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Mol Cell Biol, June 1998, p. 3509-3517, Vol. 18, No. 6
Laboratory of Immunology, National Institute
on Aging, National Institutes of Health, Baltimore, Maryland
21224-68231;
Greater Baltimore Medical
Center, Baltimore, Maryland 212042;
Washington University School of Medicine, St. Louis, Missouri
631103; and
National Cancer
Institute, National Institutes of Health, Bethesda, Maryland
208924
Received 19 September 1997/Returned for modification 6 January
1998/Accepted 2 March 1998
The Bcl2 family of proteins plays a significant role in regulation
of apoptosis. In this study, the microtubule-damaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2
hyperphosphorylation and apoptosis in MCF-7 and
MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or
vincristine induced increased expression of Bax, while overexpression
of Bcl2 in these cell lines counteracted the effects of low doses of
these drugs. In addition, paclitaxel- and vincristine-induced
activation of cyclic AMP (cAMP)-dependent protein kinase
(protein kinase A [PKA]) induced Bcl2
hyperphosphorylation and apoptosis, which were
blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This
finding suggests that activation of PKA due to microtubule damage is
an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs
caused growth arrest in G2-M phase of the cell cycle and
had no effect on p53 induction, suggesting that
hyperphosphorylation mediated inactivation of
Bcl2 and apoptosis without the involvement of p53. By
comparison, the DNA-damaging drugs methotrexate and doxorubicin had no
effect on Bcl2 hyperphosphorylation but induced p53
expression. Interestingly, paclitaxel or vincristine induced activation
of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of
Bcl2 hyperphosphorylation. These data suggest that
there may be a signaling cascade induced by agents that disrupt or
damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to
apoptosis), the cellular response to DNA damage.
Apoptosis plays an important role in
a wide variety of physiological processes and, when dysregulated,
contributes to the pathogenesis of many diseases, including
cancer, autoimmunity, and neurodegenerative disorders
(16, 41). While the regulation of apoptosis has been
extensively studied, little is known about the mechanisms of cell
survival or death. Among the growing number of genes that regulate
apoptosis induced by a wide variety of stimuli is the Bcl2
family of genes (2, 21, 33). Some proteins within this
family, including Bcl2 and Bcl-XL, inhibit
apoptosis, while others, such as Bax and Bak, promote
apoptosis. Bcl2 and related antiapoptotic proteins seem to
dimerize with a proapoptotic molecule, Bax, and modulate the
sensitivity of a cell to apoptosis (30, 35).
Apoptosis is an active and gene-directed form of cell death with
well-characterized morphological and biochemical features (16). Recent evidence suggests that the activation of a
cascade of cysteine proteases of the interleukin-1 Recently, it has been shown that Bcl2 may protect cancer cells from
apoptosis induced by a variety of anticancer agents (4, 11, 32). The precise mechanism of the Bcl2-induced
multidrug resistance is unknown. Microtubule-stabilizing
agents such as paclitaxel and docetaxel (11,
22) and microtubule-disrupting drugs such as vincristine,
vinblastine, and colchicine have antimitotic and
apoptosis-inducing activity (8). Human leukemic,
breast cancer, and prostate cancer cells exposed to
paclitaxel express a phosphorylated form of
Bcl2 and undergo apoptosis, suggesting that
phosphorylation of Bcl2 may inhibit Bcl2 function. In
addition, Bcl2 phosphorylation appears to inhibit its
binding to Bax, since less Bax was observed in an immunocomplex with
Bcl2 in taxol-treated cancer cells (12).
