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• W2027312705 abstract "The retinoblastoma tumor suppressor (RB) is functionally inactivated in many human cancers. Classically, RB functions to repress E2F-mediated transcription and inhibit cell cycle progression. Consequently, RB ablation leads to loss of cell cycle control and aberrant expression of E2F target genes. Emerging evidence indicates a role for RB in maintenance of genomic stability. Here, mouse adult fibroblasts were utilized to demonstrate that aberrant DNA content in RB-deficient cells occurs concomitantly with an increase in levels and chromatin association of DNA replication factors. Furthermore, following exposure to nocodazole, RB-proficient cells arrest with 4 n DNA content, whereas RB-deficient cells bypass the mitotic block, continue DNA synthesis, and accumulate cells with higher ploidy and micronuclei. Under this condition, RB-deficient cells also retain high levels of tethered replication factors, MCM7 and PCNA, indicating that DNA replication occurs in these cells under nonpermissive conditions. Exogenous expression of replication factors Cdc6 or Cdt1 in RB-proficient cells does not recapitulate the RB-deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells elevated ploidy and bypassed the response to nocodazole-induced cessation of DNA replication in a manner analogous to RB loss. Collectively, these results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy. The retinoblastoma tumor suppressor (RB) is functionally inactivated in many human cancers. Classically, RB functions to repress E2F-mediated transcription and inhibit cell cycle progression. Consequently, RB ablation leads to loss of cell cycle control and aberrant expression of E2F target genes. Emerging evidence indicates a role for RB in maintenance of genomic stability. Here, mouse adult fibroblasts were utilized to demonstrate that aberrant DNA content in RB-deficient cells occurs concomitantly with an increase in levels and chromatin association of DNA replication factors. Furthermore, following exposure to nocodazole, RB-proficient cells arrest with 4 n DNA content, whereas RB-deficient cells bypass the mitotic block, continue DNA synthesis, and accumulate cells with higher ploidy and micronuclei. Under this condition, RB-deficient cells also retain high levels of tethered replication factors, MCM7 and PCNA, indicating that DNA replication occurs in these cells under nonpermissive conditions. Exogenous expression of replication factors Cdc6 or Cdt1 in RB-proficient cells does not recapitulate the RB-deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells elevated ploidy and bypassed the response to nocodazole-induced cessation of DNA replication in a manner analogous to RB loss. Collectively, these results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy. The retinoblastoma tumor suppressor (RB) 3The abbreviations used are: RB, retinoblastoma protein; GFP, green fluorescent protein; Ad, adenovirus; GFP cells, Ad-GFP vector-infected cells that are RB-proficient; GFP-CRE or CRE cells, Ad-GFP-CRE-infected cells that are RB-deficient; PCNA, proliferating cell nuclear antigen; pre-RC, prereplication complex; BrdUrd, bromodeoxyuridine; PI, propidium iodide; PIPES, 1,4-piperazinediethanesulfonic acid; CSK, cytoskeletal. is a critical regulator of cell cycle and cellular proliferation, and its functional inactivation occurs in a variety of human cancers (1Weinberg R.A. Cancer Surv. 1992; 12: 43-57PubMed Google Scholar, 2Kaelin Jr., W.G. Ann. N. Y. Acad. Sci. 1997; 833: 29-33Crossref PubMed Scopus (23) Google Scholar, 3Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (230) Google Scholar). In quiescent, senescent, or differentiated cells, RB is hypophosphorylated, wherein it interacts with a host of cellular proteins to repress transcription of genes required for cell cycle progression (4Bartek J. Bartkova J. Lukas J. Curr. Opin. Cell. Biol. 1996; 8: 805-814Crossref PubMed Scopus (367) Google Scholar). Specifically, RB can interact with the E2F family of transcription factors and either directly inhibit transcription of cell cycle genes or mediate transcriptional repression through the recruitment of co-repressors (e.g. HDAC1 or SWI/SNF) (5Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (604) Google Scholar, 6Giacinti C. Giordano A. Oncogene. 