From the Laboratory of Molecular Pharmacology,
Division of Basic Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892 and the § Department of
Microbiology and Immunology, Albert Einstein College of Medicine,
Bronx, New York 11724
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ABSTRACT |
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In this study, we investigated the molecular basis of the ability of staurosporine to induce G1 arrest in murine embryonic fibroblasts (MEFs). We used MEFs from transgenic mice lacking several negative regulators of the G1/S phase transition including cells from mice lacking p53, p21, retinoblastoma (Rb), or p16 genes. We found that p53 function was not essential for staurosporine-induced G1 arrest. In contrast, MEFs from mice lacking Rb genes showed approximately a 70% reduced capacity to arrest in the G1 phase following staurosporine treatment. In support of a role for Rb in staurosporine-induced G1 arrest, rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4R24C exhibited approximately a 50% reduced G1 arrest response to staurosporine. The role of Rb in determining the degree of staurosporine-induced G1 arrest did not depend on the function of the cyclin-dependent kinase inhibitors p16 or p21 because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine exposure. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.
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INTRODUCTION |
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Staurosporine, a broad spectrum protein kinase inhibitor, has been shown to induce a G1 and G2 phase arrest in normal cells, and importantly the G1 arrest response has been shown to be selectively lost in a variety of tumor cell lines (1, 2). The mechanistic basis underlying the loss of the G1 arrest response in tumor cells is presently unresolved. In this study we focused our attention on the importance of the p53 and/or retinoblastoma (Rb)1 tumor suppressor proteins in the mechanism of staurosporine-induced G1 arrest. These studies were prompted by the findings that the p53 and/or Rb pathways are commonly altered in human tumor cells (3-6), raising the possibility that such disruptions might account for the lack of staurosporine-induced G1 arrest in tumor cell lines.
Interestingly, the effects of staurosporine on the cell cycle are reminiscent of DNA damage in that both induce a G1 and G2 arrest in normal cells, but the G1 arrest is lost in many tumor cell lines (1, 2). The p53 tumor suppressor is essential for G1 arrest induced by ionizing radiation and other DNA damaging agents, as well as G1 arrest following suppression of ribonucleoside triphosphate pools (3, 7). In fact, loss of p53 activity by gene mutation, gene deletion, or overexpression of dominant-negative acting factors such as mutant-p53, Mdm2, or the human papillomavirus E6 protein blocks G1 arrest following DNA damage (8-10). Although the lack of DNA damage-induced G1 arrest in tumor cells has been linked to p53 dysfunction, a role for p53 in staurosporine-induced G1 arrest still remains to be established. An important mediator of p53-induced G1 arrest is the p21Waf1/Cip1 gene product (p21) (11, 12). The p21 protein binds to and inhibits a variety of G1/S phase cyclin-dependent kinases (Cdk), which in turn are prevented from phosphorylating and inactivating the Rb protein (13). Indeed, studies conducted in the bladder carcinoma cell line 5637 indicated that p21 may participate in staurosporine-induced G1 arrest (14). Definitive evidence of this possibility was, however, not obtainable in these earlier studies.
Previous observations have indicated that staurosporine promotes the hypophosphorylation of Rb in cell types susceptible to G1 arrest (2, 14, 15). Hypophosphorylated Rb would retain cells in G1 phase by virtue of its ability to repress genes that are regulated through E2F-dependent consensus elements in their promoters (16, 17). When Rb function is inactivated through Rb gene mutation, gene deletion, expression of certain viral, or cellular proteins, the cells lose the capacity to G1 arrest in response to some negative regulators of the G1/S phase transition, including the Cdk inhibitor protein p16 (5, 9, 18-20). Previous studies in bladder carcinoma cells, which lack Rb and fail to undergo staurosporine-induced G1 arrest (14), showed that ectopic expression of wild-type Rb delayed progression in the G1 phase, and such cells were also sensitized to staurosporine-induced G1 arrest. This study suggested a potential role for Rb in staurosporine-induced G1 arrest, but definitive evidence of this possibility was not obtainable in these earlier studies.
