Oncogenic RAS Induces Accelerated Transition through G2/M and Promotes Defects in the G2 DNA Damage and Mitotic Spindle Checkpoints
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m511690200
ISSN1083-351X
AutoresJeffrey A. Knauf, Bin Ouyang, Erik S. Knudsen, Kenji Fukasawa, George F. Babcock, James A. Fagin,
Tópico(s)BRCA gene mutations in cancer
ResumoActivating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRASV12 increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRASV12 or HRASV12 effector mutants were presynchronized at the G1/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRASV12 accelerated the G2/M phase by ∼4 h and promoted bypass of the G2 DNA damage and mitotic spindle checkpoints. Accelerated passage through G2/M and bypass of the G2 DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G2 DNA damage checkpoint, because cells arrested appropriately in G2 despite conditional expression of HRASV12,S35 or BRAFV600E. By contrast to the MAPK requirement for radiation-induced G2 arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G2 progression and regulation of the G2 DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G2/M. Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRASV12 increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRASV12 or HRASV12 effector mutants were presynchronized at the G1/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRASV12 accelerated the G2/M phase by ∼4 h and promoted bypass of the G2 DNA damage and mitotic spindle checkpoints. Accelerated passage through G2/M and bypass of the G2 DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G2 DNA damage checkpoint, because cells arrested appropriately in G2 despite conditional expression of HRASV12,S35 or BRAFV600E. By contrast to the MAPK requirement for radiation-induced G2 arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G2 progression and regulation of the G2 DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G2/M. Human tumors, including those of the thyroid (1.Namba H. Matsuo K. Fagin J.A. J. Clin. Investig. 1990; 86: 120-125Crossref PubMed Scopus (123) Google Scholar, 2.Aeschimann S. Kopp P.A. Kimura E.T. Zbaeren J. Tobler A. Fey M.F. Studer H. J. Clin. Endocrinol. 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Mutations of all three RAS genes are found in benign and malignant follicular neoplasms and in follicular variant papillary thyroid carcinomas and are believed to be one of the early steps in thyroid tumor formation (4.Lemoine N.R. Mayall E.S. Wyllie F.S. Farr C.J. Hughes D. Padua R.A. Thurston V. Williams E.D. Wynford-Thomas D. Cancer Res. 1988; 48: 4459-4463PubMed Google Scholar, 5.Lemoine N.R. Mayall E.S. Wyllie F.S. Williams E.D. Goyns M. Stringer B. Wynford-Thomas D. Oncogene. 1989; 4: 159-164PubMed Google Scholar, 6.Namba H. Rubin S.A. Fagin J.A. Mol. Endocrinol. 1990; 4: 1474-1479Crossref PubMed Scopus (297) Google Scholar, 7.Esapa C.T. Johnson S.J. Kendall-Taylor P. Lennard T.W. Harris P.E. Clin. Endocrinol. 1999; 50: 529-535Crossref PubMed Scopus (188) Google Scholar, 8.Suarez H.G. du Villard J.A. Severino M. Caillou B. Schlumberger M. Tubiana M. Parmentier C. Monier R. Oncogene. 1990; 5: 565-570PubMed Google Scholar, 9.Karga H. Lee J.K. Vickery Jr., A.L. Thor A. Gaz R.D. 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Although not as widely appreciated, oncoproteins such as RAS have also been proposed to promote tumor progression through induction of genomic instability. For example, Finney and Bishop (15.Finney R.E. Bishop J.M. Science. 