2006; 25: 5220-5227Crossref PubMed Scopus (852) Google Scholar). During progression into the cell cycle, RB is inactivated by phosphorylation, thereby disrupting the RB-mediated transcriptional repressor complexes (7Ma D. Zhou P. Harbour J.W. J. Biol. Chem. 2003; 278: 19358-19366Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus, it is widely accepted that RB functions to regulate cell cycle transitions, particularly relating to entry into S phase. Recent studies have identified roles for RB in modulating DNA replication during the S phase of the cell cycle (8Wikman H. Kettunen E. Expert Rev. Anticancer Ther. 2006; 6: 515-530Crossref PubMed Scopus (58) Google Scholar, 9Stevaux O. Dyson N.J. Curr. Opin. Cell. Biol. 2002; 14: 684-691Crossref PubMed Scopus (350) Google Scholar, 10Angus S.P. Mayhew C.N. Solomon D.A. Braden W.A. Markey M.P. Okuno Y. Cardoso M.C. Gilbert D.M. Knudsen E.S. Mol. Cell. Biol. 2004; 24: 5404-5420Crossref PubMed Scopus (36) Google Scholar, 11Chew Y.P. Ellis M. Wilkie S. Mittnacht S. Oncogene. 1998; 17: 2177-2186Crossref PubMed Scopus (74) Google Scholar). DNA replication requires the sequential assembly of a prereplication complex (pre-RC) that comprises multiple proteins, including the origin recognition complex, Cdc6 (cell division cycle 6) protein, Cdt1 (cell division transition 1 protein), and the minichromosome maintenance family of proteins (MCM2 to -7) (12Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar, 13Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (341) Google Scholar, 14Stillman B. Science. 1996; 274: 1659-1664Crossref PubMed Scopus (431) Google Scholar). Together, assembly of the prereplication complex on chromatin specifies origins that are competent for the initiation of DNA replication (15Stillman B. FEBS Lett. 2005; 579: 877-884Crossref PubMed Scopus (111) Google Scholar, 16Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (754) Google Scholar). The regulation of pre-RC assembly is tightly controlled and is a critical means through which DNA replication is restricted to a single round per cell cycle (17Blow J.J. Dutta A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 476-486Crossref PubMed Scopus (532) Google Scholar, 18Machida Y.J. Hamlin J.L. Dutta A. Cell. 2005; 123: 13-24Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 19Takeda D.Y. Dutta A. Oncogene. 2005; 24: 2827-2843Crossref PubMed Scopus (155) Google Scholar). Subsequent to origin activation, additional factors are recruited to initiate DNA replication (e.g. Replication Protein A and primase) (20Fanning E. Klimovich V. Nager A.R. Nucleic Acids Res. 2006; 34: 4126-4137Crossref PubMed Scopus (402) Google Scholar). As replication forks mature, DNA polymerases (e.g. DNA polymerase delta) and associated processivity factors (e.g. proliferating cell nuclear antigen (PCNA)) are recruited to facilitate replication of the genome (21Diffley J.F. Curr. Biol. 2004; 14: R778-786Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 22Diffley J.F. Labib K. J. Cell Sci. 2002; 115: 869-872Crossref PubMed Google Scholar). The influence of RB on the functional activity of the pre-RC remains unclear. Studies utilizing overexpression of active alleles of RB demonstrate that RB does not a priori inhibit pre-RC assembly. Rather, it functions downstream to actively inhibit the retention of PCNA within the replication machinery (23Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar). Additionally, other studies have identified a role for RB in the maintenance of genome integrity. The impact of RB loss on genomic instability was initially observed in HPV-infected fibroblasts, where E7-medated inactivation of RB led to impaired chromosomal integrity (24White A.E. Livanos E.M. Tlsty T.D. Genes Dev. 1994; 8: 666-677Crossref PubMed Scopus (357) Google Scholar). Subsequent studies show that cellular stresses, such as DNA damage or exposure to cytotoxic compounds, coupled with RB loss also elevate cell ploidy (25Avni D. Yang H. Martelli F. Hofmann F. ElShamy W.M. Ganesan S. Scully R. Livingston D.M. Mol. Cell. 2003; 12: 735-746Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 26Di Leonardo A. Khan S.H. Linke S.P. Greco V. Seidita G. Wahl G.M. Cancer Res. 1997; 57: 1013-1019PubMed Google Scholar, 27Khan S.H. Wahl G.M. Cancer Res. 1998; 58: 396-401PubMed Google Scholar, 28Lan Z. Sever-Chroneos Z. Strobeck M.W. Park C.H. Baskaran R. Edelmann W. Leone G. Knudsen E.S. J. Biol. Chem. 2002; 277: 8372-8381Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 29Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar). Polyploidy is a likely precursor of aneuploidy, which in turn is a hallmark of most tumors (30Bharadwaj R. Yu H. Oncogene. 