In this study we investigated the mechanistic basis of staurosporine-induced G1 arrest by using murine embryonic fibroblasts (MEFs) as a model system. Our choice of MEFs in this respect was fueled by the availability of cells from transgenic mice lacking important negative regulators of the G1/S phase transition. Such a model system enabled us to determine the importance of p53, p21, Rb, and p16 in the staurosporine-induced G1 arrest mechanism in this cell type. Here we report that p53 function was not essential for staurosporine-induced G1 arrest in MEFs. In contrast, the Rb protein played an important role in determining the degree of G1 arrest observed in the first cell cycle following exposure to staurosporine. Supporting evidence of a role for Rb in staurosporine-induced G1 arrest was provided by the observation that rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4R24C exhibited a reduced G1 arrest response to staurosporine. Staurosporine did not induce a Rb-dependent G1 arrest in the first cell cycle by solely acting through the Cdk inhibitor proteins p16 or p21, because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine treatment. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.
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MATERIALS AND METHODS |
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Chemical Treatments--
Staurosporine was obtained from Sigma
and prepared as a 500 µM stock solution in
Me2SO, and aliquots were stored at 20 °C until needed.
Nocodazole (NOC) was purchased from Sigma.
Cell Culture--
Early passage MEFs and rat embryonic
fibroblasts were grown at 37 °C in 95% air/5% CO2 in
Dulbecco's modified Eagle medium (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 50 units/ml penicillin G, 50 µg/ml streptomycin (Life Technologies,
Inc.). The wild type/wild type and p16 /
MEF cells were graciously
supplied by Dr. Ronald A. DePinho (Albert Einstein College of Medicine,
New York, NY), whereas the Rb
/
and p53
/
MEFs cells were
generously provided by Drs. Allan Bradley and Larry Donehower,
respectively (Baylor College of Medicine, Houston, TX). The p21
/
MEFs were a kind gift from Dr. Chuxia Deng (NIDDK, National Institutes
of Health, Bethesda, MD). Rat embryonic fibroblasts (REF52)
overexpressing either the vector or cyclin D1/Cdk4R24C were
generously provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY). Cell counts were determined using a Coulter
counter with Channelyzer attachment to monitor cell size (Coulter
Electronics, Hialeah, FL).
Flow Cytometry-- Samples were prepared for flow cytometry essentially as described previously (21). Briefly, cells were washed with 1 × phosphate-buffered saline, pH 7.4, and then fixed with ice-cold 70% ethanol. Samples were then washed with 1 × phosphate-buffered saline and stained with propidium iodide 60 µg/ml (Sigma) containing RNase 100 µg/ml (Sigma) for 30 min at 37 °C. Cell cycle analysis was performed using a Becton Dickinson flourescence-activated cell analyzer and Cell Quest version 1.2 software (Becton Dickinson Immunocytometry Systems, Mansfield, MA). For each sample at least 10,000 cells were analyzed, and quantitation of the cell cycle distribution was performed using the ModFit LT version 1.01 software (Verity Software House Inc., Topsham, ME).
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RESULTS |
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Staurosporine-induced G1 Arrest Does Not Operate through a p53/p21-dependent Pathway-- We initially investigated if staurosporine-induced G1 arrest required the function of the p53 tumor suppressor. For this purpose we investigated the effects of p53 gene disruption on the ability of MEFs to arrest in G1 and G2 phases of the cell cycle following staurosporine treatment. Dose-response curves (5-50 nM STP) were performed to establish the dose of staurosporine that completely inhibited cell division within 24 h of addition to the different MEFs we studied. Choosing this dose enabled us to focus on events associated with the actions of staurosporine on the first cell cycle. The maximal growth inhibitory dose (50 nM) was then chosen to investigate cell cycle responses to staurosporine treatment (Fig. 1B). In the case of MEFs from normal littermates we found that staurosporine induced both a G1 and a G2 arrest, and this was evident within 24 h of staurosporine addition (Fig. 1, A and C). Comparable cell cycle responses were observed in MEFs from mice lacking p53 genes (Fig. 1, A and C), suggesting that p53 function was not essential for staurosporine-induced G1 or G2 arrest.