1993; 260: 1524-1527Crossref PubMed Scopus (112) Google Scholar) reported that replacement of a normal Hras gene with an activated mutant Hras by homologous recombination in rat1 fibroblasts is not in itself sufficient to induce transformation but rather requires secondary changes such as gene amplification events, including amplification of the mutant Ras allele. This study supports the concept that RAS may serve as a mutator gene under physiological conditions, because the mutant HRAS protein in these experiments was expressed under the control of its own promoter. The ability of activated RAS to promote chromosomal instability is also supported by studies demonstrating that expression of the human HRAS oncogene in p53-null cells leads to premature entry of cells into the S phase, increased permissivity for gene amplification, and generation of aberrant chromosomes within a single cell cycle (16.Denko N. Stringer J. Wani M. Stambrook P. Somat. Cell Mol. Genet. 1995; 21: 241-253Crossref PubMed Scopus (36) Google Scholar, 17.Denko N.C. Giaccia A.J. Stringer J.R. Stambrook P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5124-5128Crossref PubMed Scopus (189) Google Scholar, 18.Saavedra H.I. Fukasawa K. Conn C.W. Stambrook P.J. J. Biol. Chem. 1999; 274: 38083-38090Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 19.Wani M.A. Xu X. Stambrook P.J. Cancer Res. 1994; 54: 2504-2508PubMed Google Scholar, 20.Agapova L.S. Ivanov A.V. Sablina A.A. Kopnin P.B. Sokova O.I. Chumakov P.M. Kopnin B.P. Oncogene. 1999; 18: 3135-3142Crossref PubMed Scopus (48) Google Scholar). Oncogenic RAS has also been shown to produce chromosome aberrations in rat mammary carcinoma cells (21.Ichikawa T. Kyprianou N. Isaacs J.T. Cancer Res. 1990; 50: 6349-6357PubMed Google Scholar), rat prostatic tumor cells (22.Ichikawa T. Schalken J.A. Ichikawa Y. Steinberg G.D. Isaacs J.T. Prostate. 1991; 18: 163-172Crossref PubMed Scopus (23) Google Scholar), and a human colon carcinoma cell line (23.de Vries J.E. Kornips F.H. Marx P. Bosman F.T. Geraedts J.P. ten Kate J. Cancer Genet. Cytogenet. 1993; 67: 35-43Abstract Full Text PDF PubMed Scopus (20) Google Scholar). The demonstration by Agapova et al. (20.Agapova L.S. Ivanov A.V. Sablina A.A. Kopnin P.B. Sokova O.I. Chumakov P.M. Kopnin B.P. Oncogene. 1999; 18: 3135-3142Crossref PubMed Scopus (48) Google Scholar) that expression of activated HRAS promotes bypass of G2 DNA damage checkpoint in p53 mutant cells suggests that oncogenic RAS-induced genomic instability may potentially be due to a relaxation of this checkpoint. The effectors downstream of RAS that are required for this effect have not been fully elucidated. Activation by RAS of the RAL guanine nucleotide exchange factor is responsible for dampening the G2 arrest induced by ethyl methanesulfonate in p53-deficient MDAH041 fibroblasts (24.Agapova L.S. Volodina J.L. Chumakov P.M. Kopnin B.P. J. Biol. Chem. 2004; 279: 36382-36389Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). On the other hand, activation of the RAS downstream effectors MEK2 3The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; BrdUrd, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; dox, doxycycline; PI, propidium iodide; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; Gy, gray(s); FACS, fluorescence-activated cell sorter.3The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; BrdUrd, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; dox, doxycycline; PI, propidium iodide; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; Gy, gray(s); FACS, fluorescence-activated cell sorter. and ERK are required for exit from DNA damage-induced G2 cell cycle arrest (25.Abbott D.W. Holt J.T. J. Biol. Chem. 1999; 274: 2732-2742Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and the transition from G2 into M (26.Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 27.Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Crossref PubMed Scopus (157) Google Scholar), respectively.A role for the MEK/ERK pathway in G2/M is further supported by the observation that activated ERK associates with the mitotic apparatus of somatic mammalian cells (28.Shapiro P.S. Vaisberg E. Hunt A.J. Tolwinski N.S. Whalen A.M. McIntosh J.R. Ahn N.G. J. Cell Biol. 1998; 142: 1533-1545Crossref PubMed Scopus (193) Google Scholar, 29.Zecevic M. Catling A.D. Eblen S.T. Renzi L. Hittle J.C. Yen T.J. Gorbsky G.J. Weber M.J. J. 