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An Important Role for the Retinoblastoma Protein in Staurosporine-induced G1 Arrest in Murine Embryonic Fibroblasts

1998; Elsevier BV; Volume: 273; Issue: 7 Linguagem: Inglês

10.1074/jbc.273.7.3803

1083-351X

Michael S. Orr, William C. Reinhold, Lijia Yu, Nicole Schreiber‐Agus, P M O'Connor,

Epigenetics and DNA Methylation

In this study, we investigated the molecular basis of the ability of staurosporine to induce G1arrest 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. In this study, we investigated the molecular basis of the ability of staurosporine to induce G1arrest 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. 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 (1Crissman H.A. Gadbois D.M. Tobey R.A. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7580-7584Crossref PubMed Scopus (127) Google Scholar,2Schnier J.B. Gadbois D.M. Nishi K. Bradbury E.M. Cancer Res. 1994; 54: 5959-5963PubMed Google Scholar). The mechanistic basis underlying the loss of the G1arrest response in tumor cells is presently unresolved. In this study we focused our attention on the importance of the p53 and/or retinoblastoma (Rb) 1The abbreviations used are: Rb, retinoblastoma; STP, staurosporine; MEF, murine embryonic fibroblasts; Cdk, cyclin-dependent kinase; NOC, nocodazole; p21, p21Waf1/Cip1.1The abbreviations used are: Rb, retinoblastoma; STP, staurosporine; MEF, murine embryonic fibroblasts; Cdk, cyclin-dependent kinase; NOC, nocodazole; p21, p21Waf1/Cip1. 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 (3Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6667) Google Scholar, 4Tam S.W. Theodoras A.M. Shay J.W. Draetta G.F. Pagano M. Oncogene. 1994; 9: 2663-2674PubMed Google Scholar, 5Goodrich D.W. Wang N.P. Qian Y.W. Lee E.Y. Lee W.H. Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (612) Google Scholar, 6Horowitz J.M. Park S.H. Bogenmann E. Cheng J.C. Yandell D.W. Kaye F.J. Minna J.D. Dryja T.P. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2775-2779Crossref PubMed Scopus (487) Google Scholar), 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 (1Crissman H.A. Gadbois D.M. Tobey R.A. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7580-7584Crossref PubMed Scopus (127) Google Scholar, 2Schnier J.B. Gadbois D.M. Nishi K. Bradbury E.M. Cancer Res. 1994; 54: 5959-5963PubMed Google Scholar). 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 (3Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6667) Google Scholar, 7Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2288) Google Scholar). 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 (8Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 9Slebos R.J. Lee M.H. Plunkett B.S. Kessis T.D. Williams B.O. Jacks T. Hedrick L. Kastan M.B. Cho K.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5320-5324Crossref PubMed Scopus (345) Google Scholar, 10Fan S. Smith M.L. Rivet D.J. n. Duba D. Zhan Q. Kohn K.W. Fornace Jr., A.J. O'Connor P.M. Cancer Res. 1995; 55: 1649-1654PubMed Google Scholar). Although the lack of DNA damage-induced G1arrest 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) (11Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1925) Google Scholar, 12Brugarolas J. Chandrasekaran C. Gordon J.I. Beach D. Jacks T. Hannon G.J. Nature. 1995; 377: 527-552Crossref PubMed Scopus (1136) Google Scholar). 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 (13Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3201) Google Scholar). Indeed, studies conducted in the bladder carcinoma cell line 5637 indicated that p21 may participate in staurosporine-induced G1 arrest (14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar). 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 G1arrest (2Schnier J.B. Gadbois D.M. Nishi K. Bradbury E.M. Cancer Res. 1994; 54: 5959-5963PubMed Google Scholar, 14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar, 15Kwon T.K. Buchholz M.A. Chrest F.J. Nordin A.A. Cell Growth Differ. 1996; 7: 1305-1313PubMed Google Scholar). 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 (16Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1361) Google Scholar, 17Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4273) Google Scholar). 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 (5Goodrich D.W. Wang N.P. Qian Y.W. Lee E.Y. Lee W.H. Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (612) Google Scholar, 9Slebos R.J. Lee M.H. Plunkett B.S. Kessis T.D. Williams B.O. Jacks T. Hedrick L. Kastan M.B. Cho K.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5320-5324Crossref PubMed Scopus (345) Google Scholar, 18Koh J. Enders G.H. Dynlacht B.D. Harlow E. Nature. 1995; 375: 506-510Crossref PubMed Scopus (521) Google Scholar, 19Lukas J. Parry D. Aagaard L. Mann D.J. Bartkova J. Strauss M. Peters G. Bartek J. Nature. 1995; 375: 503-506Crossref PubMed Scopus (861) Google Scholar, 20Medema R.H. Herrera R.E. Lam F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6289-6293Crossref PubMed Scopus (407) Google Scholar). Previous studies in bladder carcinoma cells, which lack Rb and fail to undergo staurosporine-induced G1 arrest (14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar), 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 G1arrest 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. 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. 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). Samples were prepared for flow cytometry essentially as described previously (21O'Connor P.M. Jackman J. Jondle D. Bhatia K. Magrath I. Kohn K.W. Cancer Res. 1993; 53: 4776-4780PubMed Google Scholar). 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). 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.1 B). 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. The role of the cyclin-dependent kinase inhibitor p21, an important mediator of p53-induced G1 arrest (11Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1925) Google Scholar, 22Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3139) Google Scholar, 23el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7867) Google Scholar, 24Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5198) Google Scholar) that can also be activated through p53-independent mechanisms (25Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar), was also investigated. When MEFs from mice lacking p21 genes were exposed to staurosporine (50 nm) for 24 h, they underwent both a G1 and a G2 arrest (Fig. 1, A and C). These results suggested that p21 was similar to p53 in the respect that it also was not essential for staurosporine-induced G1 or G2 arrest. Thus, although it has previously been suggested that p21 participates in the mechanism by which staurosporine induces G1 arrest (14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar), our studies in MEFs show that p21 is not essential for G1 arrest in the first cell cycle of this cell type. 