Download Inhibition of Leukemic Cell Growth by the Protein

(CANCER RESEARCH 55. 3716-3720.
September 1. 1W]
Advances in Brief
Inhibition of Leukemic Cell Growth by the Protein Kinase C Activator Bryostatin 1
Correlates with the Dephosphorylation of Cyclin-dependent Kinase 21
Clement Asiedu, Joseph Biggs, Michael Lilly, and Andrew S. Kraft2
Division of Henialolofiy/Oficologv.
Universitv of Alabama, iïirniinghain. Altihatna .Õ5294¡C.A., J. B., A. S. K. l ami Medical Oncolo^\.
Scalile VA Medical Center. Unirersitv of
Wasiiiii/itim.
Scuttle.WaiHngun98108\M. LI
Abstract
Bryostatin 1 is a natural antineoplastic agent that activates protein
kinase C. Treatment of U937 human leukemic cells with bryostatin 1
caused a 60% reduction in cell growth, whereas another protein kinase C
activator, phorbol my ristate acetate (PMA), completely inhibited U937
cell growth. Both bryostatin 1 and I'MA induced inhibition of cyclindependent kinase 2 (cdk2) activity. The first phase of cdk2 inhibition
correlated with the transient induction of p21, a known inhibitor of cdk2.
In contrast, the second phase of cdk2 inhibition correlated with the
dephosphorylation of cdk2 on threonine-160, which must be phosphorylated for cdk2 activity. The level of growth inhibition induced by these two
compounds correlated with the degree of cdk2 dephosphorylation
as
follows: bryostatin 1, 60%; PMA, 100%.
role in regulating the growth inhibition induced by PKC-activating
agents.
The molecular basis of the growth-inhibitory effect of bryostatin 1
is unknown. To determine its mechanism of growth inhibition and to
further examine the differences between the effects of bryostatin 1 and
PMA, the activity of cdk2 in U937 cells was studied. Both compounds
induced inhibition of cdk2 activity; the early phase of inhibition
correlated with a transient induction of p21, and the later phase
correlated with the dephosphorylation of cdk2 on both tyrosine and
threonine residues. The level of cdk2 dephosphorylation on threonine160 paralleled the degree of growth inhibition of human leukemic
cells induced by bryostatin 1 and PMA, suggesting that cdk2 dephos
phorylation and inactivation may be important in the growth-inhibi
tory and antitumor activity of bryostatin 1.
Introduction
Bryostatin 1, a naturally occurring macrocyclic lactone, is an anticancer compound isolated from the marine bryozoan. ungula neritina
(1). With both in vitro and in vivo anticancer activity against murine
tumors (2), bryostatin 1 has undergone human Phase 1 testing (3). It
induces the translocation of PKC1 to the plasma membrane (4), leads
to the phosphorylation of specific protein substrates (5), and eventu
ally induces PKC degradation (6). Although it modulates PKC in a
similar fashion to the tumor-promoting phorbol esters (7), it is not a
tumor promoter (8). As with phorbol esters, treatment of HL-60 cells
with bryostatin 1 induces their macrophage-like differentiation, but
unlike PMA which completely inhibits HL-60 cell growth, bryostatin
I only reduces the growth rate of these cells (9).
Treatment of U937 cells with PMA causes them to arrest in the late
O, phase of the cell cycle (10). Progression through the cell cycle is
regulated by cdks, the activation of which involves both the binding
of a cyclin partner (11) and phosphorylation and dephosphorylation of
specific threonine/tyrosine residues (12). For example, cdk2 is acti
vated by the phosphorylation of threonine-160 and dephosphorylation
of tyrosine-15 and threonine-14 (13). Active cyclin E/cdk2 complexes
are necessary for the G,-S progression of the cell cycle (11). Recent
evidence suggests that the p53-inducible protein p21 (WAF1/CIP1) is
an important downstream effector of p53-mediated G, arrest that
regulates the activity of cdk2 (14, 15). Treatment of human leukemic
cells with PMA has been shown to activate transcription of p21 by
p53-indepcndent mechanisms (16), suggesting that p21 may play a
Received 6/27/95; accepted 7/20/95.