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These results strongly support a role for MEK and ERK in regulating the progression of cells through G2 and mitosis, suggesting that inappropriate activation of these RAS effectors in cells expressing oncogenic RAS could potentially disrupt the orderly transition of these cells through these latter cell cycle stages, which are critical for maintaining genomic integrity.We previously showed that acute expression of HRASV12 increases the frequency of chromosome misalignment, multiple spindle formation, centrosome amplification, and generation of micronuclei within the first few cell cycles after activation in rat thyroid PCCL3 cells (31.Saavedra H.I. Knauf J.A. Shirokawa J.M. Wang J. Ouyang B. Elisei R. Stambrook P.J. Fagin J.A. Oncogene. 2000; 19: 3948-3954Crossref PubMed Scopus (152) Google Scholar). These cells are not transformed and have wild type p53 genes. Rapid induction of these chromosomal abnormalities by RAS is consistent with a disruption of progression of cells through G2/M and/or alteration of the integrity of critical checkpoints needed to ensure genomic stability. Here we investigated this possibility by presynchronizing PCCL3 thyroid cells with conditional expression of HRASV12 or HRASV12 effector mutants at the G1/S boundary, followed by activation of expression of RAS mutants and release from the G1 block into a radiation-induced G2 arrest or a nocodazole-activated mitotic checkpoint. This allowed us to follow the progression of cells through G2/M as well as the G2 DNA damage and mitotic spindle checkpoints and explore the contribution of RAS effectors to this effect.MATERIALS AND METHODSCell Lines—The well differentiated rat thyroid cell line PCCL3 was propagated in H4 complete medium, which consisted of Coon's modification of Ham's F-12 medium containing 5% fetal bovine serum, glutamine (286 μg/ml), apo-transferrin (5 μg/ml), hydrocortisone (10 nm), insulin (10 μg/ml), thyrotropin (10 mIU/ml), penicillin, and streptomycin. The following cell lines have been previously described: rtTA, PCCL3 cells stably expressing the reverse tetracycline transactivator rtTA (32.Wang J. Knauf J.A. Basu S. Puxeddu E. Kuroda H. Santoro M. Fusco A. Fagin J.A. Mol. Endocrinol. 2003; 17: 1425-1436Crossref PubMed Scopus (58) Google Scholar); Ras-25, PCCL3 cells with doxycycline (dox)-induced expression of HRASV12 (31.Saavedra H.I. Knauf J.A. Shirokawa J.M. Wang J. Ouyang B. Elisei R. Stambrook P.J. Fagin J.A. Oncogene. 2000; 19: 3948-3954Crossref PubMed Scopus (152) Google Scholar, 33.Shirokawa J.M. Elisei R. Knauf J.A. Hara T. Wang J. Saavedra H.I. Fagin J.A. Mol. Endocrinol. 2000; 14: 1725-1738Crossref PubMed Scopus (48) Google Scholar); PC-BRAFV600E-6 PCCL3 cells with dox-induced expression of constitutively active BRAF mutant, BRAFV600E (34.Mitsutake N. Knauf J.A. Mitsutake S. Mesa Jr., C. Zhang L. Fagin J.A. Cancer Res. 2005; 65: 2465-2473Crossref PubMed Scopus (175) Google Scholar), and MEK1–65 PCCL3 cells with dox-induced expression of the constitutively active MEK1 mutant, MEK1S217E/S221E (31.Saavedra H.I. Knauf J.A. Shirokawa J.M. Wang J. Ouyang B. Elisei R. Stambrook P.J. Fagin J.A. Oncogene. 2000; 19: 3948-3954Crossref PubMed Scopus (152) Google Scholar, 33.Shirokawa J.M. Elisei R. Knauf J.A. Hara T. Wang J. Saavedra H.I. Fagin J.A. Mol. Endocrinol. 2000; 14: 1725-1738Crossref PubMed Scopus (48) Google Scholar). Using the same approach, we created PCCL3 cells with dox-inducible expression of the previously described RAS effector mutants (35.Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). Briefly, we subcloned the HRASV12,S35, HRASV12,G37, or HRASV12,C40 cDNAs (gifts from Kenji Fukasawa, University of Cincinnati) into pUHG10-3, downstream of seven repeats of a tet operator sequence and a minimal cytomegalovirus promoter. These constructs were co-transfected into rtTA cells with pTK-hygro using Lipofectamine 2000 (Invitrogen), and clones were selected based on the absence of expression under basal conditions and strong induction by dox.Reagents—FITC-conjugated anti-BrdUrd IgG was purchased from Pharmigen (San Diego, CA). Antibodies to phospho-MEK1/2 (sc-7995), MEK2 (sc-524), ERK1 (sc-94), phospho-ERK1/2 (sc-7383), HRAS (SC-520), cyclin B1 (SC-245), HDAC1 (SC-6298), and glyceraldehyde-3-phosphate dehydrogenase (SC-20357) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti phospho-MEK1/2 (9121S) goat polyclonal IgG was from Cell Signaling Technology (Beverly, MA). Thymidine, nocodazole, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide, thyrotropin, insulin, apo-transferrin, and hydrocortisone were purchased from Sigma, and PD98059 and wortmannin were from Calbiochem. Coon's modification of Ham's F-12 medium was from Irvine Scientific (Irvine, CA). Fetal bovine serum, penicillin-streptomycin, and glutamine were purchased from Invitrogen.Monitoring Cell Cycle Progression by FACS Cell Synchronization—To synchronize cells in G1/S, cells were plated into 60-mm tissue culture dishes at ∼50% confluence in H4 medium and incubated at 37 °C in 5% CO2 for 24 h. The medium was then replaced with fresh H4 medium containing 4 mm thymidine, and the cells were incubated for 14 h. The cells were then washed twice with PBS, H4 medium was added, and the cells were incubated for 9 h. The medium was then replaced with fresh H4 medium containing 4 mm thymidine, and the cells were incubated in the absence or presence of 1 μg/ml dox for 14 h. The cells were released by washing twice with PBS and then adding fresh H4 medium containing 10 μm BrdUrd with or without 1 μg/ml dox. After 2 h the cells were washed with PBS to remove BrdUrd, and fresh H4 medium with or without 1 μg/ml dox was added. To induce the G2 DNA damage checkpoint cells were irradiated 4 h after release cells with 10 Gy of x-rays or 15 Gy of γ-rays (Faxitron cabinet x-ray irradiator or Cesium irradiator, respectively). To induce the mitotic spindle checkpoint, nocodazole was added 5 h after release from the G1/S block to a final concentration of 0.4 μg/ml. At the indicated times after release, the cells were washed with PBS, harvested by trypsinization, fixed in 5 ml of cold (–20 °C) 70% ethanol, and incubated overnight at 4 °C.Cell Cycle Analysis—The fixed cells were pelleted by centrifugation and resuspended in 50 μl of 0.85% NaCl. To denature DNA, 2 ml of 2 m HCl was added, and the cells were incubated for 20 min at room temperature. The cells were then pelleted by centrifugation, resuspended in 1 ml of 0.1 m sodium borate (pH 8.6), and washed one time with PBS. The cells were then resuspended in 10 μl of FITC-conjugated anti-BrdUrd IgG (Pharmigen, San Diego, CA) and incubated for 1 h at room temperature with mixing. Five hundred microliters of propidium iodide (PI) staining solution (50 μg/ml PI, 50 μg/ml RNase A) was added, and the number of BrdUrd-positive cells in S, G2/M, and G1 was determined by FACS analysis using a Coulter EPICS XL flow cytometer (Miami, FL) at an excitation range of 488 nm (argon laser) and a 525 BP filter for FITC and 620 BP for propidium iodide.Monitoring Cell Cycle Progression by Manually Counting Mitotic Cells—The cells were synchronized in G1/S with thymidine as described above, except BrdUrd was not added to the releasing medium. Where indicated nocodazole was added 5 h after release to a final concentration of 0.4 μg/ml to induce the mitotic spindle checkpoint. At the indicated times the cells were harvested by trypsinization, washed with PBS, spotted onto a microscope slide, and fixed by incubating with ethanol/acetic acid (19:1) for 20 min at room temperature. The cells were then incubated for 5 min with PBS containing 0.2% Triton X-100, PBS, and then PBS containing 2 μg/ml DAPI. The slides were washed and mounted in Vectorshield (Vector Laboratories, Burlingame, CA). The number of mitotic cells (characterized by condensed chromosome structures observed in prophase through telophase) and nonmitotic cells was determined by manually counting using a fluorescent microscope from a sufficient