The objectives of this study were (i) to determine if overexpression of
Bcl2 in MCF-7 and MDA-MB-231 cells can protect the cell from
apoptosis induced by paclitaxel or vincristine and
(ii) to ascertain the functional consequences of the
phosphorylation of Bcl2. We demonstrated that
overexpression of Bcl2 counteracts the apoptotic effects of low doses
of paclitaxel or vincristine but has no effect against high
doses of these anticancer drugs. Furthermore, microtubule-damaging
drugs (paclitaxel, vincristine, and vinblastine) induced
apoptosis, caused growth arrest in G2-M phase of
the cell cycle, induced caspase 3 activation as well as PARP
degradation, but did not induce p53. In addition, microtubule-damaging drugs led to hyperphosphorylation of Bcl2 through
protein kinase A (PKA), whereas DNA-damaging drugs did not. In
contrast, DNA-damaging drugs induced p53 but had no effect on Bcl2
hyperphosphorylation, indicating that
microtubule-damaging drugs induce apoptosis through a
p53-independent mechanism. Interestingly, nocodazole, a reversible microtubule poison, does not activate PKA, does not induce Bcl2 hyperphosphorylation, and does not induce
apoptosis. This finding suggests that activation of PKA due to
microtubule damage is an important event in Bcl2
hyperphosphorylation and induction of apoptosis. However, some forms of microtubule damage activate a
signal transduction pathway that ultimately leads to
apoptosis.
Reagents.
Paclitaxel (Taxus brevifolia), vincristine,
vinblastine, nocodazole, methotrexate, doxorubicin, etoposide,
vanadate (sodium orthovanadate), Hoechst 33258, cyclic AMP
(cAMP), kemptide, and forskolin were purchased from Sigma Chemical Co.,
St. Louis, Mo. Anti-Bcl2 antibody and anti-p53 antibody were purchased
from Oncogene Science, Uniondale, N.Y. Anti-Bax antibody and anti-Yama
(caspase 3) antibody were purchased from Transduction Laboratories,
Lexington, Ky. Anti-PARP antibody was purchased from Santa Cruz
Biotechnology Inc., Santa Cruz, Calif. Enhanced chemiluminescence
Western blot detection reagents were purchased from Amersham Life
Sciences Inc., Arlington Heights, Ill.
[32P]orthophosphoric acid (specific activity, 3,000 Ci/mmol) was purchased from NEN Life Science Products, Boston,
Mass. L-[35S]methionine (specific
activity, 1,000 Ci/mmol) and [ Cells and culture conditions.
The human breast cancer cell
lines MCF-7 and MDA-MB-231 and the prostate carcinoma cell line DU145
were obtained from the American Type Culture Collection, Rockville, Md.
Cells were cultured in either Dulbecco modified Eagle medium (MCF-7) or
RPMI 1640 (MDA-MB-231 and DU145) tissue culture medium (BioWhittaker
Inc., Walkersville, Md.) supplemented with 10% fetal calf serum and a
1% penicillin-streptomycin mixture. Clones were obtained by limiting
dilution.
Expression constructs and transfection.
Viable cells (2 × 106) were transfected with a mixture of Lipofectin
reagent (GIBCO BRL) and either plasmid pSFFVneo-Bcl2 or plasmid
pSFFV-neo. Transduced cells were selected in medium containing 1 mg of
G418 (Geneticin; GIBCO BRL) per ml for 4 weeks. We selected six clones
each of MCF-7 and MDA-MB-231 cells and observed little clonal variation
in the observed effects. Lysates were evaluated for p26Bcl2 expression
by immunoblot analysis (data not shown). MCF-7 cells were stably
transfected with either vector alone or a CrmA expression construct
(39). For glutathione S-transferase (GST)-Bcl2
fusion protein, full-length Bcl2 cDNA was cloned into pGEX-4T-2
expression vector (Pharmacia Biotech Inc., Piscataway, N.J.). This
vector was expressed in bacteria, and recombinant fusion protein was
purified from bacterial lysates by gravity-flow affinity chromatography
using a Bulk GST purification kit (Pharmacia Biotech Inc.).
MTT assay.
