetramethyl
cells
FMC7
and
After
were
antimouse
with
reagents.
antigens
FITC-
with
specific
FITC-
reagents
isoanti-
specific
2
or TRITC-
Monoclonal
and
with
or A chain
K
with
incubation
stained
Ig
Tac
AL).
labeling
Ig
tests
or
by double
TRITC-conjugated
Surface
goat
Birmingham,
determined
by using the
earlier.’2
immunofluorescence
(FITC)-
(TRITC)-conjugated
expression and
in detail
by direct
isothiocyanate
thiocyanate
was performed
as described
antibodies
of the
RF-series
4’
were kindly provided by Dr iun Minowada (Fujisaki Cell Center, Okayama, iapan) and Professor George ianossy (Royal Free Hospital, London), respectively. The composite surface marker phenotypes
of the patients
Separation cells,
studied
of
plastic-adherent
suspension
cells
on Nunc
minutes
are
listed
After
cells. tissue
at 37#{176}C. The
in Table
Ficoll
were culture cell
of mononuclear
by incubation
plates
resulting
1
t
2.
separation
depleted
ET AL
(Roskilde,
population
of the
Denmark)
3
cell
4
5
6
4
5
6
Days
for 90
was T cell depleted
15
by
rosetting for 45 minutes at 4#{176}C with neuraminidase-treated sheep RBC (TCS, Berkshire, UK). Nonrosetting cells (B lymphocytes) were harvested from the interface of a second Ficoll gradient. The numbers
of
residual
labeling
of this
Leu-M3
(CD14)
Cell
population
1640
supplemented
Sussex,
UK),
10%
flat-bottomed
at
96-well
Co2 atmosphere (without further (control);
(b) (c) TPA
experiments
than
it was
I .0 #zmol/L
on
were
from
plates
formation
found
plates overnight
UK),
x
in 5%
reagents
were
Adherence
were
visually
substrate
I 87 higher
of cells assessed
were
exam-
Sewickley,
PA)
with standard
May-
to culture
in the
wells
microtiter
goat
was
used.
produced,
alkaline
assay
Dynatech,
Billinghurst,
antihuman
1gM
light
antibodies
chain
antihuman for
phosphate For
plates
C
hours.
(Sigma
determination
of the
used
For
specific
Fig 2.
the
monoclonality
of the
step.
K
All samples
Ig
or A were
tested for 1gM and IgG production. The optical density of individual wells was determined with a Multiscan MC (Titertek, Flow Lab). DNA and RNA incorporation assays. The cultures were pulsed with
I zCi/well
tional,
of 3H-uridine
Amersham,
were
harvested
tion
of
3H-uridine
UK) with
or 3H-thymidine
for one and
a Multimash into
RNA
six hours, 2000
and
(Amersham respectively.
(Dynatech),
3H-thymidine
and into
InternaThe
cells
incorporaDNA
of B-Cu cells to continuous incubation with A23187. and TPA plus A23187 in various concentrations measured at 24-hour intervals over six days. (A) 3H-thymidine incorporation (patients 1 to 1 3). Uptake of 3Hthymidine was significantly greater in the double-treated cultures than in the cultures treated with TPA or A231 87 alone on days 3 and 4 (P = .025). (B) 3H-uridine incorporation (patients 1 to 13). Uptake of 3H-uridine was significantly greater in the doubletreated cultures than in the cultures treated with TPA or A231 87 alone on days 2 and 3 (P = .007). (C) 1gM production (patient nos. 1 to 3 and 5 to 1 3). 1gM production of the double-treated cells was significantly greater than in cultures treated with TPA or A231 87 alone on days 4. 5. and 6 (P < .0001). D. Control; 0. TPA; 0. TPA + 0.7 MM A23187; C. TPA + 0.5 tM A23187; 0. TPA + 0.3 sM A23187; #{149}. 0.5 sM A23187. medium
for 1gM Substrate
as the second
3
were
visualization
antihuman
2
Days
Weybridge,
Phosphatase goat
I
(ELISA). UK)
(Dako,
antibodies
two
phosphatase-conjugated were
C.
and
of 1gM or IgG produced
immunosorbent
added
p-nitrophenyl
Tablets)
3
microscope.
rabbit
phosphatase-conjugated were
Days
was 0.3 to 0.7
changes
The amount
with
of A23
range
and stained
(MicroElisa,
(Sigma)
B
added:
or goat antihuman IgG chain-specific antiserum (Sigma). incubation of culture supernatants for two hours, alkaline
IgG
5
and
106/mL
in a humidified
concentrations
by an enzyme-linked
Microtiter
or
(Nunc)
2
(Shandon-Elliott,
phase-contrast
precoated
of
Morphological
oflgproduction.
measured
UK) After
or
(Sera-Lab,
Uxbridge,
the optimal
slides
of clusters an inverted
calfserum
following
that
cytotoxic;
solution.
Analysis was
(CDI4),
were continuously incubated of medium) for six days and
The
all cell suspensions
GrOnwald-Giemsa under
10
by
TPA (Sigma Chemical Co. Dorset, UK) at (2 x i0- mol/L) plus A23187 (Sigma) in and (d) A23l87 at 0.5 zmol/L. In prelimi-
cytocentrifuge
prepared
fetal concentration
Mmol/L A23 187. Morphological evaluation. med
determined MY4
(GIBCO,
a cell
intervals.
concentrations;
nary
(CD13),
at 37#{176}C.The cells addition or exchange
mol/L;
various
MCS-2
were
(CD3)
microwell
at 24-hour
none
cells
heat-inactivated
(GIBCO)
2 x 1O
T
penicillin/streptomycin
L-glutamine
(a)
with
and Leu-4
with
harvested
and
as described earlier. and in vitro stimulation. Cells were cultivated in medium (FLow Laboratories, Rickmansworth, UK)
culture
RPMI
monocytes
was
Response TPA.
alone.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
SYNERGISTIC
ACTION
measured
by
UltroBeta,
Bromma,
OF
standard
Statistical
A23
liquid
AND
ON B-CLL
TPA
scintillation
counting
1539
1210
plasm.
groups
often plasm
(LKB
Sweden).
