Hypoxia Inhibits G1/S Transition through Regulation of p27 Expression
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m010189200
ISSN1083-351X
AutoresLawrence B. Gardner, Qing Li, Michele S. Park, W. Michael Flanagan, Gregg L. Semenza, Chi V. Dang,
Tópico(s)Epigenetics and DNA Methylation
ResumoMammalian cellular responses to hypoxia include adaptive metabolic changes and a G1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G1/S checkpoint, we observed that the retinoblastoma protein is essential for the G1/S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G1 checkpoint, suggesting that it is a key regulator of G1/S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G1/S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension. Mammalian cellular responses to hypoxia include adaptive metabolic changes and a G1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G1/S checkpoint, we observed that the retinoblastoma protein is essential for the G1/S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G1 checkpoint, suggesting that it is a key regulator of G1/S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G1/S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension. hypoxia-inducible transcription factor-1 cyclin-dependent kinase cyclin-dependent kinase inhibitor retinoblastoma protein mouse embryo fibroblast embryonal stem cell green fluorescent protein bromodeoxyuridine propidium iodide immunoprecipitate kilobase pair base pair Cellular hypoxia is an environmental stress with important implications in developmental biology, normal physiology, and many pathological conditions, including cancer. Normal tissues display an oxygen gradient across a distance of 400 μm from a blood supply; tumors often have disordered and diminished vascularization, and hypoxia occurs in tumor tissue that is >100–200 μm away from a functional blood supply (1Arteel G.E. Thurman R.G. Yates J.M. Raleigh J.A. Br. J. Cancer. 1995; 72: 889-995Crossref PubMed Scopus (255) Google Scholar, 2Dunn J.F. Swartz H.M. 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Ratcliffe P. Moons L. Jain R.K. Collen D. Keshet E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar, 7Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2044) Google Scholar). Cells may also respond to hypoxia by diminishing their proliferative rates. Both invasive and noninvasive studies of a variety of normal tissues and tumors suggest that hypoxic cells may be viable but nonproliferating (8Raleigh J.A. Zeman E.M. Calkins D.P. McEntee M.C. Thrall D.E. Acta Oncol. 1995; 34: 345-349Crossref PubMed Scopus (29) Google Scholar, 9Kennedy A.S. Raleigh J.A. Perez G.M. Calkins D.P. Thrall D.E. Novotny D.B. Varia M.A. Int. J. Radiat. Oncol. Biol. Phys. 1997; 37: 897-905Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 10Raleigh J.A. Calkins-Adams D.P. Rinker L.H. Ballenger C.A. Weissler M.C. Fowler Jr., W.C. Novotny D.B. 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Nature. 1996; 379: 88-91Crossref PubMed Scopus (2167) Google Scholar, 16Amellem O. Sandvik J.A. Stokke T. Pettersen E.O. Br. J. Cancer. 1998; 77: 862-872Crossref PubMed Scopus (33) Google Scholar, 17Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (196) Google Scholar). The best characterized molecular event necessary for the G1/S phase transition is phosphorylation of the retinoblastoma protein (RB) by specific cyclin-dependent kinase (CDK)-cyclin complexes (18Plana-Silva M.D. Weinberg R.A. Curr. Opin. Cell Biol. 1997; 9: 768-772Crossref PubMed Scopus (206) Google Scholar). CDK activity can be inhibited by cyclin-dependent kinase inhibitors (CDKIs), such as p27 and p21, which then promote RB hypophosphorylation. There is also evidence that CDKIs may promote a G1 arrest that is RB-independent (19Arap W. Knudsen E. Sewell D.A. Sidransky D. Wang J.Y. Huang H.J. Cavenee W.K. Oncogene. 