2004; 23: 2016-2027Crossref PubMed Scopus (433) Google Scholar, 31Sen S. Curr. Opin. Oncol. 2000; 12: 82-88Crossref PubMed Scopus (228) Google Scholar, 32Storchova Z. Pellman D. Nat. Rev. Mol. Cell. Biol. 2004; 5: 45-54Crossref PubMed Scopus (620) Google Scholar), and loss of RB can lead to poly- and aneuploidy (29Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar, 33Hernando E. Nahle Z. Juan G. Diaz-Rodriguez E. Alaminos M. Hemann M. Michel L. Mittal V. Gerald W. Benezra R. Lowe S.W. Cordon-Cardo C. Nature. 2004; 430: 797-802Crossref PubMed Scopus (459) Google Scholar, 34Zheng L. Flesken-Nikitin A. Chen P.L. Lee W.H. Cancer Res. 2002; 62: 2498-2502PubMed Google Scholar, 35Zheng L. Lee W.H. Adv. Cancer Res. 2002; 85: 13-50Crossref PubMed Scopus (48) Google Scholar). Although several studies report the effect of RB loss on cell proliferation and its impact on cancer, few studies document the consequence of RB deficiency on DNA replication control and its subsequent effect on cancer. It is known that loss of RB enables aberrant DNA replication in the presence of DNA damage or under conditions of mitotic blockade (36Eguchi T. Takaki T. Itadani H. Kotani H. Oncogene. 2007; 26: 509-520Crossref PubMed Scopus (54) Google Scholar, 37Bosco E.E. Knudsen E.S. Nucleic Acids Res. 2005; 33: 1581-1592Crossref PubMed Scopus (20) Google Scholar). Such aberrant replication is linked with secondary forms of DNA damage and has been hypothesized to compromise genome integrity (27Khan S.H. Wahl G.M. Cancer Res. 1998; 58: 396-401PubMed Google Scholar, 29Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar, 38Mayhew C.N. Bosco E.E. Solomon D.A. Knudsen E.S. Angus S.P. Methods Mol. Biol. 2004; 281: 3-16PubMed Google Scholar). Whether these effects involve a direct action of RB on chromatin or indirect effects in transcriptional control have yet to be conclusively determined. Furthermore, the mechanisms through which RB loss bypasses normal controls over replication, the critical effectors in this process, and how they contribute to tumorigenesis are unknown. Given that RB controls replication, which in turn determines cell ploidy, it is critical to determine the consequences of RB loss on these phenomena. In the present study, we investigated the impact of RB loss on the DNA replication machinery and show that RB deficiency has profound effects on the establishment of the prereplication complex and enables inappropriate rounds of DNA replication. Furthermore, the consequences of RB loss on cell ploidy can be phenocopied by exogenous E2F expression in RB-proficient cells. The current findings indicate that transcriptional control is important in the deregulation of DNA replication that is observed upon RB loss and that deregulation of DNA synthesis resulting from loss of RB contributes to genomic imbalance. Isolation of Primary RbloxP/loxP Mouse Adult Fibroblasts (MAFs), Cell Culture, and Adenoviral Infections—Primary fibroblasts were isolated from the peritoneal fascia of Rb floxed mice and cultured as previously described (29Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar). All cells used in this study were between passages 2 and 4 unless indicated otherwise. Primary RbloxP/loxP MAFs were infected with adenoviruses expressing the control GFP vector alone (Ad-GFP) or GFP and Cre recombinase (Ad-GFP-CRE). The Ad-GFP-CRE infection rendered conditional RB knock-out cells due to the activity of the Cre recombinase. After infection, cells were cultured at least 3 days before experimentation. Detection of Recombination and RB Knockdown in MAFs—Genomic DNA was