RB loss promotes aberrant ploidy by deregulating levels and activity of DNA replication factors.
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.
Srinivasan
Seetha V
SV
Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, Ohio 45267, USA.
Mayhew
Christopher N
CN
Schwemberger
Sandy
S
Zagorski
William
W
Knudsen
Erik S
ES
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D050684:E2F Transcription Factors / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation; D051379:Mice; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000235:genetics*; D011003:Ploidies; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D016160:Retinoblastoma Protein / Q000172:deficiency*; D016196:S Phase
C103706:CDC6 protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D016160:Retinoblastoma Protein; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7