1997; 15: 2013-2020Crossref PubMed Scopus (35) Google Scholar, 20Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2051) Google Scholar). Hypoxia-induced arrest is associated with hypophosphorylation of RB (16Amellem O. Sandvik J.A. Stokke T. Pettersen E.O. Br. J. Cancer. 1998; 77: 862-872Crossref PubMed Scopus (33) Google Scholar, 21Krtolica A. Krucher N.A. Ludlow J.W. Oncogene. 1998; 17: 2295-2304Crossref PubMed Scopus (71) Google Scholar) and, as opposed to hypoxia-induced apoptosis, appears to be independent of p53 induction (17Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (196) Google Scholar, 22Graeber T.G. Peterson J.F. Tsai M. Monica K. Fornace A.J.J. A. J., G. Mol. Cell. Biol. 1994; 14: 6264-6272Crossref PubMed Google Scholar). A direct role for HIF-1α in regulating the cell cycle in hypoxia has not been clearly demonstrated (6Carmeliet P. Do Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshet E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar, 7Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2044) Google Scholar), and the events leading to the hypoxic hypophosphorylation of RB, and indeed the very relevance of RB phosphorylation status in hypoxia-induced G1 arrest, have not been well delineated. We therefore sought to characterize the molecular mechanisms responsible for hypoxia-induced growth arrest, and the role of HIF-1 in this response. Many previous studies exploring the effect of hypoxia on the cell cycle have been limited by the use of transformed and/or immortalized cell lines, which may have altered cell cycle regulators and/or other mutations (23Green S.L. Giaccia A.J. Cancer J. Sci. Am. 1998; 4: 218-223PubMed Google Scholar). In this ambiguous genetic background, and without the ability to manipulate regulators of the G1/S transition, conclusions regarding the significance of cell cycle regulators in hypoxia-induced G1 arrest have not been definitive. We elected to first identify cell cycle regulatory elements that are altered by hypoxia in immortalized fibroblasts, and then study the effect of hypoxia on the cell cycle of wild-type murine embryo fibroblasts (MEFs) and primary fibroblasts deficient in key regulators of the G1/S checkpoint. We demonstrate that RB and p27 play important roles in the hypoxia-induced G1 arrest of primary fibroblasts. We then used immortalized fibroblasts for further molecular manipulation to show that hypoxia transcriptionally induces p27 and causes a G1 arrest in an HIF-1-independent manner. These observations suggest a molecular mechanism for hypoxia-induced cell cycle regulation. Rat1a fibroblasts, NIH-3T3 fibroblasts, Balb-3T3 fibroblasts, and mouse embryo fibroblasts (MEFs) null for RB, p53, p21 (obtained from Dr. Tyler Jacks (24Herrera R.E. Sah V.P. Williams B.O. Makela T.P. Weinberg R.A. Jacks T. Mol. Cell. Biol. 1996; 16: 2402-2407Crossref PubMed Scopus (272) Google Scholar)), and p27 (25Luo Y. Marx S.O. Kiyokawa H. Koff A. Massague J. Marks A.R. Mol. Cell. Biol. 1996; 16: 6744-6751Crossref PubMed Scopus (204) Google Scholar) and their wild-type counterparts were cultured in Dulbecco's modified Eagle's medium containing 3.7 g/liter bicarbonate, 1.6 g/liter glucose and supplemented with penicillin/streptomycin and 10% fetal calf serum (Life Technologies, Inc.). All MEFs were used before passage 14. Embryonal Stem (ES) cells were prepared and cultured in Dulbecco's modified Eagle's medium with 4.6 