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 indicale this fact.
1This work was supported by N1H CA 42533 and ACS grant DHP 83 to ASK.
CA45672 and a grant from the Veterans Administration Research Program to ML. and
ACS grant IRG-f>6-32 to JB.
- To whom requests for reprints should be addressed, at Division of Hematology/
Oncology. University of Alabama/Birmingham.
558T Lurleen B. Wallace Tumor
Institute, 1824 Sixth Avenue South. Birmingham, AL 35294-3300.
* The abbreviations used are: PKC', protein kinase C; PMA. phorbol myristate acetate;
cdk, cyclin-dependent
kinase.
Materials and Methods
Cell Culture. U937 human myeloid leukemic cells obtained from the
American Type Culture Collection were grown in DMEM supplemented with
10% heat-inactivated bovine calf serum (GIBCO/BRL, Gaithersburg, MD) at
37°Cin 5% CO,. Cell growth was analyzed by seeding exponentially growing
U937 cells at 1 X IO5 cells/ml in triplicate. Cells were left untreated or were
cultured in the presence of either 200 nM bryostatin 1 or PMA. Cells were
counted in a Coulter counter model ZM.
Immunoprecipitation
and Kinase Assays. Cells were lysed in 0.5%
NP40 lysis buffer containing protease and phosphatasc inhibitors, as described
(17). cdk2 immunoprecipitates were prepared from equivalent amounts of
protein, and the immune complexes were assayed for histone HI kinase
activity, essentially as described (18).
Northern Blotting. Isolation of total RNA and Northern blotting analysis
were carried out according to procedures described previously (19). 32Plabeled p21 (2.1 kb Noll fragment from plasmid pc-WAFl-S)
and a-tubulin
(1.5 kb Rvfl fragment) cDNAs were used as probes.
p21 PCR. Total RNA was isolated and subjected to reverse tnmscriptionPCR to simultaneously amplify p21 and a-tubulin sequences, using standard
conditions. The four primers used were: WAF 5', 5'-CAGCTGAGCCGGGACTG; WAF 3', 5'-CGCGCTTCCAGGACTGCAGG;
actin 5', 5'-CTGGCAAATGCACACACC,
and actin 3', .V-GACAGACCCGCAAGACAA.
Western Blotting. Proteins from cdk2 immunoprecipitates were resolved
by SDS-PAGE and transferred to Immobilon P membrane. cdk2 and antiphosphotyrosine blots were blocked in Tris-buffered saline (TBS)/0.1% Tween
20/5% bovine serum albumin and incubated for l h in blocking buffer con
taining anti-cdk2 antiserum (a gift of Dr. V. Kidd, St. Jude Children's Research
Hospital) at a dilution of 1:500, and the 4G10 antiphosphotyrosine antibody
(UBI, Lake Placid, NY) at a dilution of 1:1000, respectively. After washing
three times for 10 min each in TBS/0.5% NP40, blots were incubated for 45
min with second antibody in blocking buffer and washed. p21 blots were
blocked in PBS/1% nonfat Carnation milk and washed three times for 10 min
each in TBS/0.5% BSA. After incubating with anti-p21 polyclonal antibody
(Pharmingen, San Diego, CA), 1:1000 dilution in PBS/1% BSA for 1 h, the
membrane was washed and incubated in PBS/1% nonfat Carnation milk for 30
min. Blots were washed and then incubated with second antibody for 45 min
in PBS/1% BSA and then washed extensively. All blots were developed with
Renaissance chemiluminescence reagent (NEN Dupont, Boston. MA).
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PKC ACTIVATORS
INDUCE CDK2 DBPHOSPHORYIATION
In Vivo 32P Labeling.