Cells (103) were seeded into 96-well
tissue culture plates. Viable cells were quantitated by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay (7). In brief, cells were treated with anticancer drugs for 18 h (see the figure legends). Before
harvesting, 20 µl of MTT solution (5 mg/ml) was added for
3 h to each well followed by 100 µl of 10% sodium dodecyl
sulfate (SDS) and 0.01 N HCl. Viability was quantitated by measuring
A570, using an enzyme-linked immunosorbent assay
(ELISA) plate reader with a reference wavelength of 650 nm. The mean
absorbance of four culture wells per time point was plotted. The
percentage of cell survival was defined as (mean
A650 of treated
wells/A650 of untreated control wells) × 100.
In vitro and in vivo phosphorylation.
In
vitro phosphorylation was performed on GST-Bcl2
protein. Briefly, 50 µg of GST-Bcl2 fusion protein was used in a
final reaction volume of 50 µl with PKA buffer (10 mM Tris-HCl
[pH 7.5], 0.25 mM dithiothreitol, 20 mM
MgCl2, 100 µM ATP, 20 µCi
[
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Involvement of Microtubules in the Regulation of Bcl2
Phosphorylation and Apoptosis through Cyclic AMP-Dependent
Protein Kinase
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-converting enzyme (ICE)/ced3 (caspase) family may exert a pivotal role in the execution of apoptosis (25). Mammalian cells express at least
10 such caspases, which cleave after aspartate residues (9).
Overexpression of these caspases induces apoptosis, whereas
their inhibition suppresses apoptosis (24, 25, 28).
These caspases require proteolytic cleavage for their activation
(10, 40). In turn, caspases cleave a variety of vital
cellular substrate(s) including poly(ADP-ribose) polymerase
(PARP) during apoptosis (39). Although PARP
degradation has been observed (19), activation of caspase 3 by cleavage has not been demonstrated to occur during drug-induced apoptosis in breast cancer cells.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]ATP (specific
activity, 3,000 Ci/mmol) were purchased from ICN Pharmaceuticals, Inc.,
Irvine, Calif. Protein phosphatase I (PPase I; catalytic subunit) and
protein tyrosine phosphatase (PTPase) were purchased from Boehringer
Mannheim, Indianapolis, Ind. Okadaic acid and phorbol 12-myristate
13-acetate (PMA) were purchased from GIBCO BRL, Grand Island, N.Y. PKA
inhibitor Rp diastereomers of cAMP (Rp-cAMP) (TEA-salt) was purchased
from Biolog Life Sciences Institute, La Jolla, Calif. Caspase
inhibitors, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk), and
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)
were purchased from Enzyme Systems Products, Livermore, Calif. The
protein concentration was determined with the bicinchoninic acid
reagent (Pierce, Rockford, Ill.).
-32P]ATP). The reaction was initiated by the
addition of PKA and incubated for 30 min at 30°C. Phosphorylated Bcl2
was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (12%
gel), transferred to nitrocellulose (NC) membranes, and visualized by
autoradiography.
Western blot analysis. Cells (106) were plated in 10-cm-diameter dishes and incubated with drugs as described in the figure legends. Cells were washed twice with PBS, scraped, and centrifuged, and the cell pellets were lysed in lysis buffer (0.1 M NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.5% sodium deoxycholate, 20 mM Tris-HCl [pH 7.4]) containing proteolysis inhibitors, vortexed, passed through a 22-gauge needle for 10 min, allowed to stand for 30 min at 4°C, and centrifuged at 750 × g for 20 min; the resulting supernatants were used as cell lysates. Lysates were subjected to SDS-PAGE (12% gel), blotted onto an NC membrane, probed with antibody, and visualized with an enhanced chemiluminescence kit.
Immunoprecipitation. Cells were washed twice with ice-cold PBS before harvesting and centrifuged, and the cell pellets were lysed in lysis buffer (see above). Fifty micrograms of total protein was immunoadsorbed with 1 µg of antibody, followed by protein A-Sepharose. The immunoadsorbed pellets were washed five times with washing buffer and finally resuspended in SDS sample buffer. The immunoprecipitates were subjected to SDS-PAGE, transferred onto an NC membrane, dried, and autoradiographed.
