Date of Completion
Graduate School of Biomedical Sciences, Program in Immunology/Virology
Apoptosis; Cell Cycle Proteins; Retinoblastoma Protein; Signal Transduction; Transcription Factors; Tumor Suppressor Proteins; Academic Dissertations; Dissertations, UMMS
Proper regulation of cellular proliferation is critical for normal development and cancer prevention. Most, if not all, cancers contain mutations in the Rb/E2F pathway, which controls cellular proliferation. Inactivation of the retinoblastoma protein (Rb) can occur through Rb loss, mutation, or inactivation by cellular or viral oncoproteins leading to unrestrained proliferation. This occurs primarily by de-repression and activation of the E2F transcription factors, which promote the transition of cells from the G1 to S phase of the cell cycle. In order to protect against loss of growth control, the p53 tumor suppressor is able to induce programmed cell death, or apoptosis, in response to loss of proper Rb cell cycle regulation.
E2F1 serves as the primary link between the Rb growth control pathway and the p53 apoptosis pathway. While the pathway(s) linking E2F1 to p53 activation and apoptosis are unclear, it has been proposed that E2F1 activates p53-dependent apoptosis by transactivation of p19ARF leading to inhibition of Mdm2-promoted degradation of p53. We tested this hypothesis, and found that p19ARF is not required for E2F1-induced apoptosis. Instead, we find that expression of E2F1 leads to covalent modifications of p53 that correlate with p53 activation and are required for apoptosis.
The observation that E2F1 induces covalent modification of p53 is consistent with the p53 modifications observed following DNA damage. We therefore hypothesized that E2F1 may be activating components of the DNA damage response to activate p53 and kill cells. Consistent with the DNA damage response, we find that E2F1-induced apoptosis is compromised in cells from patients with the related disorders ataxia telangiectasia and Nijmegen breakage syndrome, lacking functional Atm and Nbs1 gene products, respectively. E2F1-induced apoptosis and p53 modification also requires the human checkpoint kinase Chk2, another component of the DNA damage response. We find that the commitment step in E2F1-induced apoptosis is the induction of Chk2.
Having found that E2F1 requires DNA damage kinases to induce apoptosis, we next examined events upstream of kinase activation. To this end, we observe relocalization of the DNA damage repair MRN complex (composed of Mre11, Nbs1, and Rad50) to nuclear foci specifically following expression of E2F1. Expression of E2F1 also induces relocalization of the DNA damage recognition proteins γH2AX and 53BP1 to nuclear foci, consistent with the location of these complexes observed following DNA double strand breaks. As a consequence of activating some or all of these DNA damage signaling proteins, expression of E2F1 blocks cell cycle progression in diploid human fibroblasts. The observed block in cell cycle progression is found to be, in part, due to activation of a p21-dependent cell cycle checkpoint.
The E7 protein from the oncogenic human papillomavirus (HPV) is able to bind to and inactivate members of the Rb family. HPV infects quiescent, non-cycling cells that lack expression of DNA replication machinery that is essential for replication of the viral genome. By expression of the E7 protein, HPV is able to bypass normal Rb-mediated growth control and induce quiescent cells to enter S phase where the host cell DNA replication enzymes are present for viral replication. We find that expression of E7 can also result in apoptosis that is dependent specifically on E2F1. Additionally, E7-induced apoptosis, like E2F1-induced apoptosis, requires Atm, Nbs1, and Chk2. Expression of E7, like that of E2F1, induces E2F1-dependent covalent modification of p53 that correlates with apoptosis induction.
These findings demonstrate that deregulation of the Rb/E2F growth control pathway leads to activation of an apoptosis program with some similarity to the pathways activated by DNA damage. Our observations suggest that E2F1 not only functions as a sensor for deregulation of Rb, but may also play an important role in regulating cellular growth control in response to other oncogenic stimuli.
Rogoff, HA. Pathways Linking Deregulated Proliferation to Apoptosis: a Dissertation. (2004). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 63. http://escholarship.umassmed.edu/gsbs_diss/63
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