g/liter glucose supplemented with nonessential amino acids, insulin, monothioglycerol, serum, and HEPES as described (7Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2044) Google Scholar). AIN4 cells were cultured in Improved MEM-Zinc Option supplemented with fetal calf serum, insulin, and hydrocortisone (26Li Q. Dang C.V. Mol. Cell. Biol. 1999; 19: 5339-5351Crossref PubMed Scopus (112) Google Scholar). For all experiments, 1 × 105 cells were plated in 10-cm dishes and incubated in 20% O2 at 37 °C for 24 h. The media were then changed and supplemented with 25 mm HEPES (pH 7.55). To render cells hypoxic, dishes were placed in a modular incubator chamber (Billups-Rothenberg), flushed with 95% N2, 5% CO2, and incubated at 37 °C. This resulted in ∼0.1–0.5% O2 after several hours. After 32 h, cells were released from hypoxia and quickly scraped in ice-cold phosphate-buffered saline, and analyses were performed as described below. For CoCl2 experiments, cells were incubated with CoCl2 (200 μm) for 32 h prior to analysis. Each 10-cm dish was plated with 1 × 105 cells and 24 h later transfected with Lipofectin (Life Technologies, Inc.); after a 24-h incubation in 20% O2, cells were rendered hypoxic or incubated in 20% O2 for an additional 24 h prior to analysis. The HIF-1α expression vector, pCEP4/HIF-1α, was described previously (5Jiang B. Zheng J.Z. Leung S.W. Roe R. Semenza G. J. Biol. Chem. 1997; 272: 19260-19523Google Scholar). For the cotransfection experiments, Rat1a fibroblasts were transfected with 10 μg of pCEP4/HIF-1α (or pCEP4) and 1 μg of green fluorescent protein (GFP) expression plasmid (pEGFP-N1, CLONTECH). HIF-1 function was assessed by a construct containing a hypoxia-responsive element from the human erythropoietin gene subcloned 5′ to an SV40 promoter luciferase reporter (pGL2, Promega) (5Jiang B. Zheng J.Z. Leung S.W. Roe R. Semenza G. J. Biol. Chem. 1997; 272: 19260-19523Google Scholar). The murine p27 promoter and deletion constructs, subcloned into the pGL2 basic luciferase vector (Promega), were the generous gift of Dr. Sehng-Cai Lin (27Zhang Y. S. C.L. Biochim. Biophys. Acta. 1997; 1353: 307-317Crossref PubMed Scopus (31) Google Scholar) and the 249-602 deletion construct was created by digesting the 1.1-kb promoter with EagI. The p53-responsive p21 promoter/luciferase reporter construct was obtained from Dr. Bert Vogelstein (28el-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 (7916) Google Scholar). Luciferase assays (Promega) were performed, and the data were normalized for total protein. Balb-3T3 cells were plated at 2 × 105/10-cm plate and 24 h later transfected with 30 nm of p27 antisense TGGCTCTCXTGCGCC or missense TGGCTCXCTTGCGCC oligonucleotides, where C =5MeC and X = G-clamp (Gilead), in the presence of 2.5 μg/ml GS3815 cytofectin (Glen Research). After 5 h buffered media were added, and cells were incubated in 20% O2 or rendered hypoxic for an additional 32 h. Recombinant adenoviruses containing full-length human p27 in the antisense direction and, as control, GFP alone were prepared using the AdEasy method (29He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3246) Google Scholar) and titered so that >90% of infected cells expressed GFP and no toxicity was noted. AIN4 cells, plated at 1 × 105/10-cm plate, were infected with virus for 6 h in the presence of 2% fetal calf serum, and 24 h later media were exchanged and exposed rendered hypoxic or incubated in 20% O2 for 48 h. After 32 h, cells were released from hypoxia and quickly scraped in ice-cold phosphate-buffered saline, washed, and suspended in a buffer containing sucrose and trisodium citrate. Samples were then incubated for 10 min consecutively with trypsin/Nonidet P-40/spermine tetrahydrochloride, trypsin inhibitor/ribonuclease/and propidium iodide (PI) as described (30Vindelov L.L. Christensen I.J. Nissen N.I. Cytometry. 