Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Fred Dick

Abstract

Cellular division is primarily controlled at the G1 to S-phase transition of the cell cycle by the retinoblastoma tumor-suppressor protein (pRB). The ability of pRB to restrict S-phase entry is primarily attributed to the repression of E2F transcription factors required to upregulate cell cycle target genes necessary for cellular division. Interestingly, while pRB is disrupted in the vast majority of human cancers, mutations typically target upstream regulators of pRB leading to inactivation through hyperphosphorylation. The rarity of direct pRB mutations suggests that the regulation of the cell cycle by pRB may involve additional mechanisms outside of E2F repression, as this could to be eliminated via point mutations. Indeed, the Rb1G/G mouse model developed by Cecchini et al., which lacks the ability to form pRB-E2F complexes, showed minimal phenotypic alterations. As described in chapter 2, pRB can stabilize p27 in the absence of pRB-E2F interaction, maintaining cell cycle control. Importantly, the loss of pRB-E2F interactions in addition to the loss of p27 leads to a defective DNA damage response, and ultimately pituitary tumor development. The minimal region of pRB necessary to elicit a cell cycle arrest is the pRB large pocket which contains 3 distinct binding surfaces. Using synthetic mutants of pRB we show that all three of these sites play a role in regulating the cell cycle both in vitro and in vivo. Finally, to understand E2F independent pRB-mediated tumor-suppression, Rb1G/G mice were intercrossed with mice harboring oncogenic KrasG12D, or deletions of p21 or p53. While KrasG12D expression induced tumorigenesis was unaffected by the Rb1G mutation, the phenotype of p53 null animals was exacerbated by the Rb1G mutation. Interestingly, the loss of p21 in Rb1G/G mice showed no tumor development despite the overlapping function with p27. While it is unclear why there is a discrepancy in phenotype between Rb1G/G mice lacking p21 and those lacking p27, p27 has non-canonical functions which may be contributing to tumor development. Taken together this work describes E2F independent functions of pRB in cell cycle control and tumor suppression and provides a rationale for the unusual disruption of pRB in human cancers by hyperphosphorylation.

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