RB silencing compromises the DNA damage-induced G2/M checkpoint and causes deregulated expression of the ECT2 oncogene

Abstract

As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB( þ )-and (À)-matched cells using short hairpin RNA. In the RB(À) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(À) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(À) cells resulted in cytokinesis arrest, suggesting that RB(À) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

“…27,28 Proper RB function is required for maintenance of G2/M arrest following DNA damage and disruption of RB accelerated G2/M progression in the presence of DNA damage by elevating E2F activity and the expression of a mitotic regulatory genes. 29 Our results demonstrated that CARF suppression led to hypophosphorylated RB and downregulation of E2F1, suggesting that CARF inhibition may also activate this pathway to bring about growth arrest. However, although Saos-2 cells, which lack RB, were resistant to CARF-inhibition induced cell death, restoration of RB to Saos-2 did not revert the phenotype, which indicated that RB is not sufficient to revert the apoptosis induced by CARF suppression.…”

E2F4 Function in G2 Maintaining G2-arrest to Prevent Mitotic Entry with Damaged DNA

“…Our results are consistent with previous studies based on combined cross-linking DNA immunoprecipitation and microarray experiments, 52 which have identified some of the same targets we have uncovered as E2F4 target genes with a putative role in various phases of the cell cycle including G 2 and M. 53 Similar targets were independently identified in studies examining specific p130 or Rb-regulated gene expression. 24 At the same time, Rb-deficient U2OS cells showed an analogous dependence on E2F4-regulated genes, 48 suggesting a context-dependence for p130 or Rb requirement for regulating mitotic entry. From our results and other studies we conclude that in response to DNA damage, these tumor Figure 3.…”

“…56,57 Interestingly, a number of E2F4-bound genes originally identified through array analyses were also bound by E2F1, 53 an observation extended to the promoters of genes regulated during G 2 and M containing distinct E2F4 and E2F1 binding sites. 52,58 Some of these promoters may display collaborative binding of several E2F family members to the same elements, such as in case of ect-2, 48 while others distinct positive and negative E2F-dependent regulatory elements. 58 In addition, E2F-dependent transcriptional activity may require binding of other transcription factors, such NF-Y or Myb to regulate the transcription of Cdk1.…”

“…Stathmin, 47 HEC, and Ect-2, are involved in spindle formation. 48 The abundance of these targets and their individual role proposed in the DNA damage response is continuously expanding. For example, p130 regulates telomere length by interacting with RINT-1, previously identified as a Rad50-interacting protein.…”

RB's original CIN?: Figure 1.

“…Accumulating evidence has indicated that loss of RB function may play a role in this process; for example, by inducing defects during the replication of DNA, and potentially by causing abnormal segregation of chromosomes during mitosis (Kennedy et al 2000;Foijer et al 2005;Eguchi et al 2007). Embryonic stem cells (ESCs), which are largely devoid of a G1 checkpoint, provide a good system to investigate a potential role for the RB pathway in G2/M (Conklin and Sage 2009).…”