isolated from either GFP (RB-proficient) or GFP-CRE (RB-deficient) MAFs using the Dneasy kit (Qiagen). To detect Cre-mediated recombination, PCR analysis of Rb exon 19 was done as described previously (39Mayhew C.N. Bosco E.E. Fox S.R. Okaya T. Tarapore P. Schwemberger S.J. Babcock G.F. Lentsch A.B. Fukasawa K. Knudsen E.S. Cancer Res. 2005; 65: 4568-4577Crossref PubMed Scopus (86) Google Scholar). The efficiency of the Rb deletion was also confirmed by Western blot analysis of target protein deregulation. BrdUrd/Propidium Iodide Flow Cytometry Analysis Staining—RB-proficient and RB-deficient MAFs were plated (2 × 106 cells/10-cm2 dish) in 10-cm2 dishes. Eighty percent confluent cells were treated with 100 ng/ml nocodazole or Me2SO control. After 24 h, the cells were washed and labeled with BrdUrd (Amersham Biosciences) for 2 h, trypsinized, and fixed in 70% ethanol overnight. Fixed cells were resuspended in 2 n HCl for 30 min at 37 °C. Cells were washed with PBS and resuspended in 1 ml of 0.5% FBS plus 0.5% Tween 20 in PBS. Anti-BrdUrd-fluorescein isothiocyanate (BD Pharmingen) was added to the cell suspension and incubated for 30 min. Propidium iodide (50 μg/ml) and RNase A (80 μg/ml) were added to the cell suspension and allowed to stain overnight at 4 °C. Samples were analyzed for BrdUrd/propidium iodide (PI) by bivariate flow cytometry analysis. Flow Cytometry—RB-proficient and RB-deficient MAFs were plated as described above. Two days post-plating, cells were treated with either Me2SO or 100 ng/ml nocodazole (Calbiochem) for 24 h. Cells were then trypsinized (both the floating and the adherent cells were collected) and fixed with 70% ethanol. Fixed cells were treated with RNase (80 μg/ml) and stained with propidium iodide (50 μg/ml). Cells were processed for flow cytometric analysis as previously described (39Mayhew C.N. Bosco E.E. Fox S.R. Okaya T. Tarapore P. Schwemberger S.J. Babcock G.F. Lentsch A.B. Fukasawa K. Knudsen E.S. Cancer Res. 2005; 65: 4568-4577Crossref PubMed Scopus (86) Google Scholar). Modfit software was used to determine the percent of cells with >4 n DNA content. Preparation of Cell Lysates and Western Blotting—RB-proficient (GFP) and RB-deficient (GFP-CRE or CRE) MAFs were plated (2 × 106/ 10-cm2 dish). Cells were then lysed in radioimmune precipitation buffer (supplemented with protease inhibitors), sonicated, and quantified (Pierce). Lysates were boiled and subjected to SDS-PAGE electrophoresis. Specific proteins MCM7, PCNA, Cdc6, lamin B, Cdk4, cyclin A, cyclin E, Replication Protein A (Santa Cruz Biotechnology), and Cdt1 (a gift from Dr. Jeanne Cook) were detected by Western blotting. BrdUrd Incorporation Assay—Cells were plated on coverslips and treated with Me2SO or nocodazole (100 ng/ml) for 24 h. Cells were washed and labeled with BrdUrd (Amersham Biosciences) for 6 h to detect DNA synthesis. Labeled cells were probed with anti-BrdUrd antibody as described previously (40Bosco E.E. Mayhew C.N. Hennigan R.F. Sage J. Jacks T. Knudsen E.S. Nucleic Acids Res. 2004; 32: 25-34Crossref PubMed Scopus (80) Google Scholar). Biochemical Fractionation to Isolate Chromatin-bound Proteins—The isolation of chromatin-associated proteins was performed as described previously (41Braden W.A. Lenihan J.M. Lan Z. Luce K.S. Zagorski W. Bosco E. Reed M.F. Cook J.G. Knudsen E.S. Mol. Cell. Biol. 2006; 26: 7667-7681Crossref PubMed Scopus (25) Google Scholar). Briefly, cells were treated with Me2SO or nocodazole (100 ng/ml), washed with PBS, and trypsinized. The cell pellet was resuspended in ice-cold cytoskeletal (CSK) buffer (10 mm PIPES, pH 6.8, 100 mm NaCl, 300 mm sucrose, 1 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.2% Triton X-100). Total cell lysates were generated by lysing one-third of the cells in CSK buffer. The remaining cells were extracted twice with cold CSK buffer on ice. The suspension was then centrifuged, and the pellet was resuspended in CSK buffer. All samples were quantified and processed for blotting of the indicated proteins. Immunofluorescence Assay to Visualize Chromatin-bound Replication Factors—Cells were plated on coverslips, and one set was utilized to detect total proteins. The second set was subjected to CSK