Comparisons
analysis.
were made
1 87
by using
paired
between
treatment
t testing.
ent cells analyzed Less
After
populations.
(monocytes, contained
than
cells
(pan-T) or for monclonal cytic antigens (MCS-2,
were
the
which
cases
and
and
samples of 98%.
with
a clear,
“Hof”-like
toid
features
were
positive
for Leu-4
or RFT-2
TPA and 0.5 imol/L A23 187. 3H-thymidine incorporation.
HCL
associated
were
with
Normal B cells were obtained healthy adults. However, these not
represent
cells
the any
express
positive
but
not
for
for
42%
cells
Morphological tion, control
by
B-CLL
which
B cells.’4
populations
RFB7
cells
is limited).
CD5,
blood
normal
stained
to B-CLL results
cell marker
peripheral
monocyte-depleted
the
blood B cells
The
T cell-
contained
2A,
and to
cases
of B-PLL
could
not
be triggered
hours cells
incubawere
3H-uridine rized in Fig
incorporation
into
small
the activation
with
clumps
or aggregates similar
growth
plus
were
A23l87
formed
nonadherent.
B-PLL
cul-
growth pattern of HCL2: the
alone or displayed
as described cells became
with the promi-
of incubation
with
On light microscopy, exposed cells remained Table
3.
trypsin
elsewhere strongly
3H-Thymidine With
None(control)
x 108mo1/L) O.7umoI/LA23187
TPA
+
TPA
+ 0.5zmoI/LA23187
Malignant
1,318
±
1,207
13,828
to the double the cells from
4 in In
stimulus patient
did no. 3
3H-thymidine
(Table
In the experiments were stimulated
cells
that
was
at day
with varying to 3H-uridine
significantly
one of the inducers 3 with
alone
summaeither
with
increased
(P
0.7 imol/L
over
.007).
=
A23
response of cells from patient no. I 5 (Bthe same magnitude as seen in the B-CLL
uptake occurred 3 in B-CLL
413
±
381
A23187
amounts The on
B-Ph,
on day 2, whereas cells. HCL cells did
of 3H-uridine (Table effect of the combined
Ig production
was
and HCL Cells
at Days
assessed
it was uninot incorpo-
5). action
of TPA
in an
ELISA
3 or 4 of Treatment
Plus *23187 B-PU. (Patient 1 5)
In
-
3) 94
±
240
50,473
54,762
178
± 4,159
63,840
87,693
256
1,397
66,354
51,764
143
(mean
±
(mean
Counts
per minute
from triplicate wells). ± SD from three donors).
13 patients).
486t
Normal B Cells 16)
384
per minute
229t
HQ. (Patient
328
iOO
The
I 87 plus
45,726
per
(mean
to day
not responsive.
±
at 4 x 10 cells per well and normal SD from
incorpo-
242
nd, not done.
minute
were
the
maximum
43t
tCounts
Counts
B-PU. (Patient 14)
154
159±
cells were cultured
3 as opposed
cells
at
used,
the
to incorporate
on day
by B-Cu. and TPA
±
1,288±
A23187(0.5moI/L)
and
177
2,478
TPA+O.3zmol/LA23187
Abbreviation:
1- 1 3)
(Patients
RNA
was seen
rate significant lgproduction.
*23187.
B-CLL
TPA(2
formly
Incorporation TPA.
peak
3H-uridine
solution.
of the Maximal
Treatment
peaked
higher
at day
=
cultures,3H-uridine incorporation of cultures from patient no. 14 (B-PLL) was about four- to sixfold higher (Table 5). In both cases of B-PLL, the maximum response in terms of
control B-CLL cells and A23 187round with slightly increased cyto-
Comparison
and
alone or TPA in combination The combined agents led
Although the PLL) was ofabout
adherent and developed very long, thin, branched, cytoplasmic extensions. These cells could not be detached by vigorous shaking or pipetting with culture medium but required two cycles
or A23187 of A23187.
highest TPA.
changes.
HCL cells treated with either TPA combination ofTPA and calcium ionophore nent changes in their for TPA treatment
plus TPA
a severalfold
HCL
of
alone (P of prolifera-
3). In addition,
response alone;
incorporation. 2B, B-CLL
Cells showed
was
The
I and 4 the that to TPA
clumps. tures
the double
concentration
showed
cells
samples.
TPA doses
that
A23l87
(Table
of B-PLL B-CLL
were with large
TPA
cells,
4).
After 72 to 96 and A23l87-treated
with
zmol/L
of 3H-thymidine
round, small, and nonadherent. TPA-exposed cells either adherent to the surface of the culture dish cytoplasmic elongations or clustered in densely packed, treated
in cells treated 3). Maximal
at 0.5
patient nos. not surpass
(CD2O).
changes. cells
In B-CLL
to both
with TPA stimulation
most
28%
plasmacy-
exposed
found Table
(Fig
response
on
These
in cells
incorporation
Both
(and
is detected
zone.
pronounced
increased the uptake over the 3H-thymidine
ration
B-CLL
cytowith
evenly dispersed with was deeply basophilic
perinuclear
most
day 4. At the highest ionophore response was less pronounced.
of do
shaped,
TPA and A231 87 into DNA significantly
tion occurred
these
irregularly
stimulus with 3H-thymidine .025)
FMC7,
specific
from the peripheral blood-borne normal
counterparts of
the pan-T
of normal
