Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Kapil N. Bhalla

Second Advisor

Daniel Fernandes

Third Advisor

James Noris

Fourth Advisor

Clifford Schweinfest

Fifth Advisor

Robert Stuart

Sixth Advisor

Mark Wilingham

Abstract

High expression of p26Bcl-2 in patient-derived AML cells has been associated with poor response to chemotherapy including Ara-C. It has been well established that Bcl-2 over expression blocks apoptosis or programmed cell death induced by a wide variety of stimuli including chemotherapeutic drugs. However, the exact mechanism of action of Bcl-2 in promoting this blockade is still largely unknown. To determine the types of Ara-C-induced DNA damage with which Bcl-2 specifically interferes, HL-60/neo and HL-60/Bcl-2 cells were created via retroviral-mediated transfection of the bcl-2 gene. These transfectants served as in vitro AML cell models that expressed different levels of p26Bcl-2. The clone with the highest Bcl-2 levels contained 5- to 10-fold greater Bcl-2 by Western blot and immunofluorescence as compared to HL-60/neo cells. HL-60/Bcl-2 cells are resistant to Ara-C-induced apoptosis, which was evident in HL-60/neo cells as internucleosomal and high molecular weight DNA fragmentation, as well as by the activation of the cysteine protease cascade of apoptosis. Differences in apoptosis in the two cell types was correlated with differences in the loss of cell viability as measured by the MTT (3-[3,5-dimethylthiazol-2-yl]-2,5-diphenyltetrasolium bromide) assay. Proximal steps in Ara-C metabolism including intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation, Ara-C-induced DNA strand breaks (by the alkaline elution assay) and Ara-C-induced inhibition of DNA synthesis (by measurement of [H3]-TdR incorporation), were not significantly different between HL-60/neo and HL-60/Bcl-2 cells. Bcl-2 expression was studied in cells surviving over time after Ara-C treatment. Various clones of HL-60/Bcl-2 cells were generated by limiting dilution of transfected HL-60/Bcl-2 cells. Regardless of their endogenous Bcl-2 levels, following Ara-C treatment, the non-apoptotic (surviving) cells of various clones exhibited further transcriptional up-regulation of bcl-2 mRNA and p26Bcl-2 levels detected by RNase protection assay, and Western blot analysis or flow cytometry, respectively. HL-60/neo cells that had survived the initial Ara-C treatment were exposed to a second dose of Ara-C, to determine whether the induction in the Bcl-2 levels in these cells were biologically relevant. This was confirmed by demonstrating a reduced reduced cytotoxicity of the second dose of Ara-C by the MTT assay. Whether increased Bcl-2 levels in HL-60/Bcl-2 cells promote increased repair of non-lethal Ara-C-induced DNA damage was also addressed. Following Ara-C treatment, HL-60/neo as well as HL-60/Bcl-2 cells exhibited equivalent rates of repair of non-lethal DNA damage, as assessed by the comparisons of unscheduled DNA synthesis, and assessment by PCR of the repair of the damage of the c-myc genomic DNA template. Therefore, these data indicate that while Bcl-2 does not block early steps of Ara-C metabolism, Ara-C-induced DNA damage or its repair, it does block the induction of Ara-C-induced apoptosis by inhibiting the conversion of Ara-C-induced early and potentially reparable DNA damage into lethal DNA fragmentation characteristic of apoptosis.

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