extraction to retain only the chromatin-bound proteins, as previously described (41Braden W.A. Lenihan J.M. Lan Z. Luce K.S. Zagorski W. Bosco E. Reed M.F. Cook J.G. Knudsen E.S. Mol. Cell. Biol. 2006; 26: 7667-7681Crossref PubMed Scopus (25) Google Scholar). All cells were methanol-fixed and probed with the indicated antibodies. Cells were visualized using a fluorescent microscope. Ectopic Expression of Ad-Cdc6, Ad-Cdt1, Ad-E2F1, and Ad-E2F2 in RB-proficient Cells and Dominant Negative E2F-A/B in RB-deficient Cells—GFP cells (RB-proficient) were plated and infected with virus, at least 1 × 1011 plaque-forming units/10-cm2 dish for 24–30 h, as previously described (41Braden W.A. Lenihan J.M. Lan Z. Luce K.S. Zagorski W. Bosco E. Reed M.F. Cook J.G. Knudsen E.S. Mol. Cell. Biol. 2006; 26: 7667-7681Crossref PubMed Scopus (25) Google Scholar). Cells were then treated with Me2SO control or nocodazole and processed for flow cytometry, CSK extraction, and Western blotting as described above. RB-deficient cells were plated (2 × 106/10-cm2 dish) and transfected with either H2B-GFP plasmid or H2B-GFP and E2F-A/B using Fugene transfection agent. Forty-eight hours later, cells were treated with Me2SO control or nocodazole (100 ng/ml). Twenty-four hours after treatment, cells were labeled with BrdUrd for 6 h and processed for BrdUrd incorporation as described above. Microscopy and Digital Image Acquisition—A DIX digital camera (Nikon) attached to a Microphot FXA upright microscope (Nikon) with a ×63 or ×100 dippable Zeiss objective was used to visualize and photograph cells. NikonView5 software was used to transfer images. For the immunofluorescence images, an ORCA-ER Hamamatsu Avioplan 2 imaging microscope was used with a ×100 dippable Zeiss objective and Hoechst and Rhodamine Zeiss filters. Metamorph software was used to acquire images. RB Loss Deregulates Expression of Replication Factors and Results in Aberrant Ploidy—To investigate the acute loss of RB on replication control, MAFs were cultured from the peritoneal fascia of RbloxP/loxP mice. The cells were then infected with recombinant adenoviruses expressing either GFP vector alone (Ad-GFP) or GFP and Cre-recombinase (Ad-GFP-CRE). The infection of these cells with Ad-GFP-CRE resulted in the efficient deletion exon 19 of Rb, as detected by genomic PCR (Fig. 1A). This deletion event is well characterized to produce an unstable, nonfunctional protein, resulting in efficient ablation of the RB protein (39Mayhew C.N. Bosco E.E. Fox S.R. Okaya T. Tarapore P. Schwemberger S.J. Babcock G.F. Lentsch A.B. Fukasawa K. Knudsen E.S. Cancer Res. 2005; 65: 4568-4577Crossref PubMed Scopus (86) Google Scholar). In this manner, MAFs that are RB-proficient (GFP) and RB-deficient (CRE) were obtained. Consistent with previous observations (29Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar), the acute loss of RB results in a 7-fold increase in the percentage of cells that exhibit >4 n DNA content (Fig. 1, B (flow cytometry trace) and C (combined average from three experiments)). Coordinate analyses of DNA replication (via BrdUrd incorporation) and DNA content reveal that the 2 n population exhibits pronounced BrdUrd incorporation (Fig. 1D). Since the majority of these cells are cycling, diploid cells, there is a high degree of BrdUrd incorporation in the 2 n fraction. However, a small fraction of these cells have begun to acquire >4 n DNA content (Fig. 1D, boxed region); among these cells, a greater proportion of RB-deficient cells are BrdUrd-positive when compared with RB-proficient cells (Fig. 1D, boxed regions, compare BrdUrd-positive cells with >4 n DNA in GFP and CRE populations). This result, coupled with the discrete peaks at 4 and 8 n (Fig. 1B), suggests that the increased ploidy is the result of a complete round of DNA replication. To determine if aberrant DNA replication in RB-deficient cells was associated with changes in DNA replication factors, the levels of these factors were determined by immunoblotting. In RB-deficient cells, components of the prereplicative complex, such as Cdc6, Cdt1, and MCM7 were greatly increased as compared with RB-proficient cells (Fig. 1E, compare lanes 1 and 2). In addition, levels of