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The Rb Protein Is an Important Mediator of Staurosporine-induced
G1 Arrest in the First Cell Cycle--
Having established
that the p53 and/or p21 gene products were not essential for
staurosporine-induced G1 arrest, we next turned our
attention to the role of the Rb pathway in the staurosporine-induced G1 arrest mechanism. For this purpose we conducted studies
in MEFs with intact versus disrupted Rb genes. Again,
dose-response curves (5-50 nM STP) were performed to
establish the dose of staurosporine that completely inhibited cell
division within 24 h of addition. The maximal growth inhibitory
dose (50 nM) was then chosen to investigate the cell cycle
responses following addition of staurosporine (Fig.
2). In the case of MEFs from normal
littermates, we found that staurosporine induced both a sustained
G1 and a G2 arrest, and this arrest was evident
within 24 h of addition (Fig. 1, A and C).
In contrast, MEFs lacking Rb almost completely lost the ability to
G1 arrest in the first cell cycle following exposure to
staurosporine (Fig. 2, A and C). Cell cycle
analysis of Rb /
MEFs 24 h after a chronic exposure to 50 nM staurosporine showed that the G1 population
was 14 ± 3% (n = 3) compared with 41 ± 5%
(n = 3) for the vehicle-treated control cells. This
amounted to approximately a 70% reduction in the degree of
G1 arrest seen in the presence of staurosporine
(p < 0.01, Student's t test).
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Staurosporine-induced G1 Arrest in the First Cell Cycle Does Not Require the Function of the p16 Tumor Suppressor Gene-- Because the Cdk inhibitor p16 has previously been shown to induce an Rb-dependent G1 arrest in mammalian cells (18-20), we investigated whether staurosporine might act through p16 to induce G1 arrest in MEFs. Our investigations showed, however, that MEFs from mice lacking p16 genes underwent a combined G1 and G2 arrest comparable with that observed in wild-type MEFs (Fig. 2, A and C). These results suggested that p16, a well recognized regulator of Rb function, was not essential for staurosporine-induced G1 arrest in MEFs.
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DISCUSSION |
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In this study we investigated the mechanistic basis of staurosporine-induced G1 arrest in murine cells derived from transgenic mice lacking important negative regulators of the G1/S phase transition. Such a model system enabled us to investigate the importance of the p53, p21, Rb, and p16 genes in the mechanism through which staurosporine induces G1 arrest in otherwise normal cells. We found that in contrast to the p53 tumor suppressor, the Rb protein was an important mediator of the degree of staurosporine-induced G1 arrest observed in the first cell cycle. Further evidence implicating Rb in the mechanism of staurosporine-induced G1 arrest was evidenced in rat embryonic fibroblasts stably overexpressing cyclin D1-Cdk4R24C. Such cells exhibited a reduced G1 arrest response to staurosporine compared with empty vector control cells. Staurosporine did not, however, induce an Rb-dependent G1 arrest by acting through the Cdk inhibitor proteins p16 or p21, because MEFs lacking these genes were still capable of G1 arrest following staurosporine treatment. Our studies provide evidence of an important role for Rb in determining the degree of staurosporine-induced G1 arrest.
Our finding that MEFs lacking p53 still underwent a combined G1 and G2 arrest in response to staurosporine provided evidence that in this cell type, p53 was not essential for staurosporine-induced G1 arrest. Because the Cdk inhibitor, p21, can be induced by p53-independent mechanisms (25) and because p21 has previously been implicated in the mechanism by which staurosporine induces G1 arrest (14), we also examined the importance of p21 in staurosporine-induced G1 arrest. However, we again found that MEFs lacking p21 still underwent G1 arrest in response to staurosporine, indicating that in this cell type, p21 was not essential for the G1 arrest response.
Studies indicating the common occurrence of Rb disruptions in tumor
cells and studies indicating that a tumor cell line overexpressing a Rb
transgene underwent G1 arrest following staurosporine
exposure (14) prompted us to also test the role of Rb in
staurosporine-induced G1 arrest. We found that Rb was
indeed an important mediator of the degree of G1 arrest
observed following exposure to staurosporine: approximately 70% of the
Rb /
cells escaped G1 arrest when exposed to
staurosporine, whereas in contrast virtually all of the cells with
intact Rb remained arrested in the G1 phase (>95%). The
function of the tumor suppressor, p16, was not essential for
staurosporine-induced G1 arrest because MEFs lacking the
p16 gene still underwent G1 arrest. Approximately 30% of
Rb
/
MEFs remained in the G1 phase in the presence of
staurosporine. This residual subpopulation could conceivably represent
an early G1 fraction of cells that arrest through a
Rb-independent mechanism; however, further studies outside the scope of
this report will be needed to investigate any early G1
effects of staurosporine. In conclusion we show that Rb function is
important for the majority of G1 arrest seen in the first
cell cycle and that investigations aimed at determining the mechanism
through which staurosporine maintains Rb in an active form could
ultimately provide the target of staurosporine as it relates to Rb.