Exponentially growing U937 cells were treated with
200 nM bryostatin 1 for various periods of time. [12P]Orthophosphate labeling
and cdk2 irnmunoprecipitation
(17, 20).
were performed,
as described
cells also demonstrated inhibition of cdk2 activity (Fig. \B). PMA
treatment of U937 cells induced a 70-80% drop in cdk2 activity
within 4-8 h, followed by a total inhibition of activity by 24-72 h.
previously
These data suggest that the degree of bryostatin 1 and PMA inhibition
of cdk2 activity correlates with the level of growth inhibition induced
by these agents.
Recent studies demonstrate that cdk2 activity is inhibited by p21
(14). PMA treatment of U937 cells stimulates an increase in p21,
suggesting that p21 may be important in the regulation of the cell
cycle in response to PMA (16). To determine whether bryostatin 1
also induced p21, U937 cells were treated for varying periods of time,
and p21 mRNA expression was analyzed by Northern blot analysis.
Bryostatin 1 caused a dramatic transient induction of p21 mRNA (Fig.
2A). p21 mRNA expression peaked at 4 h and declined thereafter until
no message was detectable by 24-48 h (in contrast to tubulin control),
Results
To evaluate the effect of bryostatin 1 on human leukemic cell
growth, U937 cells were cultured in the presence of either bryostatin
1 (200 nM) or PMA (200 nM), and cell numbers were quantitated for
5 consecutive days. Although PMA completely inhibited U937 cell
growth, bryostatin 1 caused only a 60% reduction in cell growth up to
day 5 (Fig. 1/4). Even though bryostatin 1 was growth inhibitory,
U937 cells continued to divide slowly in the presence of this com
pound. Similar growth results with bryostatin 1 have been reported
previously (9).
The transit of cells through the cell cycle is controlled by cyclin/cdk
complexes. Treatment of U937 cells with PMA causes them to arrest
in the late G, phase of the cell cycle (10). Because active cdk2 is
important in regulating the G,-S transition, the levels of cdk2 activity
were evaluated after bryostatin 1 or PMA treatment. cdk2 was immunoprecipitated from treated U937 cells, and cdk2 activity was meas
ured in vitro using histone HI as a substrate. Bryostatin 1 treatment
consistently induced a biphasic reduction but never completely inhib
ited cdk2 activity (Fig. IB). From baseline, bryostatin 1 caused a 45%
reduction in cdk2 activity by 4 h, a 27% reduction at 8 h, and a 63%
reduction at 24 h, where it remained up to 72 h. PMA-treated U937
(Fig. 2A).
Since bryostatin 1 is an anticancer agent that potentially will be
used to treat patients with solid tumors and leukemias, it was of
interest to determine whether bryostatin 1 also induced p21 in normal
bone marrow and fresh leukemic cells. Using reverse transcribed
cDNA and PCR, bryostatin 1 is shown to induce p21 mRNA in normal
bone marrow cells, FACS purified CD34+ cells, and peripheral blood
cells from a patient with chronic myelogenous leukemia (Fig. 2B). These
findings demonstrate that the effects of bryostatin 1 are not limited to a
cell line in culture but are also seen with fresh patient samples.
To evaluate the levels of p21 protein in U937 cells after treatment
1.2-1-0.8--•Control-•PMA—A—
Bryostatin
1
•55
0.6O
0.4Fig. I. Comparison of the effects of bryostatin 1
and PMA on cell growth and cdk2 kinase activity.
A, bryostatin I and PMA inhibit growth of U937
cells. Cells were treated with 200 n\i bryostatin 1 or
PMA in triplicates and counted on the days indi
cated. The average values are shown. H. bryostatin
I and PMA inhibit cdk2 kinase activity. Total cell
protein (2(M) fig) was subjected to cdk2 immunoprecipitation and histone HI phosphorylation
assays.