In vitro translation assay. Hemagglutinin (HA)-tagged human Bax was obtained from J. M. Hardwick, Johns Hopkins School of Medicine, Baltimore, Md. (5). In brief, BglII fragments of HA-tagged human Bax were cloned into the BglII site of pSG5. In vitro translation of HA-tagged protein was performed as instructed by the manufacturer (TNT, T7 Quick Coupled Transcription/Translation System; Promega, Madison, Wis.).
cAMP-dependent protein kinase assay.
PKA was measured in
cell pellets that were lysed and Dounce homogenized before the assay
was performed with ATP, [-32P]ATP,
MgCl2, kemptide, and Tris-HCl with or without cAMP
(43). The incubation mixture was spotted on phosphocellulose
filters and washed with phosphoric acid; then the radioactivity was
measured. The PKA ratio is defined as the ratio of activity measured in the absence and in the presence of 8 µM cAMP. Since cAMP fully activates PKA, this ratio is a measure of how much PKA is activated relative to the total amount of PKA present.
Nuclear morphology. To assay nuclear morphology (apoptotic nuclei), cells were washed with PBS, fixed with 70% ethanol for 1 h, and stained with 1 mM Hoechst 33258 for 30 min (7). The nuclear morphology of cells was visualized by a fluorescence microscope (Olympus BH2). Fluorescent nuclei were screened for normal morphology (unaltered chromatin), and apoptotic nuclei comprising those with fragmented (scattered) and condensed chromatin were counted. Apoptosis was expressed as the percentage of apoptotic nuclei/103 nuclei.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Induction of apoptosis by paclitaxel and vincristine in breast cancer cells. To evaluate the responsiveness of breast epithelial carcinoma to paclitaxel or vincristine, we first analyzed the viability of MCF-7 and MDA-MB-231 cells after exposure to various concentrations of drug. Both paclitaxel and vincristine inhibited viability of MCF-7 and MDA-MB-231 cells in a dose-dependent manner (data not shown). Similarly, paclitaxel or vincristine induced apoptosis in MCF-7 and MDA-MB-231 cells in a dose-dependent manner (Fig. 1). We next examined if overexpression of Bcl2 could counteract the apoptotic effects of paclitaxel or vincristine. The MDA-MB-231 cell line does not express endogenous Bcl2 as tested by Northern and Western blot analyses (data not shown). Overexpression of Bcl2 slightly attenuated the effect of low doses of paclitaxel or vincristine on apoptosis but had no effect on higher doses of these drugs (Fig. 1 and 2). In addition, paclitaxel or vincristine caused growth arrest at the G2-M stage of the cell cycle in MCF-7 and MDA-MB-231 cells (data not shown).
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Hyperphosphorylation of Bcl2 and induction of Bax expression by anticancer drugs. We next investigated the effects of paclitaxel, vincristine, or okadaic acid (a serine/threonine phosphatase inhibitor) on phosphorylation of Bcl2 and induction of Bax. Paclitaxel, vincristine, or okadaic acid induced Bcl2 hyperphosphorylation (caused a mobility shift) in MCF-7 cells (Fig. 3A, lanes 2 to 4). DU145, a prostate cancer cell line that does not express endogenous Bcl2 or Bax, was used as a negative control (Fig. 3A to C, lanes 5). Figure 3B clearly indicates more than 50% reduction of Bax protein in the immunocomplexes precipitated by anti-Bcl2 antibody in paclitaxel-, vincristine-, or okadaic-acid treated cells (Fig. 3B, lanes 2 to 4). We further confirmed this hypothesis by immunoprecipitating cell lysates with anti-Bax antibody and immunoblotting the complex with anti-Bcl2 antibody (Fig. 3D). Only hypophosphorylated Bcl2 was immunoprecipitated with anti-Bax antibody (Fig. 3D). These experiments suggest that hyperphosphorylated Bcl2 is incapable of forming heterodimers with Bax (Fig. 3B and D). We next examined if paclitaxel, vincristine, or okadaic acid could induce Bax protein in MCF-7 cells. Treatment of cells with paclitaxel, vincristine, or okadaic acid resulted in induction of Bax protein (Fig. 3C, lanes 2 to 4). Thus, these agents appear to exert both inhibitory effects on the antiapoptotic Bcl2 protein and stimulatory effects on the levels of the proapoptotic Bax protein.