1983; 3: 328-331Crossref PubMed Scopus (360) Google Scholar). PI and forward light scattering were detected by using a Coulter EPICS 752 flow cytometer equipped with MDADS 11 software, version 1.0. Cell cycle distribution profiles were determined with a curve fitting program ELITE version 3.0 (Coulter). To assess directly the rate of DNA synthesis, cells were exposed to media containing BrdUrd (10 μm) for 30 min. Cells were then trypsinized and removed from hypoxia. Nuclei were prepared and stained with a fluorescein isothiocyanate-labeled and BrdUrd antibody, and total DNA was stained with propidium iodide and analyzed as described (26Li Q. Dang C.V. Mol. Cell. Biol. 1999; 19: 5339-5351Crossref PubMed Scopus (112) Google Scholar). Wild-type MEF controls from RB, p53, and p27 wild-type animals all showed similar degrees of hypoxia-induced growth arrest and were averaged together. Cells were released from 32 h of hypoxia, quickly washed with ice-cold phosphate-buffered saline, and then resuspended in a solution containing 1% SDS and boiled. Protein was quantitated by the BCA method (Pierce), and equal amounts of total cellular protein were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis (26Li Q. Dang C.V. Mol. Cell. Biol. 1999; 19: 5339-5351Crossref PubMed Scopus (112) Google Scholar). RB antibody was obtained from PharMingen, and cyclin A, D, and E were from Upstate Biotechnology, Inc.; for immunoblots, p27, p53, and p21 were from Santa Cruz Biotechnology, and HIF1α was from Novus Biologicals. Coomassie Blue staining of total cellular protein confirmed equal protein loading. For immunoprecipitation (IP) experiments, cyclin A (H432) and CDK2 (M2) antibodies were obtained from Santa Cruz Biotechnology, and cyclin E antibody (Ab1) was obtained from Neomarkers. 100 μl of protein A-Sepharose beads (Amersham Pharmacia Biotech) were loaded with 20–40 μg of antibodies in IP buffer (50 mm Tris-HCl (pH 7.4), 250 mm NaCl, 0.5 mmNa3VO5, 20 mm β-glycerophosphate, 15 mm phosphatase substrate p-nitrophenyl phosphate, protease inhibitor Complete tablets (Roche Molecular Biochemicals), and 0.1% Nonidet P-40). Lysates from cells subjected to hypoxic or nonhypoxic culture conditions for 32 h were lysed with IP buffer, sonicated, and solubilized for 30 min at 4 °C, and protein concentration was assayed with the BCA kit (Pierce). Washed protein A beads were incubated with 300 μg of lysate for 2 h at 4 °C, and the beads were then washed three times with IP buffer. Histone kinase assays were performed by incubating the beads in buffer containing 50 mm Tris (pH 7.4), 1 mmCaCl2, 5 mm MgCl2, 0.5 mm Na3VO5, 20 mmβ-glycerophosphate, 15 mm phosphatase substratep-nitrophenyl phosphate, protease inhibitor Complete tablets (Roche Molecular Biochemicals), 50 μm ATP, 0.2 μg of purified histone H1 (Roche Molecular Biochemicals), and 10 μCi of [γ-32P]ATP at 30 °C for 30 min. The reaction was stopped by the addition of 50 μl of 2× Laemmli buffer and heating for 95 °C for 5 min. The products were separated by SDS-polyacrylamide gel electrophoresis, and autoradiography was performed. To assess the amount of CDK2 protein in the lysates, CDK2 was immunoprecipitated with a goat anti-CDK2 antibody, and the blot was probed with a rabbit anti-CDK2 antibody. After 28 h of hypoxic or nonhypoxic conditions, medium was changed to contain 0.5 μg/ml actinomycin D (Sigma), a concentration at which no toxicity was seen after 12 h of incubation. Total RNA was harvested at 0, 30, 120, and 240 min after actinomycin D addition under continued hypoxic or nonhypoxic conditions using Trizol (Life Technologies, Inc.) and the supplier's protocol. 