number of randomly selected fields to obtain at least 5,000 cells.Western Blotting—The cells were synchronized in G1/S with thymidine as described above. At the indicated times the cells were harvested, washed with cold PBS, resuspended in buffer A (20 mm Tris-HCl pH 7.4, 135 mm NaCl, 2 mm EDTA, 1% Triton X-100, 25 mm β-glycerophosphate, 10% glycerol, 1 mm sodium orthovanadate, sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml E-64), and incubated for 20 min on ice. The cells were lysed by repeatedly passing through a 16-gauge needle. The lysates were centrifuged, the supernatant was collected, and protein concentrations were determined using Coomassie protein assay as directed by manufacturer (Pierce).Nuclear Fractionation—The cells were collected, washed twice with PBS, and resuspended in buffer B (10 mm Tris, pH 7.4, 2 mm MgCl2, 1 mm EDTA, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml E-64). The cells were incubated for 15 min on ice, passed through a 16-gauge needle, and centrifuged at 5,000 × g for 8 min at 4 °C. The supernatant was collected (cytosolic fraction), and the protein concentrations were determined using Coomassie. The pellets were washed twice with buffer B, suspended in SDS-PAGE gel loading buffer (10% SDS, 25% glycerol, 0.1% bromphenol blue, 0.75 m Tris-HCl, pH 8.8), incubated for 5 min at 95 °C, and loaded on to an SDS-polyacrylamide gel. Immunoblotting was performed as previously described (36.Knauf J.A. Elisei R. Mochly-Rosen D. Liron T. Chen X.N. Gonsky R. Korenberg J.R. Fagin J.A. J. Biol. Chem. 1999; 274: 23414-23425Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).Cyclin B1 Kinase Assay—Protein A/G plus agarose conjugate was incubated with anti-cyclin B1 for 2 h at 4°C. The anti-cyclin B1/A/G plus agarose solution was incubated overnight at 4 °C with 400 μg of total cell lysate prepared in buffer A. The immunoprecipitate was washed three times with buffer A and then once with kinase buffer (25 mm HEPES, pH 7.2, 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm NaVO3, 1 mm dithiothreitol, 10 mm NaF, 2 μg/μl of histone H1; Calbiochem). The immunoprecipitate was resuspended in kinase buffer and 32PγATP (0.25 μCi) was added. The reaction mixture was incubated at 30 °C for 30 min, and the reaction was stopped by the addition of SDS-PAGE loading buffer at 98 °C for 5 min. The agarose conjugate was removed by centrifugation, and the supernatant was loaded on to a SDS-PAGE gel. The gel was exposed to x-ray film, and the band intensity was determined by densitometry using a Kodak image station.RESULTSHRASV12 Expression Accelerates Transition through G2/M—To examine the effects of activated RAS on progression through G2/M, we used Ras-25 cells, PCCL3 cells with dox-inducible expression of HRASV12. Effects of HRASV12 on G2/M were determined by first synchronizing cells at the G1/S boundary by double thymidine treatment and then inducing expression of HRASV12 by the addition of dox for 14 h prior to release. Cell cycle progression was then monitored as described under "Material and Methods." PCCL3 cells expressing HRASV12 had an ∼2-h delay in progression through S phase (Fig. 1) but had an accelerated transition through G2/M because they entered the next G1 ∼2 h sooner. Thus, expression of HRASV12 resulted in a net acceleration of G2/M of ∼4 h. The abnormal duration of G2/M is illustrated by the flattened G2/M peak seen between 8–12 h after release (Fig. 1A, upper panels). Treatment of the parental line, rtTA (PCCL3 cells that only express the reverse tetracycline transactivator), with dox did not significantly affect the progression of cells through S or G2/M (Fig. 1A, lower panels). Acute expression of HRASV12 did not affect the ability of cells to synchronize at the G1/S border or to release from the double thymidine block (data not shown), indicating that the differences were not due to the effects of HRASV12 on cell synchronization. To confirm the accelerated transition through G2/M, the cells were