We speculate on some potential targets through which staurosporine might act to induce a Rb-dependent G1 arrest in Fig. 5. We represent Rb in an equilibrium between its hyperphosphorylated (relatively inactive) state and its hypophosphorylated (active) state. This equilibrium is governed by cyclin-dependent kinases (28) and probably a type 1 phosphatase, respectively (29, 30). Rb kinases may include cyclin D1-Cdk4, cyclin D1-Cdk6, cyclin E-Cdk2, or cyclin A-Cdk2, all of which are known to directly phosphorylate Rb (17, 31, 32). Cyclin E-Cdk2 and cyclin A-Cdk2 are probably not the G1 phase targets of the action of staurosporine because microinjection of neutralizing antibodies to either cyclin E or cyclin A induces a Rb-independent G1 arrest (33, 34). Also, overexpression of a dominant-negative Cdk2 transgene induces a Rb-independent G1 arrest, providing evidence against a role for Cdk2 in staurosporine-induced G1 arrest (35). This leaves cyclin D1-Cdk4 open as a possible target, and it has been reported that microinjection of anti-cyclin D1 antibodies or cyclin D1 antisense cDNA into cells induces G1 arrest in cells containing wild-type Rb but not defective Rb (4). Furthermore, staurosporine does inhibit cyclin D1-Cdk4 kinase activity in vitro in the dose range used in our G1 arrest experiments (50% inhibitory concentration of approximately 20 nM, data not shown), and we found that overexpression of cyclin D1-Cdk4R24C in REF52 cells reduced the ability of staurosporine to induce G1 arrest compared with the empty vector control cells. Such results, although suggestive of cyclin D1-Cdk4 as the direct target of the actions of staurosporine, are not by themselves conclusive. Indeed, staurosporine in G1 phase might induce a Rb-dependent G1 arrest through the activation of a phosphatase that dephosphorylates and activates Rb (Fig. 5). This could occur if staurosporine inhibited a kinase that normally negatively regulated this Rb phosphatase (Fig. 5). There is evidence that a protein phosphatase type 1 can be inactivated by serine/threonine phosphorylation, and this same class of phosphatases has been implicated in the dephosphorylation of Rb (29, 30). It will be interesting to determine if the phosphorylation status and activity of this type 1 phosphatase is affected by staurosporine.
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In the different MEFs we studied, we did not observe any obvious differences in the ability of cells to arrest in the G2 phase 24 h after staurosporine treatment. This indicated that p53, p21, Rb, or p16 were not essential components of the induction of this G2 arrest response. A possible target of the action of staurosporine in G2 has been presented before as the Cdc2 kinase (36).
In summary, our results highlight the importance of the Rb protein in the mechanism through which staurosporine induces G1 arrest in the first cell cycle. Continued investigation of the actions of staurosporine on Rb phosphorylation status could uncover the route and eventual target of the actions of staurosporine as it relates to Rb.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Ronald DePinho for the kind gift of the murine embryonic fibroblasts from mice lacking p16 genes. We are grateful to Drs. Allan Bradley and Larry Donehower for the kind gift of murine embryonic fibroblasts lacking either Rb or p53 genes and Dr. Chuxia Deng for the kind gift of murine embryonic fibroblasts from mice lacking p21 genes. We are also indebted to Dr. Scott Lowe for the gracious donation of rat embryonic fibroblasts overexpressing cyclin D1-Cdk4R24C.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 5D09, National Cancer Institute, NIH, Bethesda, MD 20892. Tel.: 301-435-2848; Fax: 301-402-0752; E-mail: po18c{at}nih.gov.
1 The abbreviations used are: Rb, retinoblastoma; STP, staurosporine; MEF, murine embryonic fibroblasts; Cdk, cyclin-dependent kinase; NOC, nocodazole; p21, p21Waf1/Cip1.
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REFERENCES |
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