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). To delineate whether staurosporine-induced G1 arrest was a transient or sustained event, we chronically exposed either wild-type or Rb −/− MEFs to staurosporine for up to 72 h and performed flow cytometric analysis at 24-h intervals over this time course. As indicated in Fig. 3, staurosporine induced a sustained G1 arrest in wild-type MEFs, whereas a substantial proportion of Rb −/− cells were able to escape the G1 arrest in the first cell cycle. These cells went on to arrest at the G2/M junction, a response that did not require Rb function. Interestingly, although Rb disruption enabled approximately 70% of the original G1 population to escape the G1 arrest normally induced by staurosporine, approximately 30% of the G1 fraction of Rb −/− MEFs still remained in G1 phase over this time course. This result suggested that a subpopulation of G1 cells, presumably in early G1 phase, might still be capable of G1 arrest despite the absence of Rb genes (see “Discussion”). We also investigated the effects of staurosporine in REF52 cells infected with viruses expressing the Rb-inactivating kinase complex, cyclin D1-Cdk4. A Cdk4 mutant (Cdk4R24C) that is resistant to the inhibitory influence of p16 was used in these studies (26Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3855) Google Scholar, 27Wolfel T. Hauer M. Schneider J. Serrano M. Wolfel C. Klehmann-Hieb E. De Plaen E. Hankeln T. Meyer zum Buschenfelde K. Beach D. Science. 1995; 262: 1281-1284Crossref Scopus (987) Google Scholar). Our view here was that if we could inactivate Rb through cyclin D1-Cdk4-mediated hyperphosphorylation, these cells would show a reduced capacity to G1 arrest in response to staurosporine. In support of a role for Rb in the staurosporine-induced G1arrest mechanism, we found that REF52 cells expressing cyclin D1-Cdk4R24C were less susceptible to staurosporine-induced G1 arrest as compared with the empty vector control cells (Fig. 4). Cell cycle analysis of REF52 cells 24 h after a chronic exposure to staurosporine showed that approximately 49 ± 11% (n = 3) of the original G1 population of cyclin D1-Cdk4R24Coverexpressing cells remained in G1 phase compared with 96 ± 5% (n = 3) for the empty vector control cells. This amounted to approximately a 50% reduction in the degree of staurosporine-induced G1 arrest in cyclin D1-Cdk4R24C overexpressing cells as compared with empty vector control cells (p < 0.01, Student'st test). Because the Cdk inhibitor p16 has previously been shown to induce an Rb-dependent G1 arrest in mammalian cells (18Koh J. Enders G.H. Dynlacht B.D. Harlow E. Nature. 1995; 375: 506-510Crossref PubMed Scopus (521) Google Scholar, 19Lukas J. Parry D. Aagaard L. Mann D.J. Bartkova J. Strauss M. Peters G. Bartek J. Nature. 1995; 375: 503-506Crossref PubMed Scopus (861) Google Scholar, 20Medema R.H. Herrera R.E. Lam F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6289-6293Crossref PubMed Scopus (407) Google Scholar), 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. 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 G1arrest 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 G1arrest. 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 (25Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar) and because p21 has previously been implicated in the mechanism by which staurosporine induces G1 arrest (14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar), 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 (14Schnier J.B. Nishi K. Goodrich D.W. Bradbury E.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5941-5946Crossref PubMed Scopus (64) Google Scholar) 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 G1effects 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 (28Hunter T. Pines J. Cell. 1994; 79: 573-582Abstract Full Text PDF PubMed Scopus (2147) Google Scholar) and probably a type 1 phosphatase, respectively (29Alberts A.S. Thorburn A.M. Shenolikar S. Mumby M.C. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 388-392Crossref PubMed Scopus (140) Google Scholar, 30Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar). 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 (17Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4273) Google Scholar, 31Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (867) Google Scholar, 32Akiyama T. Ohuchi T. Sumida S. Matsumoto K. Toyoshima K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7900-7904Crossref PubMed Scopus (193) Google Scholar). 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 (33Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1042) Google Scholar, 34Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1107) Google Scholar). 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 (35van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (968) Google Scholar). 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 (4Tam S.W. Theodoras A.M. Shay J.W. Draetta G.F. Pagano M. Oncogene. 1994; 9: 2663-2674PubMed Google Scholar). 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 G1arrest 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 (29Alberts A.S. Thorburn A.M. Shenolikar S. Mumby M.C. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 388-392Crossref PubMed Scopus (140) Google Scholar, 30Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar). It will be interesting to determine if the phosphorylation status and activity of this type 1 phosphatase is affected by staurosporine. In the different MEFs we studied, we did not observe any obvious differences in the ability of cells to arrest in the G2phase 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 (36Gadbois D.M. Hamaguchi J.R. Swank R.A. Bradbury E.M. Biochem. Biophys. Res. Commun. 1992; 184: 80-85Crossref PubMed Scopus (130) Google Scholar). 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. 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.