Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Cancer Biology

Major Professor

Thomas M. Guadagno, Ph.D.

Keywords

SiRNA, Spindle, Centrosome, Kinetochores, Aneuploidy

Abstract

The MAP kinase pathway is well known for its key roles in regulating cell proliferation and cell cycle progression. MAP kinases have also been implicated in mitotic functions, however these functions are less-well understood. Recent studies from our laboratory used Xenopus egg extracts to identify B-Raf as an essential activator of the MAPK cascade during mitosis. Therefore, the first objective of my dissertation research was to determine if B-Raf has functional significance during mitosis in human somatic cells. Using RNA interference against B-Raf and various immunofluorescence techniques, I show that B-Raf: (1) localizes to and is phosphorylated at a key mitotic structure, (2) is critical for proper mitotic spindle assembly and chromatin congression, (3) is important for the engagement of microtubules with kinetochores during mitosis, and (4) is necessary for activation of the spindle assembly checkpoint. It has been demonstrated that B-Raf is a prominent oncogene, constitutively activated in the vast majority of melanomas and other cancers. I hypothesized that oncogenic B-Raf expression perturbs mitosis and causes aneuploidy. First, we show that oncogenic B-Raf expression correlates with mitotic abnormalities in human melanoma cells and that spindle defects are induced when oncogenic B-Raf is ectopically expressed. Further, using FISH and karyotype analysis, I demonstrate that oncogenic B-Raf drives aneuploidy and chromosome instability in primary, immortalized, and tumor cells. In summary, my dissertation studies elucidate novel roles for B-Raf in mammalian mitosis. In addition, my studies show for the first time that oncogenic B-Raf disrupts mitosis causing chromosomal instability. I propose that oncogenic B-Raf-induced chromosome instability contributes to tumorigenesis.

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