Date of Award

Summer 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Martin Carroll, MD

Abstract

Chronic myeloid leukemia (CML) is a two-stage disease caused by the p210 BCR-ABL protein. BCR-ABL is a constitutively activated tyrosine kinase that activates signaling pathways to enhance proliferation and inhibit apoptosis. BCR-ABL kinase activity directly causes CML chronic phase, the first stage of CML, but the role of BCR-ABL in transitioning to blast crisis (BC), the second stage of disease, remains under investigation. Given that CML BC is marked by additional chromosomal abnormalities, the role of BCR-ABL in contributing to genomic instability through effects on DNA damage and DNA repair processes has been studied. We investigated what impact BCR-ABL has on the development of DNA double strand breaks (DSBs) and mis-repair in a cell system that is unable to undergo apoptosis. We determined that the failure of cells to undergo apoptosis after DNA damage leads to genomic instability, which is not further increased by BCR-ABL expression. We expressed BCR-ABL in a mouse hematopoietic cell line null for the pro-apoptotic proteins Bax and Bak (DKO). Both DKO cells and the BCR-ABL-expressing cell line (DBA) fail to undergo apoptosis after γ-irradiation (IR). DKO cells are dependent on interleukin-3 (IL-3) for growth and proliferation, but expression of BCR-ABL renders them IL-3 independent. We compared the induction of DSBs after IR and determined that unlike apoptosis-competent cell lines, which demonstrate increased DSB formation in the presence of BCR-ABL, there was no difference in DSB formation comparing DKO to DBA cells. This suggests that the Bax/Bak-mediated induction of apoptosis may be important in the DNA damage response. We also evaluated the level of misrepair in DKO and DBA cells after IR using spectral karyotype (SKY) analysis. We determined that DKO and DBA cells showed a similar accumulation of new chromosomal abnormalities, including gains and losses of chromosomes as well as translocations, after IR and repair. These results suggest that the BCR-ABL-mediated effects on DNA damage and repair pathways may occur through inhibition of apoptosis rather than or in addition to direct effects on DNA repair pathways. We postulate that BCR-ABL shifts the apoptotic threshold, allowing a group of cells with an increased amount of damage to survive.

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