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cAMP-dependent protein kinase is involved in Bcl2 phosphorylation. We next examined if the cAMP-dependent protein kinase (PKA) pathway is involved in Bcl2 phosphorylation. Forskolin, an adenylate cyclase activator and 8-Cl-cAMP, a site selective cAMP analog, induced Bcl2 hyperphosphorylation in MCF-7 cells (Fig. 4A). Since MDA-MB-231 cells do not natively express Bcl2, it was of interest to determine if MDA-MB-231 cells transfected to overexpress Bcl2 would respond similarly to these drugs. Paclitaxel, forskolin, or okadaic acid induced Bcl2 hyperphosphorylation in MDA/Bcl2 (Bcl2 overexpressing MDA-MB-231 cells) (Fig. 4B). We also examined whether PKC was involved in Bcl2 phosphorylation by using a phorbol ester, PMA. PMA did not cause any change in a band mobility shift of Bcl2 (Fig. 4B). Thus, PKA rather than PKC appears to hyperphosphorylate Bcl2 even when Bcl2 is overexpressed.
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In vitro and in vivo phosphorylation of
Bcl2.
We tested whether Bcl2 could serve as a substrate for
PKA in vitro. GST-Bcl2 was incubated in the presence of
[-32P]ATP and PKA (see Materials and Methods). The
phosphorylated protein was resolved by SDS-PAGE (12%
gel), electrotransferred onto NC filters, and autoradiographed. PKA
phosphorylated the Bcl2 protein in vitro (Fig.
5A, lanes 2 to 4).
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Activation of PKA by paclitaxel and vincristine. To determine whether changes in the catalytic activity of PKA are involved following microtubule damage, we examined phosphotransferase activity in cells during paclitaxel, vincristine, nocodazole, and forskolin treatment, using kemptide as a substrate in the presence and absence of a saturating concentration of cAMP (43). This method of assay gives accurate determination of the relative levels of dissociated catalytic subunit activity and total catalytic activity. The PKA activity ratio, the ratio of activity in the absence of added cAMP to that in the presence of cAMP, measures the degree of free catalytic subunit release. As shown in Fig. 6, paclitaxel, vincristine, or forskolin induced PKA activation as early as 30 min. In contrast, nocodazole, a drug that reversibly blocks microtubule polymerization, had no effect on PKA activation (Fig. 6) and Bcl2 hyperphosphorylation (data not shown). In our previous studies, we have shown that the PKA activity ratio remained elevated during 48 h of 8-Cl-cAMP treatment in MCF-7TH cells (36). These results show that paclitaxel and vincristine treatment resulted in an increase in release of free catalytic subunit of PKA.
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Microtubule-damaging drugs hyperphosphorylate Bcl2, whereas DNA-damaging drugs do not. Because paclitaxel induces tubulin polymerization and forms extremely stable and nonfunctional microtubules (20), it was of an interest to examine if drugs inducing microtubule depolymerization also induce Bcl2 hyperphosphorylation. Vincristine and vinblastine, two vinca alkaloids that bind monomeric tubulin and prevent microtubule assembly (34), also induced Bcl2 hyperphosphorylation in MCF-7 cells (Fig. 7A). By comparison, the DNA-damaging agents methotrexate and doxorubicin failed to induce Bcl2 hyperphosphorylation (did not cause a band shift) (Fig. 7A). In addition, etoposide, which stabilizes covalent complexes between topoisomerase II and genomic DNA, did not induce Bcl2 hyperphosphorylation. Similarly, when lysate of [32P]orthophosphoric acid-labeled cells exposed to doxorubicin was immunoprecipitated with anti-Bcl2 antibody and resolved by SDS-PAGE (8 to 20% gradient gel), no hyperphosphorylated band was detected (data not shown). On the basis of these data, it appears that drugs that damage microtubule integrity induce loss of Bcl2 antiapoptotic function through hyperphosphorylation, whereas DNA-damaging drugs do not. This has been recently shown to occur in 697 leukemia cells and PC-3 prostate carcinoma cells (11).