other S phase-associated factors, such as PCNA, cyclin A, cyclin E, and Replication Protein A were also elevated (Fig. 1E). These results suggest that the elevated levels of replication factors may functionally contribute to the aberrant ploidy observed with the loss of RB. RB Loss Is Associated with Increased Chromatin Association of DNA Replication Factors—The preceding results indicate that the total protein levels of several DNA replication factors are enhanced with RB loss. Therefore, we next investigated the extent to which these proteins engage in DNA replication. Most DNA replication proteins are considered active when they are associated with chromatin. For instance, it has been determined that the chromatin-bound fraction of MCM2 to -7 denotes the presence of a functional prereplication complex (42Kubota Y. Mimura S. Nishimoto S. Masuda T. Nojima H. Takisawa H. EMBO J. 1997; 16: 3320-3331Crossref PubMed Scopus (94) Google Scholar, 43Homesley L. Lei M. Kawasaki Y. Sawyer S. Christensen T. Tye B.K. Genes Dev. 2000; 14: 913-926PubMed Google Scholar, 44Fujita M. Kiyono T. Hayashi Y. Ishibashi M. J. Biol. Chem. 1997; 272: 10928-10935Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, we assessed the relative levels of chromatin-bound replication factors in RB-proficient versus RB-deficient cells, since they represent the functional components of the replication machinery. Western blot analysis of replication factors, such as MCM7, PCNA, and Cdc6, demonstrated increased total protein levels (Fig. 2A, compare lanes 1 and 2), consistent with the preceding results. Interestingly, their chromatin-tethered levels were also significantly elevated in RB-deficient cells (Fig. 2A, compare lanes 3 and 4). Thus, components of DNA replication are differentially engaged in RB-proficient versus RB-deficient cells. In contrast, other chromatin/insoluble proteins, such as lamin B (loading control), were unaffected by RB loss. Additionally, we observed that Cdk1 was not associated with chromatin (Fig. 2A), indicating that the increased tethering of the replication factors is specific to RB loss. Since the accumulation of pre-RC proteins in the chromatin fraction was pronounced in RB-deficient cells, this suggests that assembly of the prereplication complex is deregulated with the loss of RB. These results were further validated by immunofluorescence, wherein cells were subjected to in situ extraction to remove soluble cytosolic/nucleoplasmic proteins and retain only chromatin-tethered proteins. They were probed with antibodies to MCM7, PCNA, cyclin A, Cdk1 (negative control for tethering), and lamin B (additional control) and evaluated by fluorescence microscopy (Fig. 2, B–F). RB-deficient cells exhibited higher levels of total and tethered replication proteins. Data from fractionation and immunofluorescence experiments thus demonstrate enhanced chromatin retention of replication proteins, suggesting that RB loss influences DNA replication control by altering the level and activity of components of the DNA replication machinery. RB Loss Facilitates DNA Rereplication in the Presence of a Mitotic Block—Although deregulation of DNA replication processes could contribute to alterations in genome copy, similar effects can occur as a result of high frequency mitotic failures (45Kops G.J. Weaver B.A. Cleveland D.W. Nat. Rev. Cancer. 2005; 5: 773-785Crossref PubMed Scopus (922) Google Scholar). Therefore, we sought to distinguish between the contribution of DNA replication and mitotic progression to the increased ploidy of RB-deficient cells. To this end, we used the microtubule-destabilizing agent nocodazole to block mitotic progression in RB-proficient and RB-deficient cells. In this manner, any changes in ploidy by definition would be the specific consequence of reinitiation of DNA replication. Under this condition, nocadazole significantly inhibits DNA replication in RB-proficient cells as detected by flow cytometry, and cells accumulate with 4 n DNA as expected, with minimal cells harboring >4 n DNA content (Fig. 3A). In contrast, RB-deficient cells failed to arrest following exposure to nocodazole. Furthermore, in the RB-deficient condition, ∼20% of cells harbored >4 n DNA