0.20
3
Day
B
Bryostatin 1
Rx (h)
Relative Kinase
Activity (%)
PMA
0
4
8
24 48 72
100
55
73
37
28
40
02
100
87
4
8
35
21
18 24
48 72
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PKC ACTIVATORS
INDUCE CDK2 DEPHOSPHORYLATION
Bryostatin 1 Rx (h) 0 0.5 1
2
4
8
24
48
WAF1/CIP1
Tubulin
Fig. 2. Bryostatin 1 induces expression of p21 in
hematopoielic cells. A, hryostatin 1 induces accu
mulation of p21 mRNA in U937 cells. Total RNA
isolated from bryostatin 1-treated cells was ana
lyzed by Northern blotting with 32P-labeled p21
and a-tubulin cDNA probes. B, bryostatin 1 in
duces expression of p21 in fresh myeloid leukemic
blood cells. Total bone marrow cells, partially pu
rified CD34* cells, and mononuclear cells purified
from chronic myelogenous leukemic peripheral
blood were treated with bryostatin 1. Total RNA
isolated from these cells was subjected to reverse
transcription-PCR to amplify p21 and a-actin se
quences simultaneously. PCR products were ana
lyzed in ethidium bromide-containing agarose gels. C,
p21 induced by PKC activators associates with cdk2
in vivo, U937 cells were treated with 2(X)nM bryosta
tin 1 and PMA for the indicated times. Proteins in
cdk2 immunoprecipitates were subjected to Western
blot analysis with anti-p21 polyclonal antiserum.
B
bone
marrow
Cells:
CD34+
marrow
WBC/CML
Bryostatin 1 Rx:
-WAF1/CIP1
-actin
EtBr
PMA
8
24
Bryostatin 1
Rx (h)
WAF1/CIP1
0
4
8
24
48
72
48
72
«
cdk2 activity is stimulated by threonine-160 phosphorylation and
with bryostatin l, U937 cells were treated with bryostatin 1, and cdk2
inhibited by threonine-14 and tyrosine-15 phosphorylation. Because
was immunoprecipitated. Proteins in the immunoprecipitates were
resolved by SDS-PAGE, transferred to Immobilen P membrane, and
threonine-160-phosphorylated
cdk2 runs more rapidly on SDS-PAGE
than cdk2 phosphorylated on threonine-14 and tyrosine-15, cdk2
probed with a p21 polyclonal antiserum (Pharmingen). This Western
blot demonstrates that both bryostatin 1 and PMA induced p21 protein
Western blots contain two cdk2 bands (13). Bryostatin 1 treatment
maximally at 4-8 h (Fig. 2C). p21 protein was undetectable in U937
caused a 49% decrease in the intensity of the faster-migrating threocells at 48-72 h, a time when cdk2 activity continues to decrease.
nine-160-phosphorylated
cdk2 band at 48 h (lower band), which
Similar results were obtained by directly probing Western blots of appeared to increase to 76% of control by 72 h, as determined by
cellular extracts with anti-p21 antiserum (data not shown). In com
densitometric scanning (Fig. 3/4). In comparison, PMA induced a
marked reduction in the lower threonine-160-phosphorylated
form of
parison to PMA, bryostatin 1 induced lower levels and more transient
cdk2 by 24 h and the complete loss of this phosphorylated form of
expression of p21. The induction of p21 by bryostatin 1 correlated
with the inhibition of cdk2 activity observed at 4 h. That no p21 is cdk2 by 72 h (Fig. 3fi). The difference between these agents was not
detectable in U937 cells at 24-72 h when cdk2 activity is decreased
dependent on the concentration of bryostatin 1 used, since over a wide
suggests that a second mechanism of bryostatin 1-induced inhibition
range of concentrations, 0.2-1000 nM, bryostatin 1 did not induce
of cdk2 activity must be activated.
complete cdk2 dephosphorylation (Fig. 3C) or inactivation (data not
cdk2 activity is also regulated by several phosphorylation events.
shown).