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Induction of caspase 3 activation and PARP cleavage by microtubule-damaging drugs. The effects of paclitaxel, vincristine, and vinblastine on caspase 3 protein as well as PARP levels were examined in MCF-7 and MDA-MB-231 cells (data not shown). All three drugs induced activation of caspase 3 by cleaving it to a 20-kDa subunit (data not shown). Similarly, this has been shown to occur when caspase 3 is activated by cleavage (45). In addition to caspase 3 cleavage and activation, following treatment of MCF-7 cells with paclitaxel, vincristine, or vinblastine for 18 h, p116PARP was degraded into an 85-kDa fragment (data not shown). Both the cleavage and activation of caspase 3 as well as the degradation of PARP were not observed following treatment with paclitaxel (0.01 to 10 µM) for 6 h (data not shown), the time at which Bcl2 begins to undergo hyperphosphorylation. In addition, paclitaxel-induced effects of caspase 3 and PARP were detected at 12 h of exposure to paclitaxel (data not shown), suggesting that the activation of caspase 3 protease and PARP degradation precedes the DNA fragmentation of apoptosis.
It has been shown that caspases play significant roles in execution of apoptosis induced by chemotherapeutic drugs, and inactivation of these caspases by cowpox virus gene product CrmA could block drug-induced apoptosis (9, 40). We therefore examined the effects of CrmA on paclitaxel- or vincristine-induced PARP degradation and apoptosis in MCF-7 cells. Stably transfected CrmA protected cells against paclitaxel- or vincristine-induced PARP degradation and apoptosis (data not shown). However, Bcl2 was phosphorylated in these cells (data not shown). In addition, a caspase inhibitor z-DEVD-fmk was able to block paclitaxel- or vincristine-induced caspase 3 activation in MDA-MB-231 cells (data not shown). Interestingly, caspase inhibitors z-DEVD-fmk and z-VAD-fmk were effective in blocking paclitaxel- or vincristine-induced apoptosis (as measured by nucleosome ELISA; Oncogene Research Products, Cambridge, Mass.) in MDA-MB-231 cells (data not shown). In contrast, nocodazole, a reversible inhibitor of microtubule polymerization, does not activate PKA (as shown above) and does not induce apoptosis (data not shown). These caspase inhibitors also blocked paclitaxel- or vincristine-induced PARP cleavage in MDA-MB-231 cells (data not shown).| |
DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Recent evidence suggests that inactivation of the antiapoptotic function of Bcl2 by phosphorylation occurs after treatment with the microtubule-stabilizing anticancer drug paclitaxel or the phosphatase inhibitor okadaic acid (1, 11, 14). Results of the present study extend these previous findings and demonstrate that the microtubule-disrupting drugs induce apoptosis in MCF-7 cells that is associated with Bcl2 hyperphosphorylation and loss of function while having no effect on p53. In contrast, DNA-damaging drugs have no effect on Bcl2 hyperphosphorylation but induce p53, suggesting that the actions of microtubule-damaging drugs on apoptosis are exerted independently of the p53 pathway. Interestingly, hyperphosphorylation occurred on serine/threonine residues since PPase I treatment resulted in dephosphorylation of Bcl2. Furthermore, this is the first study showing that PKA may be involved in Bcl2 hyperphosphorylation. In addition, time course experiments demonstrate that paclitaxel and vincristine induce caspase 3 activation as well as PARP degradation downstream of Bcl2 hyperphosphorylation.
