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• W1995086523 abstract "pRB family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G1 to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G1 restriction point, they are hyperphosphorylated by G1 cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G1regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells. pRB family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G1 to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G1 restriction point, they are hyperphosphorylated by G1 cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G1regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells. cyclin-dependent kinase hemagglutinin polyacrylamide gel electrophoresis serine-proline or threonine-proline motif tetracycline isopropyl β-d-thiogalactopyranoside hydroxyurea interleukin phosphate-buffered saline The decision to commit to cell division takes place at a late G1 point, termed the restriction point, that precedes the onset of DNA synthesis in the S phase. The passage through the restriction point is primarily controlled by the retinoblastoma protein (pRB) family, which consists of pRB, p107, and p130 (1Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4326) Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4986) Google Scholar, 3Beijersbergen R.L. Bernards R. Biochim. Biophys Acta. 1996; 1287: 103-120Crossref PubMed Scopus (181) Google Scholar, 4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar). The pRB family proteins, acting as negative growth regulators, share a structure termed the “pocket domain” and, through the domain, interact with multiple cellular proteins, most notably the E2F family of transcriptional factors (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 5Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar). E2Fs transactivate a set of genes whose products are critical in S phase entry and progression (5Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar, 7Johnson D.G. Schneider-Broussard R. Front. Biosci. 1998; 3: 447-448Crossref PubMed Google Scholar, 8Hurford Jr., R.K. Cobrinik D. Lee M.H. Dyson N. Genes Dev. 1997; 11: 1447-1463Crossref PubMed Scopus (382) Google Scholar). Upon complex formation, pRB family proteins neutralize the transcriptional activities of E2Fs (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar, 9Weintraub S.J. Prater C.A. Dean D.C. Nature. 1992; 358: 259-261Crossref PubMed Scopus (560) Google Scholar, 10Nevins J.R. Leone G. DeGregori J. Jakoi L. J. Cell. Physiol. 1997; 173: 233-236Crossref PubMed Scopus (176) Google Scholar). Furthermore, the pRB family-E2F complexes appear to also act as E2F site-specific transcriptional repressors (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar, 11Hamel P.A. Grill R.M. Phillips R.A. Gallie B.L. Mol. Cell. Biol. 1992; 12: 3431-3438Crossref PubMed Scopus (212) Google Scholar, 12Weintraub S.J. Chow K.N. Luo R.X. Zhang S.H. He S. Dean D.C. Nature. 1995; 375: 812-815Crossref PubMed Scopus (459) Google Scholar, 13Zhang H.S. Postigo A.A. Dean D.C. Cell. 1999; 97: 53-61Abstract Full Text Full Text PDF PubMed Google Scholar). Hence, by actively repressing E2F-dependent gene expression, the pRB family proteins prevent cell cycle progression from G1 to S. The pocket function of the pRB family proteins is considered to be negatively regulated by phosphorylation (14Mittnacht S. Curr. Opin. Genet. 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A. 1996; 93: 4633-4637Crossref PubMed Scopus (96) Google Scholar, 22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Upon mitogenic stimulation, G1 cyclin-cyclin-dependent kinase (CDK)1 complexes are activated and collaboratively phosphorylate the pRB family proteins (19Beijersbergen R.L. Carlee L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (236) Google Scholar, 20Mayol X. Garriga J. Grana X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 21Xiao Z.X. Ginsberg D. Ewen M. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4633-4637Crossref PubMed Scopus (96) Google Scholar, 22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 23Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (222) Google Scholar, 24Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1027) Google Scholar, 25Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (741) Google Scholar, 26Resnitzky D. Reed S.I. Mol. Cell. Biol. 1995; 15: 3463-3469Crossref PubMed Scopus (437) Google Scholar, 27Knudsen E.S. Wang J.Y. J. Biol. Chem. 1996; 271: 8313-8320Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 28Zarkowska T. Mittnacht S. J. Biol. Chem. 1997; 272: 12738-12746Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 29Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (859) Google Scholar). Through extensive phosphorylation, the pRB family proteins lose their ability to form complexes with E2Fs, and the released E2Fs initiate S phase entry and subsequent progression (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 8Hurford Jr., R.K. Cobrinik D. Lee M.H. Dyson N. Genes Dev. 1997; 11: 1447-1463Crossref PubMed Scopus (382) Google Scholar, 14Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (334) Google Scholar, 30Ohtani K. DeGregori J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12146-12150Crossref PubMed Scopus (537) Google Scholar). Accordingly, inactivation of the pRB family proteins appears to be an essential prerequisite for traversing the restriction point. Ectopic expression of pRB family proteins in a variety of cell types gives rise to G1 cell cycle arrest (31Goodrich D.W. Wang N.P. Qian Y.W. Lee E.Y. Lee W.H. Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (617) Google Scholar, 32Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar, 33Zhu L. van den Heuvel S. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar, 34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 35Zhu L. Enders G. Lees J.A. Beijersbergen R.L. Bernards R. Harlow E. EMBO J. 1995; 14: 1904-1913Crossref PubMed Scopus (133) Google Scholar), supporting their crucial roles in preventing S phase entry. Consistently, pRB and p130 are abundantly expressed in G0/G1-arrested cells in their active, hypophosphorylated forms (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 19Beijersbergen R.L. Carlee L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (236) Google Scholar, 20Mayol X. Garriga J. Grana X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 36Mayol X. Garriga J. Grana X. Oncogene. 1996; 13: 237-246PubMed Google Scholar). In contrast, the p107 protein levels are modulated in an opposing manner. In quiescent cells, p107 protein levels are very low, which is at least in part due to E2F-dependent transcriptional repression of the p107 gene, most probably through the pRB-E2F and/or p130-E2F repressor complex (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 37Zhu L. Zhu L. Xie E. Chang L.S. Mol. Cell. Biol. 1995; 15: 3552-3562Crossref PubMed Scopus (116) Google Scholar). As cells progress toward mid-to-late G1, the levels of p107 increase, and in S phase cells p107 becomes a predominant pocket protein, although the majority are hyperphosphorylated and hence inactivated (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 19Beijersbergen R.L. Carlee L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (236) Google Scholar, 37Zhu L. Zhu L. Xie E. Chang L.S. Mol. Cell. Biol. 1995; 15: 3552-3562Crossref PubMed Scopus (116) Google Scholar). These results indicate that low levels of p107 present during mid-to-late G1 may play an important role in traversing the restriction point in conjunction with other pocket proteins. Alternatively, p107 may play a unique role among the pocket proteins in cells that have already passed the G1 restriction point. We have previously shown that in certain hematopoietic cells, including BaF3 and 32D cells, ectopically expressed p130 inhibits the cell cycle in G1, whereas pRB, even in its phosphorylation-resistant form, fails to do so (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 38Mori A. Higashi H. Hoshikawa Y. Imamura M. Asaka M. Hatakeyama M. Oncogene. 1999; 18: 6209-6221Crossref PubMed Scopus (14) Google Scholar). Given this observation, we wished to know the role of p107, which is structurally closer to p130 than pRB (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar), in hematopoietic cell cycle regulation. We therefore generated BaF3 lymphoid cells in which wild type or a nonphosphorylatable and thus constitutively active p107 was inducibly expressed. Here we demonstrate that p107 is capable of inhibiting the S phase cell cycle progression. We further provide evidence that p107 is involved in inhibiting the cell cycle in S phase cells with DNA damage. A p107 mutant, p107ΔS/T-P, that lacks all of the potential phosphorylation sites by cyclin-CDK was generated from human p107 cDNA by multiple rounds of oligonucleotide-mediated mutagenesis with use of Chameleon site-directed mutagenesis system (Stratagene) according to the manufacturer's instructions (22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In this mutant, threonines 332, 340, 369, 385, 915, and 997 and serines 368, 515, 615, 640, 650, 749, 762, 964, 975, 988, 1009, and 1041 were substituted with alanine residues. Wild type and the phosphorylation-resistant p107 were tagged with influenza hemagglutinin (HA) epitope at the carboxyl terminus (p107HA and p107ΔS/T-P-HA). cDNAs encoding p107HA and p107ΔS/T-P-HA were inserted into a mammalian expression vector, pSP65SRα2 (39Shinobu N. Maeda T. Aso T. Ito T. Kondo T. Koike K. Hatakeyama M. J. Biol. Chem. 1999; 274: 17003-17010Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). pOPTET-BSD is an inducible cDNA expression vector having the TcIP promoter and the blasticidin-resistance gene as a drug-selection marker (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 40Hoshikawa Y. Amimoto K. Mizuguchi R. Hatakeyama M. Anal. Biochem. 