15 μg of RNA/lane was subjected to electrophoresis in 1% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with a p27 cDNA probe. Autoradiographic signals were quantitated with a PhosphorImager and normalized to 18 S RNA. Since contact inhibition and serum withdrawal are well documented to result in G1 cell cycle arrest (31Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1286-1290Crossref PubMed Scopus (1059) Google Scholar, 32Holley R.W. Kiernan J.A. Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 300-304Crossref PubMed Scopus (465) Google Scholar, 33Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (650) Google Scholar, 34St Croix B. Sheehan C. Rak J.W. Florenes V.A. Slingerland J.M. Kerbel R.S. J. Cell Biol. 1998; 142: 557-571Crossref PubMed Scopus (403) Google Scholar), all experiments were performed at low (<50%) confluency in the presence of fresh, serum-containing, buffered media. We initially studied a well characterized, easily manipulated, immortalized fibroblast cell line to identify key cell cycle components that could be further investigated. When Rat1a fibroblasts were exposed to hypoxia for approximately two doubling times (32 h), propidium iodide staining and flow cytometric analysis of cell cycle distribution revealed a significant (p < 0.005) G1arrest (Table I). To assess directly the rate of DNA synthesis, fibroblasts were rendered hypoxic for 30 h, transferred to a large hypoxic atmospheric chamber (PlasLabs), and BrdUrd labeling was performed as described (26Li Q. Dang C.V. Mol. Cell. Biol. 1999; 19: 5339-5351Crossref PubMed Scopus (112) Google Scholar). Hypoxic cells incorporated significantly less BrdUrd (44 ± 0.1% in normoxia versus 24 ± 0.3% in hypoxia) confirming a decrease in S phase under hypoxic conditions.Table IEffects of hypoxia on the cell cycle profiles of various cell linesG1SG2/M% of cellsRat1a45 ± 0.740 ± 0.915 ± 0.6Rat1a hypoxia60 ± 1.429 ± 1.511 ± 0.6Rat1a CoCl247 ± 2.336 ± 2.317 ± 0.3Rat1a/pCEP458 ± 0.136 ± 0.16 ± 0.7Rat1a/HIF1α59 ± 0.135 ± 0.16 ± 0.7Wild-type MEF47 ± 1.336 ± 1.018 ± 2.0Wild-type MEF hypoxia58 ± 0.625 ± 2.817 ± 2.4MEF Rb −/−47 ± 1.327 ± 1.723 ± 1.1MEF Rb −/− hypoxia50 ± 0.625 ± 1.823 ± 0.9MEF p53 −/−36 ± 0.352 ± 1.513 ± 1.5MEF p53 −/− hypoxia44 ± 2.044 ± 2.012 ± 0.0MEF p21 −/−50 ± 3.537 ± 4.013 ± 0.0MEF p21 −/− hypoxia66 ± 1.522 ± 1.012 ± 0.0MEF p27 −/−54 ± 3.029 ± 1.017 ± 1.0MEF p27 −/− hypoxia55 ± 2.022 ± 1.023 ± 1.0Cells were cultured, transfected, and exposed to 32 h of hypoxia, normoxia, or CoCl2 as described under “Materials and Methods.” Results of cell cycle analyses represent the average ± S.E. of 4–10 experiments performed in duplicate. Open table in a new tab Cells were cultured, transfected, and exposed to 32 h of hypoxia, normoxia, or CoCl2 as described under “Materials and Methods.” Results of cell cycle analyses represent the average ± S.E. of 4–10 experiments performed in duplicate. We then sought to identify further components of the G1checkpoint that respond to hypoxia and might contribute to cell cycle arrest. RB phosphorylation is promoted by CDK2 and CDK4 when they complex with cyclins E, A, and D. A G1 arrest occurs when these CDK activities are inhibited by the CDKIs p21, p27, or p16. After two doubling times in either normoxia or hypoxia, Rat1a fibroblasts were collected, and the expression of several proteins important for the G1-S phase transition was examined. As previously reported in other immortalized cell lines (16Amellem O. Sandvik J.A. Stokke T. Pettersen E.O. Br. J. Cancer. 