Graduate School of Biomedical Sciences
DNA Damage; E2F1 Transcription Factor; Genes, Retinoblastoma; Mutation; Academic Dissertations; Dissertations, UMMS
Life Sciences | Medicine and Health Sciences
Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that most, if not all, cancers have mutations that inactivate the Rb-mediated growth control pathway. We show here that acute inactivation of Rb by RNA interference or expression of the E7 viral oncoprotein from human papillomavirus (HPV), and the resultant deregulation of one E2F family member, E2F1, leads to DNA double strand break (DSB) accumulation. These DSBs occur independent of apoptosis induction, and activation of ATM, NBS1, p53, or MAD2, and generation of reactive oxygen species (ROS).
Although ROS accumulation is associated with DSBs generated from the mis-expression of another nuclear oncoprotein, c-MYC, we find that E2F1 does not contribute to c-MYC associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. However, we find that small changes in E2F1 levels by inhibition of c-MYC transactivated microRNAs known to limit E2F1 protein expression, lead to DSB accumulation. These results suggest that despite the DSBs arising by different mechanisms, c-MYC assists in the regulation of E2F1-associated DSBs.
We also find elevated levels of E2F1-associated DSBs in Rb mutated cancer cell lines in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are substantially lower in Rb wildtype cancer cell lines that have p16ink4 inactivated or express HPV E7. However, we show that we can manipulate DSB levels in these cancer cell lines by modulating Rb and E2F1 activity and suggest that these results may be extended to breast tumor organ culture. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 might contribute to the genetic instability associated with transformation and tumorigenesis.
Pickering, Mary Theresa, "Rb Inactivation Leads to E2F1-mediated DNA Double Strand Break Accumulation: A Dissertation" (2006). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 26.