Riordan, Fiona Anne; (1998) The role of protein kinases and protein phosphatases in the regulation of leukaemic cell apoptosis. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Although the importance of apoptosis in the field of cancer therapy is known, the signalling mechanisms underlying this process were largely uncharacterized at the commencement of this study. Therefore, in these studies, inhibitors of protein kinases and protein phosphatases were employed to investigate the role of these enzymes in the induction of apoptosis in leukaemic cells. Low concentrations of the serine/threonine phosphoprotein phosphatase (PP) inhibitor okadaic acid (OKA) induced apoptosis in proliferating cell lines and in quiescent cells from chronic lymphocytic leukaemia (CLL) patients. However, later features of apoptosis such as nuclear condensation were abrogated by treatment with N-benzyloxycarbonyl- Val-Ala-Asp-(0-methyl)fluoromethane (ZVADfmk), an inhibitor of interleukin-1- -converting enzyme (ICE)-like proteases suggesting that the down-regulation of phosphoprotein phosphatases results in ICE protease activation. Bcl-2 protein and messenger RNA (mRNA) were decreased following treatment of HL60 cells with OKA. Therefore, I determined whether bcl-2 promoter activity was altered by OKA treatment. A decrease in the read-out from a bcl-2 promoter construct was not observed, suggesting that alterations in bcl-2 mRNA stability and/or protein half-life could mediate the decrease in bcl-2 protein expression seen in HL60 cells treated with OKA. Conversely, treatment of the HL60 cell line and cells from CLL patients with y irradiation and etoposide resulted in an initial rise in PP2A-like activity which declined within several hours. Therefore, the activation of some phosphatases and the inhibition of others may have roles in the induction of apoptosis in leukaemic cells. The deregulated protein tyrosine kinase activity of the bcr/abl fusion oncoprotein, found on the Philadelphia (Ph) chromosome, abrogates apoptosis following treatment of Ph+ve leukaemic cells with DNA damaging agents. I observed that the bcr/abl-selective protein kinase inhibitor herbimycin A (HMA) synergized with etoposide or with y irradiation in several Ph+ve cell lines by dramatically increasing the induction of nuclear apoptosis and DNA fragmentation caused by these agents alone. Conversely, in Ph-ve cell lines, HMA treatment either protected cells from apoptosis or failed to affect killing induced by cytotoxic agents. Selective protein tyrosine kinase inhibitors may be of value in securing the death of Ph+ve leukaemia cells.

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