It has been demonstrated previously that Bcl2 is phosphorylated on serine residues, and of the 17 serine residues present in Bcl2, several could be the potential sites of phosphorylation by different kinases (13, 31). It is not yet known whether the same sites are hyperphosphorylated by microtubule-damaging-drugs and PKA. Thus, the effect of hyperphosphorylation on Bcl2 function may depend on the site of phosphorylation. Further studies are under way to identify the site(s) of hyperphosphorylation in Bcl2 protein by these drugs and define the relevant kinases.
Recently, it has been shown that binding of the angiotensin type 2 receptor leads to dephosphorylation of Bcl2 and apoptosis by activation of mitogen-activated protein kinase phosphatase 1 in PC12 cells (17). Furthermore, overexpression of c-Jun N-terminal kinase antagonizes an antiapoptotic action of Bcl2 in N18TG cells (31). Contrary to our findings and those of others (1, 13), it has been reported that interleukin-3 (IL-3) and erythropoietin, or the PKC activator bryostatin 1, not only suppress apoptosis but also stimulate the phosphorylation of Bcl2 in murine IL-3-dependent NSF/N1.H7 cells (18, 26). In addition, it has been reported that Bcl2 is phosphorylated on serine 70 after IL-3 or bryostatin stimulation, and Bcl2 phosphorylation is required for its antiapoptotic function. The differences observed with activators of PKA or PKC, and subsequent Bcl2 hyperphosphorylation by these drugs, may be due to differences in cell types. It is likely that multiple kinases are involved in Bcl2 hyper- or hypophosphorylation, and the specific sites of phosphorylation may determine whether Bcl2 loses or gains its antiapoptotic function.
The actions of cAMP in the regulation of various cellular functions,
including cell proliferation, differentiation, and gene induction,
through the activation of PKA are well known (20). We and
others have recently demonstrated that down-regulation of PKA type I
and up-regulation of PKA type II by cAMP analog 8-Cl-cAMP and RI
antisense induce apoptosis in several cancer cell lines
(6, 7, 23, 37). Recently, synergistic inhibition of growth
and induction of apoptosis by 8-Cl-cAMP and
paclitaxel, cisplatin, or retinoic acid in several human
cancer cells has been demonstrated (37, 42). Furthermore,
intracytoplasmic microinjection of purified PKA catalytic subunit
commits the cells to death (48). In support of this
hypothesis, PKA type II has been found to be associated with mammalian
centromeres (44), and upon microtubule disruption by
paclitaxel, vincristine, or vinblastine, activated PKA
would hyperphosphorylate Bcl2, leading to
apoptosis. Interestingly, nocodazole acts like vincristine to
block microtubule polymerization but does not activate PKA, does not
induce Bcl2 hyperphosphorylation, and does not
lead to apoptosis. This finding suggests that activation of PKA
due to microtubule damage is an important event in Bcl2
hyperphosphorylation and induction of
apoptosis.
Apoptosis can be selectively triggered in tumor cells in response to several stimuli, including anticancer drugs and Fas antigen (15, 27). Aborted apoptosis in neoplastic cells could be a critical factor in resistance both to natural defenses and to clinical therapy, particularly in cases of recurrent disease (14), and the mechanisms underlying this phenomenon have yet to be fully elucidated. Apoptosis induction in mammalian cells is controlled by an equilibrium between suppressor and inducer gene products (29). Dysregulated expression of Bcl2 is believed to contribute to neoplastic cell expansion via an antiapoptotic effect which enhances cell survival rather than by accelerating rates of cellular proliferation. Although the mode by which Bcl2 affects the process of cell death is not fully understood, recent studies indicate that the Bcl2 protein binds to other proteins with which it has amino acid sequence homology, including Bax, Bcl-XL, Bcl-XS, Mcl-1, Bik, and Bad (2, 3, 30, 35, 47). The functional significance of many of these Bcl2 family protein-protein interactions remains unclear. However, the heterodimerization of Bcl2 with Bax appears crucial in preventing Bax-mediated apoptosis (30). Bax appears to antagonize Bcl2 function, abrogating the ability of Bcl2 to prolong cell survival. In the present study, less Bax was immunoprecipitated with Bcl2 antibody in paclitaxel-, vincristine-, or vinblastine-treated cells, indicating that hyperphosphorylated Bcl2 is less able to form heterodimers with Bax. Similarly, the treatment of prostate cancer cells results in the phosphorylation of Bcl2 and in programmed cell death of cancer cells concomitantly with a reduction of heterodimer complexes with Bax (12). Thus, cell viability will be maintained either by activating apoptosis-suppressing proteins or by inhibiting apoptosis-inducing proteins.
