Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Frederick A Dick

Abstract

Condensation and segregation of mitotic chromosomes are critical processes for cellular propagation and if compromised, can lead to genomic instability. Genomic instability is known to be an active contributor to tumorigenesis, rather than being a by-product of malignant progression. The retinoblastoma protein (pRB) is the prototypic tumor suppressor. Its tumor suppressive properties are linked to its ability to negatively regulate proliferation by inhibiting E2F target gene transcription. Using a gene targeted mouse model defective for interactions mediated by the pRB LXCXE binding cleft that is distinct from E2F binding (Rb1ΔL/ΔL), I have demonstrated that LXCXE-interactions are an essential part of pRB-mediated tumor suppression. When these interactions are disrupted, cells exhibit chromosome condensation and mitotic defects that are unrelated to G1 to S-phase regulation by pRB. These defects contribute to earlier tumor formation and more aggressive pathology in Trp53+/- and Trp53-/- mouse models, revealing a new mechanism of tumor suppression, facilitated by pRB, whereby genome stability is maintained by the proper condensation of mitotic chromosomes. Subsequent study of the mechanism by which pRB facilitates genome stability suggests that a pRB-E2F1-Condensin II complex localizes to pericentromeric heterochromatin. In the absence of this complex, DNA double strand breaks are observed and persist into mitosis and the ensuing G1 phase of the cell division cycle. Moreover, haploinsufficiency of Rb1 was enough to compromise loading of Condensin II at pericentromeric DNA and elicit the same defects. Significantly, RB1+/- fibroblasts from retinoblastoma patients also exhibit DNA damage and mitotic errors. And, in cancers of mesenchymal origin, RB1+/- cells exhibit as much genomic instability as RB1-/- cells. Finally, haploinsufficiency of the LXCXE binding cleft of pRB compromises pRB-mediated tumor suppression, resulting in tumors with increased chromosome gains and losses, comparable to Rb1ΔL/ΔL mutant mice. The data presented in this thesis change our understanding of the importance of genome stability as a tumor suppressive mechanism of the retinoblastoma protein and contrary to traditional thought, suggests that haploinsufficiency of RB1 functionally contributes to tumorigenesis in humans.

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