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PKC ACTIVATORS
Bryostatin
1 Rx (h)
o
4
8
24
48
INDUCE CDK2 DEPHOSPHORYLATION
72
IgH
cdk2
cdk2-P
phosphorylation between bryostatin I and PMA were simply concen
tration dependent, U937 cells were treated for 48 h with varying
concentrations of bryostatin 1. Cellular extracts were immunoprecipitated with cdk2 antibody and evaluated by anti-phosphotyrosine
Western blot analysis. Interestingly, the highest concentration of
bryostatin 1, 1000 nM, had less of an effect on tyrosine phosphoryl
ation than 20 nM concentration (Fig. 4ß).A similar concentrationdependent effect on leukemic differentiation has been reported previ
ously (4). In comparison to bryostatin 1, the addition of both 1000 and
200 nM PMA each caused complete cdk2 tyrosine dephosphorylation
(Fig. 4B).
The phosphorylation status of cdk2 after bryostatin 1 treatment of
U937 cells was directly evaluated by [3~P]orthophosphate labeling,
Bryostatin 1
RX (h)
B
PMA Rx (h)
0248
24
0
4
8
24
PMA
48 72
24
48
72
48
cdk2-P
cdk2
cdk2-P
-
*
-
B
48h Rx (nM)
48h
Rx (nM)
-
Bryostatin
1000 200 20
1
2
-
Bryostatin
1000 200 20
1
2
PMA
0.2 1000 200
PMA
0.2 1000 200
cdk2-P
cdk2
cdk2-P
Bryostatin 1 Rx (h)
Fig. 3. PKC activators induce cdk2 dephosphorylation of threonine-160. U937 cells
were treated with 2(H) nM bryostatin 1 (A) or PMA (ß)for the indicated limes. C. U°37
cells were treated with the indicated doses of bryostatin 1 or PMA for 48 h. cdk2
immunoprecipitates were prepared and subjected to Western blot analysis with anti-cdk2
antiserum.
o
24 48 72
cdk2-P
To determine whether bryostatin 1 affected the tyrosine phospho
rylation of cdk2, cdk2 immunoprecipitates were subjected to antiphosphotyrosine Western blot analysis. Treatment of U937 cells with
Fig. 4. Bryostatin 1 and PMA stimulate dephosphorylation of cdk2. A, bryostatin 1 and
bryostatin 1 induced a partial dephosphorylation of cdk2 on tyrosine
PMA stimulate dephosphorylation of cdk2 on lyrosine-15. U937 cells were treated with
by 24 h, which was maximal but not complete by 48 h (Fig. 4/4). 200 nvi bryostatin 1 or PMA for the times shown. Cell lysates (400 ¿tgtotal protein) were
subjected to cdk2 immunoprecipitation, Inllowed by anti-phosphotyrosine Western blot.
However, 72 h after bryostatin 1 treatment, the tyrosine phosphoryl
B. bryostatin I and PMA dose response. U937 cells were treated with the indicated doses
ation of cdk2 returned, approaching levels observed in control cells
of bryostatin 1 and PMA for 48 h. cdk2 immunoprecipitates were prepared and analyzed
(Fig. 4/4). In comparison, by 24 h of PMA treatment, no tyrosine
by antiphosphotyrosine Western blotting. C, bryostatin 1 induces partial dephosphoryl
ation of cdk2. U937 cells were treated with 20(1MMbryostatin 1 for the indicated times and
phosphorylation was detectable, and this phosphorylation was not
labeled with ['-P]-orthophosphatc for 4 h. cdk2 immunoprecipitates were prepared from
regained at the 72 h time point (Fig. 4/4).
the ' ~P-labeled cell lysates. Proteins from the edk2 immunoprecipitates were resolved on
SDS-acrylamide gel and visualized by autoradiographv.
To rule out the possibility that the differences in effect on tyrosine
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PKC ACTIVATORS
followed by cdk2
decrease in cdk2
observed on both
the levels of cdk2
INDUCE CDK2 DEPHOSPHORYLATION
immunoprecipitation. Bryostatin 1 induced a 60%
phosphorylation by 48 h (Fig. 4Q. However, as
anti-cdk2 and anti-phosphotyrosine Western blots,
phosphorylation increased by 72 h.
antiserum. Dr. Bert Vogelstein (Johns Hopkins Medical Center, Baltimore.
