PROTEIN KINASE C ISOTYPES IN LEUKEMIA CELL PHYSIOLOGY

The protein kinase C enzyme family is involved in a number of cellular functions, including the control of cellular proliferation and differentiation. Human erythroleukemia (K562) cells express three PKC isotypes, alpha PKC, betaII PKC and zeta PKC. These cells can be induced to differentiate along the megakaryocytic pathway upon exposure to the PKC activator phorbol myristate acetate e (PMA). Expression of the three PKC isotypes are differentially regulated during PMA-induced cytostasis and differentiation. Alpha and zeta PKC levels increase significantly and beta II PKC levels fall dramatically upon PMA treatment. Therefore, alpha and zeta PKC levels correlate with PMA-induced cytostasis and differentiation, whereas beta II PKC levels correlate with the proliferative capacity of the cells. In order to explore isotype-specific function, we assessed the effect of overexpression and anti-sense inhibition of expression of the alpha and beta II PKC isotypes on the proliferation and differentiation potential of k562 cells. Over expression of alpha PKC leads to a gene dose-dependent decrease in the rate of proliferation and increased responsiveness to PMA whereas anti-sense inhibition of alpha PKC expression leads to relative resistance to PMA-induced cytostasis. Conversely, overexpression of betaII PKC leads to relative resistance to PMA-induced cytostasis whereas anti-sense inhibition of beta II PKC expression blocks K562 cell proliferation. these results indicate that alpha PKC is directly involved in the cytostatic and differentiative effects of PMA whereas betaII PKC is required for K562 cell proliferation. Through the use of domain switch alpha beta II PKC chimera, we have identified regions on alpha and beta II PKC required for isotype-specific function. The goals of this proposal are to i) determine the role of zeta PKC in K562 cell proliferation and differentiation through overexpression and anti-sense inhibition of expression of the enzyme, ii) define minimal molecular determinants capable of mediating the isotype specific functions of alpha and beta II PKC in-vivo through expression of chimeric alpha beta II PKC in K562 cells and iii) identify and characterize cellular proteins involved in isotype-specific signal transduction in K562 cells through interactive cloning strategies. Completion of these aims will provide important new insight into the molecular basis for PKC isotype specific function in-vivo and identify molecular targets for PKC isotype-specific signalling. Furthermore, since anti-proliferative and differentiation therapy is an important modality for the clinical treatment of human leukemia, the proposed studies may identify molecular targets i human leukemic cells of therapeutic importance.