collected, and the mitotic cells were quantified after release from the double thymidine block by staining with DAPI and visual counting of cells with condensed chromosomes (Fig. 1C). The decreased number of cells in mitosis after RAS activation points to an asynchronous passage of cells through mitosis, consistent with an abbreviated G2 and/or less likely, of the mitotic phase (Fig. 1C). A time course of cyclin B1 kinase activity in cells treated as described for Fig. 1C demonstrates lower levels of kinase activity between 8 and 12 h after release in cells expressing HRASV12 (Fig. 1D). The decrease in kinase activity corresponded closely with cyclin B1 immunoreactivity, indicating no intrinsic effect of HRASV12 on kinase activity (Fig. 1E). These results are consistent with a reduced number of cells in G2/M at any of the sampled time points, presumably because of a more rapid transit through these stages in cells expressing HRASV12. Acute HRASV12 Expression Promotes Exit from the DNA Damage and Mitotic Spindle Checkpoints—Next we determined whether inappropriate activation of HRAS resulted in abnormalities of either the DNA damage or mitotic spindle checkpoints, which could also contribute to the chromosomal abnormalities we observed after acute expression of HRASV12. To determine the effects of HRASV12 expression on the G2 DNA damage checkpoint Ras-25 cells were synchronized and released as in Fig. 1A, except that 4 h after release they were exposed to 15 Gy of ionizing radiation. The cells were harvested at the indicated times, and the cell cycle stage of the BrdUrd-positive cells was determined by FACS analysis. As shown in Fig. 2, irradiated cells that did not express HRASV12 remained in G2/M for ∼4 h longer than unirradiated cells. In the presence of dox there was a more rapid entry of BrdUrd-positive cells into the next G1, indicating that the irradiation-induced G2 arrest was partially overcome by expression of HRASV12 (Fig. 2).FIGURE 2Acute RAS activation promotes exit from the DNA damage checkpoint. Ras-25 cells were synchronized in G1/S and released as described for Fig. 1A. Four hours after release, the cells were exposed to 15 Gy of x-ray radiation (Faxitron cabinet irradiator) and maintained in culture until harvested for FACS analysis of BrdUrd-positive cells at the indicated times. A, in the absence of dox, radiated cells were delayed in G2. Expression of HRASV12 (+dox) resulted in more rapid exit from the G2 block (see 14-h FACS profile). B, impact of oncogenic RAS expression on the percentage of BrdUrd-positive cells entering the next G1 following radiation-induced DNA damage. A greater number of irradiated cells entered G1 compared with those not expressing the oncoprotein. The data points represent the means of a single experiment performed in duplicate. Similar results were obtained in two additional experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next explored the effects of HRASV12 expression on the mitotic spindle checkpoint. Nocodazole, a microtubule disruptor that arrests cells in metaphase, was added 5 h after release from the double thymidine block (a time point when most cells were in S phase) to activate the mitotic checkpoint. The cells were harvested at the indicated times, smeared on glass slides, and stained with DAPI, and mitotic cells were counted manually. In the absence of dox, nocodazole induced an accumulation of Ras-25 cells in mitosis that peaked 15 h after release and then declined slightly over the next 15 h. In cells expressing HRASV12, nocodazole induced a peak of mitotic cells at 13 h, which rapidly declined over the next 10 h (Fig. 3A). Cyclin B1, which is rapidly degraded as cells exit mitosis, had reduced activity (not shown) and was expressed at lower levels and declined earlier in HRASV12-expressing cells despite the continued presence of nocodazole (Fig. 3B). In parental rtTA cells, nocodazole produced an accumulation of cells in mitosis that
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