content (Fig. 3A), indicating that DNA rereplication does occur in these cells and enhances cell ploidy. Additionally, nocodazole-treated RB-proficient cells showed a decrease in BrdUrd incorporation, indicating cessation of DNA synthesis, whereas the RB-deficient cells bypassed the mitotic block and exhibited continued BrdUrd incorporation, indicating active, ongoing DNA synthesis (Fig. 3B, top). Determination of the percentage of BrdUrd-positive cells demonstrated that treatment with nocodazole resulted in a 3-fold decrease in the BrdUrd incorporation in RB-proficient cells, whereas the RB-deficient cells retained comparable levels of BrdUrd-positive cells either in the absence or presence of nocodazole (Fig. 3B, bottom). The aberrant ploidy occurring in RB-deficient cells was accompanied by a significant increase in micronuclei that were largely positive for BrdUrd incorporation (Fig. 3B, compare nuclei in nocodazole-treated GFP and CRE images). The numerical increase in micronuclei seen upon RB loss was further exacerbated by nocodazole treatment (Fig. 3C). Thus, RB deficiency enables DNA replication to proceed under inappropriate conditions and contributes to increased ploidy. To determine the mechanistic basis for the observed DNA rereplication, we evaluated the influence of nocodazole on the levels of DNA replication factors in RB-proficient and RB-deficient cells. In cells containing functional RB, treatment with nocodazole resulted in diminished levels of MCM7 and PCNA in both total and chromatin-associated fractions (Fig. 4A, compare lane 1 with lane 2 and lane 5 with lane 6). In contrast, the chromatin-tethered levels of MCM7 and PCNA in RB-deficient cells were retained at high levels relative to RB-proficient cells (Fig. 4A, compare lane 2 with lane 4 and lane 6 with lane 8). Additionally, nocodazole treatment did not diminish levels of these proteins in RB-deficient cells (compare lane 3 with lane 4 and lane 7 with lane 8). Evaluation of MCM7 and PCNA levels and their association with chromatin by fluorescence microscopy revealed that nocodazole-treated RB-proficient cells show decreased levels of total and tethered replication proteins (GFP in Fig. 4, B and C). However, RB loss resulted in the retention of chromatin association of these proteins even in the presence of nocodazole (CRE in Fig. 4, B and C). These results concur well with the biochemical fractionation experiments shown in Fig. 4A. Another feature of nocodazole-treated RB-deficient cells was the increased presence of micronuclei. Analysis of such cells by immunofluorescence demonstrated that they also show significant levels of MCM7 (Fig. 4D). This finding illustrates the functional consequence of RB loss on genomic integrity. Collectively, these data indicate that RB deficiency results in the abrogation of nocodazole-induced cell cycle arrest and inappropriate retention of DNA replication factors onto chromatin. Consequently, RB-deficient cells undergo DNA synthesis under nonpermissive conditions, thus leading to aberrant increases in cell ploidy and genome instability. Ectopic Cdc6 or Cdt1 Expression Does Not Recapitulate the Effects of RB Loss—The loading of MCMs onto chromatin to assemble a functional prereplication complex is dependent on the activity of Cdc6 and Cdt1, since these two proteins recruit the MCMs to sites of DNA replication (46Eward K.L. Obermann E.C. Shreeram S. Loddo M. Fanshawe T. Williams C. Jung H.I. Prevost A.T. Blow J.J. Stoeber K. Williams G.H. J. Cell Sci. 2004; 117: 5875-5886Crossref PubMed Scopus (67) Google Scholar). As shown in Fig. 1E, the levels of these proteins are significantly elevated in RB-deficient cells. These" @default.
• W2027312705 created "2016-06-24" @default.
• W2027312705 creator A5005306449 @default.
• W2027312705 creator A5022171301 @default.
• W2027312705 creator A5029801038 @default.
• W2027312705 creator A5068495574 @default.
• W2027312705 creator A5074739271 @default.
• W2027312705 date "2007-08-01" @default.
• W2027312705 modified "2023-10-06" @default.
• W2027312705 title "RB Loss Promotes Aberrant Ploidy by Deregulating Levels and Activity of DNA Replication Factors" @default.
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