1998; 272: 22355-22363Google Scholar). A cDNA encoding p107HA, p107ΔS/T-P-HA, or adenovirus E1A 12S (E1A) was subcloned into the pOPTET-BSD vector. COS-7 and SAOS-2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The 6-1 cell is a BaF3-derived mouse pro-B cell line that stably co-express tTA and LacI (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 40Hoshikawa Y. Amimoto K. Mizuguchi R. Hatakeyama M. Anal. Biochem. 1998; 272: 22355-22363Google Scholar, 41Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum and 20% WEHI-3B-conditioned medium (20% WEHI) as a source of interleukin 3 (IL-3). Stable transfectants that conditionally express p107HA, p107ΔS/T-P-HA, or E1A were created by transfecting pOPTET-BSD-p107HA, pOPTET-BSD-p107ΔS/T-P-HA, or pOPTET-BSD-E1A into 6-1 cells by electroporation as described (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 38Mori A. Higashi H. Hoshikawa Y. Imamura M. Asaka M. Hatakeyama M. Oncogene. 1999; 18: 6209-6221Crossref PubMed Scopus (14) Google Scholar). Expression of the cDNA-directed proteins in these transfectants was repressed in medium containing 1 μg/ml tetracycline (Tc) and was induced in medium containing 5 mm isopropyl β-d-thiogalactopyranoside (IPTG) for 24 h in the absence of Tc. COS-7 cells in a 100-mm plate were transfected with 10 μg of each expression plasmid using the DEAE-dextran method. SAOS-2 cells in a 100-mm plate were transfected with 20 μg of each expression plasmid using the calcium-phosphate precipitation method. The transfected cells were harvested 2 days after transfection and lysed in E1A lysis buffer (250 mm NaCl, 5 mm EDTA, 50 mm HEPES, pH 7.0, 0.5% Nonidet P-40, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml trypsine inhibitor, 0.5 μm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride (32Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar). Cell lysates were then immunoprecipitated with anti-HA mouse monoclonal antibody (12CA5) or anti-adenovirus E1A mouse monoclonal antibody (PharMingen, PM-14161A). Immunoprecipitates were recovered on protein A-Sepharose beads, washed four times with E1A lysis buffer, and eluted by boiling in SDS-containing sample buffer. Immunoprecipitates and lysates were resolved by electrophoresis on a 7.5, 10 or 12% SDS-polyacrylamide gel. Gels were transferred to polyvinyldenedifluoride membrane filters (Millipore) as described (42Mizuguchi R. Hatakeyama M. J. Biol. Chem. 1998; 273: 32297-32303Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and subjected to immunoblot analysis using anti-HA, anti-p107 (Santa Cruz Biotechnology, sc-318), anti-p130 (sc-317), anti-p53 (sc-1314), anti-p21WAF1 (Oncogene Research Products, OP79), anti-cyclin A (sc-751), anti-E2F4 (sc-512), or anti-adenovirus E1A, followed by anti-mouse IgG (Amersham Pharmacia Biotech), anti-rabbit IgG (Amersham Pharmacia Biotech), or anti-goat IgG (sc-2020) secondary antibody conjugated to horseradish peroxidase. Proteins were visualized using the ECL detection system (PerkinElmer Life Sciences). 6-1 cells and their stable transfectants were washed three times with phosphate-buffered saline (PBS) and resuspended at a density of 4×105/ml with RPMI 1640 medium containing 10% fetal calf serum for cytokine starvation. Cells were then divided into two and cultured with IL-3-depleted medium in the presence of 1 μg/ml Tc or 5 mm IPTG. Following 24 h of culture in the absence of cytokine, cells were restimulated with IL-3 at a final concentration of 200 pg/ml. At 8 h after IL-3 restimulation, cisplatin was added to the culture at a final concentration of 8 μg/ml. Cells were harvested at appropriate time points. For cell cycle synchronization at early S phase, the 6-1-derived transfectant cells were first starved for cytokine and restimulated with IL-3 in the presence of 1 μg/ml Tc as above described. After 4 h of IL-3 restimulation, they were washed twice with PBS, divided into two, and cultured with IL-3-containing medium in the presence of 1 μg/ml Tc or 5 mm IPTG. These cells were respectively treated with hydroxyurea for additional 16 h at a final concentration of 5 mm. The cells were then washed twice with PBS to remove HU and cultured with IL-3-containing medium in the presence of 200 ng/ml nocodazole and 1 μg/ml Tc or 5 mm IPTG. For flow cytometric analysis, harvested cells were washed in PBS and fixed in 80% ethanol on ice. The cells were washed again and resuspended in PBS containing 500 μg/ml RNase A for 20 min at 37 °C. The samples were incubated for another 15 min at 4 ° with propidium iodide solution (100 μg/ml propidium iodide, 0.1% sodium citrate) prior to flow cytometric analysis with a Becton Dickinson FACS Calibur. Cell cycle profiles were determined by use of the CELL Quest and ModFit cell cycle analysis software. 