MD) for supplying the p21 cDNA, and Dr. Bill Weaver for his help in
reviewing this manuscript.
References
Discussion
In this study, we have shown the following: (a) the natural antitumor agent, bryostatin 1, which like PMA is a protein kinase C
activator, induces a biphasic inhibition of cdk2 kinase activity; (b)
bryostatin 1 transiently induces p21, this induction correlates with the
early partial inhibition of cdk2 activity caused by bryostatin 1; (c) the
later partial, sustained inhibition of cdk2 kinase activity induced by
bryostatin 1 is p21-independent and is correlated with dephosphorylation of cdk2 on threonine-160; and (ci) the degree of cdk2 dephosphorylation induced by both bryostatin 1 and PMA parallels the ability of
each of these compounds to inhibit the growth of leukemic cells.
The partial inhibition of cdk2 kinase activity is sustained from 24 to
72 h after bryostatin 1 treatment, a period during which p21 is
undetectable at both the mRNA and protein levels, suggesting the
existence of at least one other inhibitory mechanism that is p21
independent. Phosphorylation of cdk2 on threonine-160 is essential
for kinase activity, while tyrosine-15 phosphorylation is inhibitory
(13). Our data demonstrate that the second phase of cdk2 inhibition by
both bryostatin 1 and PMA correlates with the loss of both threonine160 and tyrosine-15 phosphorylation. PMA induction of differentia
tion of hematopoietic cells by PMA has been shown to induce in
creases in transcription in specific protein phosphatases (21). It is,
therefore, possible that bryostatin 1 and PMA induced a dual speci
ficity phosphatase that dephosphorylated both the tyrosine and threonine residues in cdk2.
In normal cells, p21 transcription is regulated by two upstream p53
sites (14). However, PMA is capable of inducing p21 in U937 leu
kemic cells that are p53 negative (22), suggesting that additional
mechanisms exist for the regulation of this gene. Likewise, bryostatin
1 was capable of inducing p21 in U937 human myeloid leukemic cells
by a p53-independent mechanism. This result could have important
clinical ramifications since many solid tumors contain p53 mutations
and cannot respond to either X-rays or chemotherapy with a p53mediated increase in p21 (23). Because bryostatin 1 will be adminis
tered to patients as cancer chemotherapy, it will be important to
examine in patients whether solid tumors can also respond with
increases in p21. It might then be possible to combine bryostatin 1
with other modes of cancer chemotherapy.
In this report, we present evidence that suggests that another po
tential target of cancer therapy might be the induction of cdk2 dephosphorylation, leading to the inhibition of cell growth. Both bryo
statin 1 and PMA induced a dephosphorylation of cdk2, but it is
unclear why bryostatin 1 effects on dephosphorylation were weaker at
all concentrations than PMA. Bryostatin 1 causes a more rapid deg
radation of PKC (6) and regulates specific PKC isoforms differently
from PMA. Since many cancers lack additional regulators of cdks,
e.g., pl6 (24), it is of interest to determine whether the induction of
dephosphorylation by bryostatin 1 and PMA is specific for cdk2 and
whether it can also regulate the activity of other cdks, e.g., cdk4.
Acknowledgments
We thank Dr. Vincent Kidd (St. Jude Children's
Research Hospital. Memphis,
TN) for helping us with the cdk2 kinase assays and supplying the anti-cdk2
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Inhibition of Leukemic Cell Growth by the Protein Kinase C
Activator Bryostatin 1 Correlates with the Dephosphorylation of
Cyclin-dependent Kinase 2
Clement Asiedu, Joseph Biggs, Michael Lilly, et al.
Cancer Res 1995;55:3716-3720.
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Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1995 American Association for Cancer Research.