Rb Inactivation Leads to E2F1-mediated DNA Double Strand Break Accumulation: A Dissertation

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 ofRb by RNA interference or expression of the E7 viral oncoprotein from human papilomavirus (HPV), and the resultant deregulation of one E2F family member, E2Fl , leads to DNA double strand break (DSB) accumulation. These DSBs occur independent of apoptosis induction, and activation of ATM, NBSl , p53 , or MAD2 , and generation of reactive oxygen species (ROS). Although ROS accumulation is associated with DSBs generated from the misexpression of another nuclear oncoprotein, c-MYC, we find that E2Fl does not contrbute to c-MYC associated DSBs, indicating that theDSBs associated with these oncoproteins arise through distinct pathways. However, we find that small changes in E2F 1 levels by inhibition of c-MYC trans activated microRNAs known to limit E2Fl protein expression lead to DSB accumulation. These results suggest that despite the DSBs arising by different mechanisms , c MYC assists in the regulation ofE2Fl-associated DSBs. We also find elevated levels of E2F 1associated DSBs in Rb mutated cancer cell lines in the absence of an exogenous DSB stimulus. These basal, E2F I-associated DSBs are substantially lower in Rb wildtype cancer cell lines that have p 16 ink4 inactivated or express HPV E7. However, we show that we can manipulate DSB levels in these cancer cell lines by modulating Rb and E2Fl activity and suggest that these results may be extended to breast tumor organ cultJre. Thus Rb status is key to regulating both the proliferation promoting fuctions associated with E2F and for preventing DNA damage accumulation if E2Fl becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2Fl might contrbute to the genetic instability associated with transformation and tumorigenesis.