6-1 cell is a BaF3-derived mouse lymphoid cell line whose growth is totally dependent on IL-3, with the depletion of IL-3 24 h from the start of culture inducing G0/G1 cell cycle arrest (Fig.1 A, 0 h). In these growth-arrested 6-1 cells, a member of pRB family, p130, was abundantly expressed, whereas expression of p107 was very low (Fig. 1 B,lane 1). We have previously shown that the IL-3-dependent growth of the 6-1 cell is inhibited by ectopic expression of p130 but not by pRB, indicating that the cell is actively halted in G0/G1 by the accumulated p130 in the absence of IL-3 and that inactivation of p130 is an essential prerequisite to traversing the G1 restriction point in this cell (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar). Restimulation of the growth-arrested 6-1 cells with IL-3 gave rise to synchronized cell cycle reentry and progression from G1 to S phases (Fig. 1 A). At 8 h after IL-3 stimulation, most of the p130 became hyperphosphorylated (Fig. 1 B, lane 2), indicating that by this time G1 cyclin-CDKs were activated sufficiently to phosphorylate and inactivate the pRB family pocket proteins (4Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 19Beijersbergen R.L. Carlee L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (236) Google Scholar, 20Mayol X. Garriga J. Grana X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 36Mayol X. Garriga J. Grana X. Oncogene. 1996; 13: 237-246PubMed Google Scholar, 37Zhu L. Zhu L. Xie E. Chang L.S. Mol. Cell. Biol. 1995; 15: 3552-3562Crossref PubMed Scopus (116) Google Scholar). This in turn suggests that most if not all cells passed through the G1 restriction point by 8 h after the IL-3 stimulation. After 16 h, ∼80% of cells entered S phase (Fig. 1 A). By this time point, p130 became barely detectable, whereas p107 was potently induced and abundantly expressed but was hyperphosphorylated (Fig. 1 B,lane 3). 24 h after IL-3 stimulation, the cell cycle profile returned to the pattern that is typical of asynchronously growing cells (Fig. 1 A). Using this cell cycle re-entry/progression protocol, we wished to address the possible role of p107 in cell cycle regulation, particularly in S phase progression. As an initial approach, we generated 6-1-derived stable transfectants, p107-13 and p107-16, that conditionally express wild type p107 (Fig.2 A) with the use of the tetracycline/IPTG dual-regulated inducible system, in which expression of the cDNA is strongly inhibited by tetracycline and is potently activated by IPTG (34Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 40Hoshikawa Y. Amimoto K. Mizuguchi R. Hatakeyama M. Anal. Biochem. 1998; 272: 22355-22363Google Scholar). However, despite ectopic p107 expression (Fig. 2 B), we were not able to see any changes in the cell cycle entry and progression in response to elevated p107 (Fig.2 C). This may be at least in part due to insufficient expression of ectopic p107, because actively cycling 6-1 cells already express high levels of endogenous p107 (Fig. 1 B). Indeed, we could only increase the p107 levels by at most 2-fold in relation to endogenous levels (Fig. 2 B, compare the expression of total p107 between Tc- and IPTG-treated lanes at the corresponding times). Furthermore, the massive phosphorylation of p107 in response to IL-3 restimulation (Fig. 2 B) indicates that under such circumstances cells possess p107 kinase activity that is sufficient to inactivate both endogenous and exogenous p107. Accordingly, we next generated a phosphorylation-resistant p107 mutant by replacing all of the serine and threonine residues that constitute possible cyclin-CDK phosphorylation motifs (either serine-proline or threonine-proline) with nonphosphorylatable alanine residues (Fig.3 A) (22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Upon its transient expression in COS-7 cells, the phosphorylation-resistant p107, p107ΔS/T-P-HA, was detected exclusively in its quickly migrating, hypophosphorylated form (Fig. 3 B). Furthermore, it formed physical complexes with endogenous E2F4 and cyclin A in both of COS-7 cells and SAOS-2 osteosarcoma cells (Fig. 3 