1998; 77: 862-872Crossref PubMed Scopus (33) Google Scholar, 21Krtolica A. Krucher N.A. Ludlow J.W. Oncogene. 1998; 17: 2295-2304Crossref PubMed Scopus (71) Google Scholar, 35Krtolica A. Krucher N.A. Ludlow J.W. Br. J. Cancer. 1999; 80: 1875-1883Crossref PubMed Scopus (36) Google Scholar, 36Ludlow J.W. Howell R.L. Smith H.C. Oncogene. 1993; 8: 331-339PubMed Google Scholar), hypoxia induced hypophosphorylation of RB in Rat1a cells, a hallmark of G1 arrest in normal cells (Fig.1 A). This reduction in RB phosphorylation was associated with a decrease in CDK2 activity (Fig.1 A). Despite this decrease in kinase activity, the amount of immunoprecipitated CDK2 protein was unaffected by hypoxia (Fig.1 A), indicating that a modifier of CDK activity (e.g. cyclin E, A, p21, and/or p27) is altered by hypoxia. Although p53 may be induced in hypoxia by an HIF-1α-dependent manner (37An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (652) Google Scholar, 38Wenger R.H. Camenisch G. Desbaillets I. Chilov D. Gassmann M. Cancer Res. 1998; 58: 5678-5680PubMed Google Scholar), we observed only a minimal increase in p53 and actually observed a decrease in p21 under our experimental conditions (Fig. 1 A). In contrast, protein lysates of hypoxic cells revealed a decrease in cyclin E and a minimal decrease in cyclin A (Fig. 1 A). Also consistent with a decrease in CDK2 activity and RB phosphorylation, hypoxia led to a significant increase in the CDKI p27 (Fig. 1 B). The decrease in CDK2 activity and hypophosphorylation of RB associated with hypoxia that we and others observed in rat and other immortalized cell lines suggest, but do not conclusively support, a role for RB and p27 in hypoxia-induced G1 arrest. Immortalized cells may have multiple genetic abnormalities. Alterations seen in one regulatory component (e.g. p27 induction) may be the result of hypoxia-induced growth arrest, not a cause of the G1 arrest. In addition, both overexpression of p27 and serum withdrawal may induce G1 arrest in an RB-independent manner (19Arap W. Knudsen E. Sewell D.A. Sidransky D. Wang J.Y. Huang H.J. Cavenee W.K. Oncogene. 1997; 15: 2013-2020Crossref PubMed Scopus (35) Google Scholar, 20Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2051) Google Scholar, 24Herrera R.E. Sah V.P. Williams B.O. Makela T.P. Weinberg R.A. Jacks T. Mol. Cell. Biol. 1996; 16: 2402-2407Crossref PubMed Scopus (272) Google Scholar). To evaluate the significance of these hypoxia-induced changes noted in immortalized cells, hypoxia-induced arrest was examined in mouse embryo fibroblasts (MEFs). This approach allows the identification of changes that occur in primary cells with minimal genetic alterations and permits the further examination of the effects of hypoxia on isogenic MEFs deficient in key regulators of the G1 phase of the cell cycle. Similar to the immortalized fibroblasts, wild-type MEFs underwent a G1 arrest (Table I) and decreased BrdUrd uptake (Fig.2 A) when rendered hypoxic. The extent of this hypoxia-induced arrest was most apparent when wild-type MEFs were synchronized with 32 h of serum withdrawal. When re-stimulated with serum, MEFs incubated in 20% O2 showed a dramatic (>60%) percentage of S phase at 16 h, whereas serum-stimulated hypoxic MEFs increased S phase only minimally (<12%) throughout the 32-h experiment (Fig. 2 B). These observations indicate that hypoxia inhibits G1/S transition in normal primary fibroblasts. Several of the changes that we (Fig. 1 A) and others (16Amellem O. Sandvik J.A. Stokke T. Pettersen E.O. Br. J. Cancer. 