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Loss of the RB tumor suppressor contributes to genomic instability

SRINIVASAN, SEETHA V.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212166350

Abstract Details

2008, PhD, University of Cincinnati, Medicine : Cell and Molecular Biology.
The retinoblastoma tumor suppressor RB, is functionally inactivated in many human cancers. Classically, it is known that RB ablation leads to deregulated G1 and S phase leading to uncontrolled cell proliferation and tumorigenesis. However, emerging evidence suggests that the role of RB in cancer is not limited to instituting the G1/S checkpoint and extends to other processes including genome integrity. Given this background, we set out to understand the role of RB in aspects of the cell cycle such as DNA synthesis and mitosis. We then tried to apply our findings to the context of multiple tumor suppressor losses to investigate if tumor suppressor functions are non-overlapping or redundant, specifically when it comes to maintaining the normal DNA content in the cell. We used mouse adult fibroblasts, MAFs, to demonstrate that aberrant DNA content in RB deficient cells occurs concomitantly with an increase in levels and chromatin-association of DNA replication factors. Furthermore, RB-deficient cells bypass the mitotic block induced by microtubule inhibitors and accumulate cells with higher ploidy and micronuclei. To examine the mechanistic basis of our observations, we exogenously expressed replication factors Cdc6 or Cdt1 in RB-proficient cells but that did not recapitulate the RB deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells did elevate ploidy and bypassed the response to nocodazole induced cessation of DNA replication in a manner analogous to RB loss. Collectively, the above results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy. To investigate the role of RB in mitosis, we examined the protein levels of mitotic markers and observed that RB loss elevated their expression. Furthermore, loss of RB decreased the overall time taken by cells to complete mitosis and cytokinesis. The mitotic data indicates that in addition to classic functions of RB where it plays a role in preventing hyperproliferation, the RB tumor suppressor also exerts control over several aspects of the cell cycle. We then proceeded to characterize the effect of combined RB and p53 loss on cell ploidy and nocodazole-mediated cell cycle arrest. The rationale behind these experiments was that some tumors harbor losses of both RB and p53. It is known that such tumors have novel phenotypes and are generally more aggressive than tumors that have lost either RB or p53 alone. We therefore hypothesized that in a cellular model, the combined loss of both these tumor suppressors would exhibit additive or even synergistic effects when compared to cells harboring loss of either RB or p53. Our results show that combined loss of RB and p53 results in an additive increase in cell ploidy following nocodazole treatment. Surprisingly, growth after drug removal showed that RB-deficient cells were best able to sustain the polyploidy population for extended periods when compared to p53-deficient or doubly deficient cells. Also, the double null cells did not revert to their pre-nocodazole cell cycle profile after drug removal. Together, these results emphasize that RB and p53 have a complex interplay of overlapping and non-redundant functions.
Erik Knudsen, PhD (Committee Chair)
Sohaib Khan, PhD (Committee Member)
Carolyn Price, PhD (Committee Member)
John Bissler, MD (Committee Member)
Kathleen Goss, PhD (Committee Member)
141 p.
Cellular Biology; Molecular Biology
RB; cell cycle; DNA replication; mitosis; p53; ploidy; genome integrity

Recommended Citations

Citations

  • SRINIVASAN, S. V. (2008). Loss of the RB tumor suppressor contributes to genomic instability [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212166350

    APA Style (7th edition)

  • SRINIVASAN, SEETHA. Loss of the RB tumor suppressor contributes to genomic instability. 2008. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212166350.

    MLA Style (8th edition)

  • SRINIVASAN, SEETHA. "Loss of the RB tumor suppressor contributes to genomic instability." Doctoral dissertation, University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212166350

    Chicago Manual of Style (17th edition)

ucin1212166350
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© 2008, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.