C), indicating that the mutant is biologically active despite multiple point mutations (22Ashizawa S. Nishizawa H. Yamada M. Higashi H. Kondo T. Ozawa H. Kakita A. Hatakeyama M. J. Biol. Chem. 2001; 276: 11362-11370Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The cDNA encoding p107ΔS/T-P-HA was introduced into the 6-1 cells and was inducibly expressed in the stable transfectants, PRp107-14 and PRp107-24, with the use of the tetracycline/IPTG dual regulation system (Fig. 4 A). The transfectant clones were first growth-arrested in G0/G1 by IL-3 deprivation for 24 h. During IL-3 starvation, cells were also treated with Tc or IPTG. Upon IPTG treatment, the phosphorylation-resistant p107ΔS/T-P-HA was expressed in the growth-arrested cells (Fig. 4 B, lane 3), whereas it was undetectable in the same transfectant that was treated with Tc (Fig. 4 B, lane 1). Restimulation of the G0/G1 arrest cells with IL-3 in the presence of Tc (i.e. the uninduced condition) gave rise to a cell cycle progression that is basically indistinguishable from that observed with the parental 6-1 cells (Fig. 4 C, Tc; see also Fig. 1 A). In striking contrast, the same cells inducibly expressing p107ΔS/T-P-HA exhibited a severe delay of S phase entry and progression, despite the continued presence of IL-3 for 48 h (Fig. 4, C and D). To investigate the S phase inhibitory activity of the phosphorylation-resistant p107 more directly, we examined the effect of p107ΔS/T-P-HA in cells synchronized in the early S phase (Fig.5 A). G0/G1-arrested PRp107-24 cells were restimulated with IL-3 and, at 4 h after the IL-3 restimulation, the DNA synthesis inhibitor HU was added to the culture at a final concentration of 5 mm. At this time, IPTG was also added to the culture to induce p107ΔS/T-P-HA. Because these cells pass through the G1 restriction point by 8 h after the onset of IL-3 restimulation and 4 h of IPTG induction is too short to induce p107ΔS/T-P-HA protein in cells, 2T. Kondo, and M. Hatakeyama, unpublished observations. the HU-treated cells were expected to traverse from G1 to S without receiving the effect of the phosphorylation-resistant p107. The cells were treated with HU and IPTG for 16 h to induce sufficient amounts of p107ΔS/T-P-HA while arrested in the early S phase. As expected, in these HU-blocked cells, endogenous p107 molecules were totally hyperphosphorylated (Fig. 5 B, lanes 2 and4). Furthermore, the cells expressed p107ΔS/T-P-HA, whose levels were not increased by further incubation with IPTG (Fig.5 B, lanes 4 and 5). The early S phase-synchronized cells were then released from the HU block, and subsequent cell cycle progression was monitored by flow cytometry in the presence of 200 ng/ml of the tubulin inhibitor nocodazole to prevent entry into the next cell cycle. In the control cells in which expression of p107ΔS/T-P-HA was not induced, DNA synthesis was rapidly initiated upon HU release, and completion of full genome replication occurred within 18 h after the release (Fig.5 C). In striking contrast, in the presence of p107ΔS/T-P-HA, DNA synthesis was severely impaired, and most cells failed to achieve replication of full genome as late as 24 h (Fig.5 C). These results clearly demonstrate that the phosphorylation-resistant p107 is capable of inhibiting progression of the S phase. It should be noted, however, that the cells were obviously capable of initiating some degree of DNA synthesis in the presence of p107ΔS/T-P-HA, although they failed to complete genome replication (Fig. 5 C). This may indicate that chain elongation can still take place in the presence of the phosphorylation-resistant p107. Although the phosphorylation-resistant p107 is capable of inhibiting S phase progression, it remains possible that the S phase inhibitory effect is due to supraphysiologic levels of the protein expressed. To address this possibility, we examined the expression levels of the p107 mutant. Because p107ΔS/T-P-HA is detectable exclusively in its hypophosphorylated form, it is possible to compare expression levels between endogenous p107 and p107ΔS/T-P-HA. In the IPTG-treated PRp107-24 cells wherein p107ΔS/T-P-HA was inducibly expressed (Figs.4 B, lanes 3–6, and 5 B, lanes 4 and 5) and cell cycle progr" @default.
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• W1995086523 date "2001-05-01" @default.
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• W1995086523 title "Involvement of pRB-related p107 Protein in the Inhibition of S Phase Progression in Response to Genotoxic Stress" @default.
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• W1995086523 doi "https://doi.org/10.1074/jbc.m009911200" @default.
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