1998; 77: 862-872Crossref PubMed Scopus (33) Google Scholar, 21Krtolica A. Krucher N.A. Ludlow J.W. Oncogene. 1998; 17: 2295-2304Crossref PubMed Scopus (71) Google Scholar,35Krtolica A. Krucher N.A. Ludlow J.W. Br. J. Cancer. 1999; 80: 1875-1883Crossref PubMed Scopus (36) Google Scholar, 36Ludlow J.W. Howell R.L. Smith H.C. Oncogene. 1993; 8: 331-339PubMed Google Scholar) noted in immortalized cells were not apparent in primary cell lines. Specifically, we noted only mild decreases in cyclin E and cyclin A in MEFs (Fig. 1 C and data not shown). Also, in contrast to Rat1a cells, we noted minimal kinase activities associated with CDK4 and cyclin E in both hypoxic and normoxic MEFs (data not shown). However, similar to the immortalized cells that underwent a G1 arrest in hypoxia, we noted that hypoxia increased p27, decreased CDK2 activity, and led to a hypophosphorylation of RB in hypoxic MEFs (Fig. 1 C and Fig. 3, B andC). These data suggest that these changes may be key in regulating hypoxia-induced growth arrest. To better assess the significance of RB hypophosphorylation and p27 induction in hypoxia, MEFs deficient in G1 cell cycle regulators were subjected to 32 h of hypoxia. Previous studies have shown that hypoxia-induced growth arrest occurs in transformed cells with mutant or null p53 status (17Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (196) Google Scholar, 22Graeber T.G. Peterson J.F. Tsai M. Monica K. Fornace A.J.J. A. J., G. Mol. Cell. Biol. 1994; 14: 6264-6272Crossref PubMed Google Scholar). Consistent with these reports, MEFs null for p53 or p21 arrested in hypoxia to the same degree as wild-type cells (Table I and Fig. 2 A). Therefore, neither p53 nor p21 is required for hypoxia-induced G1 arrest. However, as compared with these cells, the ability of RB-null MEFs to arrest in G1 in response to hypoxia was markedly diminished (Table Iand Fig. 2 A), indicating that RB participates in this arrest. Although the decrease in cyclin E in wild-type MEFs was less dramatic than that in immortalized fibroblasts (Fig. 1, A andC), either a decrease in cyclin E and/or an increase in p27 could contribute to diminished CDK2 activity leading to RB hypophosphorylation in hypoxia. Previous studies have shown that p27 induction is necessary for the growth arrest observed in Balb-3T3 fibroblasts subjected to serum withdrawal (33Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (650) Google Scholar). To determine the extent that p27 induction is responsible for hypoxia-induced growth arrest, p27 null MEFs were rendered hypoxic for two doubling times. There was no significant change in cell cycle profile or incorporation of BrdUrd (Table I and Fig. 2 A) despite hypoxia, indicating that p27 is necessary for hypoxia-induced G1 arrest. The contribution of p27 to hypoxia-induced G1 arrest was also apparent when CDK2 activity and RB phosphorylation status were examined in p27 null fibroblasts rendered hypoxic (Fig.3, B and C). The base-line RB phosphorylation appears enhanced in the p27 null fibroblasts. Whereas hypoxia led to a 42% decrease in CDK2 activity as well as RB hypophosphorylation in wild-type MEF cells, these changes were not evident in p27 null fibroblasts. It could be argued that the increase of p27 in hypoxic wild-type MEFs is a secondary effect of cell cycle arrest and not a mediator of hypoxia-induced G1 arrest. However, p27 expression was also induced in hypoxic yet cycling RB null MEFs (Fig. 1 C), which indicates that the induction of p27 is a direct effect of hypoxia rather than a secondary feature
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