Consistent with these observations, overexpression of Bcl2 in MCF-7 and MDA-MB-231 cell lines counteracted the effects of low doses of paclitaxel or vincristine on cell survival, whereas high doses of paclitaxel or vincristine inhibited survival in both Neo- and Bcl2-overexpressing breast cancer cell lines. A previous report also has shown that Bcl2 overexpression inhibits low dose paclitaxel-induced apoptosis and significantly improves the survival of human leukemic cells (38).
It is now clear that the proteolytic degradation of specific substrates
is involved in many of the morphological and biochemical features of
apoptosis (25, 40, 45). Activation of the cysteine proteases belonging to the ICE/ced-3 family is part of the cascade of
protease activity that results in apoptosis (10,
45). We have demonstrated that paclitaxel- and
vincristine-induced apoptosis of breast cancer cells
activates at least one such protease encoded by the caspase 3 (CPP32/Yama) gene. Caspase 3 was cleaved and activated, leading to
PARP degradation. The cowpox virus CrmA and caspase inhibitors
z-DEVD-fmk and z-VAD-fmk were able to protect cells from
paclitaxel- or vincristine-induced apoptosis
and PARP degradation. Similarly, CrmA-expressing Rat-1
fibroblasts were protected from death activated by serum withdrawal
(46). Overexpression of CrmA also prevented
apoptosis due to engagement of the Fas (CD95) receptor or to
exposure to tumor necrosis factor alpha (9, 40). Time course
experiments demonstrated that the caspase 3 activation and PARP
degradation occur downstream of Bcl2 phosphorylation.
Based on our data and those of others (1, 11), it is clear that drugs that affect microtubule integrity by inhibiting polymerization or depolymerization induce loss of Bcl2 antiapoptotic function through hyperphosphorylation, whereas anticancer drugs that damage DNA do not. These experiments may provide a rationale for the use of the anticancer drugs, paclitaxel, vincristine, and vinblastine for the treatment of Bcl2-positive cancers, such as follicular lymphoma and breast, prostate, and ovarian cancer. Furthermore, these data suggest that there may be a cytoskeletal damage response pathway just as there seems to be a DNA damage response pathway. Analysis of the nature of the signaling cascade induced by the cytoskeleton-damaging agents is under way. Taken together, these studies not only increase our understanding of Bcl2 function but also elucidate general mechanisms through which intracellular signals can mediate regulatory functions relevant to disease states.
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ACKNOWLEDGMENTS |
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We are grateful to J. M. Hardwick (Johns Hopkins School of Medicine, Baltimore, Md.) for providing HA-tagged Bax expression plasmid. We thank Jim Kenny, Tony Passaniti, and Charles Filburn for critically reviewing the manuscript. We also thank Francis J. Chrest for his technical help.
This work was supported partly by a fellowship of the National Research Council, National Academy of Sciences, to R.K.S.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr., Box 28, Baltimore, MD 21224-6825. Phone: (410) 558-8480. Fax: (410) 558-8284. E-mail: rakeshs{at}vax.grc.nia.nih.gov.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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