GENE REGULATION OF IONIZING RADIATION-INDUCED APOPTOSIS

Virginia Commonwealth University, Richmond VA

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Abstract

DESCRIPTION: (Applicant's Description) The goal of this proposal is to modulate gene expression and the PKC signal transduction pathway in order to circumvent protection of human myeloid leukemia cells from radiation- induced cell death by the anti-apoptotic protein Bcl-2. To this end, human leukemia cell lines (HL-60 and U937-) stably over-expressing Bcl-2 have been isolated which exhibit significant resistance to radiation related apoptosis compared to their empty-vector counterparts. Attempts will be made to establish whether bryostatin 1, which down-regulates PKC, and UCN- 01, which directly inhibits the enzyme, can reverse radio-resistance in Bcl-2-over-expressing cells. Phosphorothioate antisense oligonucleotides (AS-ODN) will also be employed to reduce Bcl-2 expression and enhance radiosensitivity; parallel studies will examine whether exposure of cells to the combination of AS-ODN and bryostatin 1 or UCN-01 results in further potentiation of radiation-induced apoptosis and loss of clonogenic survival. The possibility that differentiation induction following radiation-induced DNA damage results in enhance apoptosis will also be tested by exposing irradiated cells to bryostatin 1 +/- the [Ca2+] I modulators A23187 and AS101. The role of Raf-1-mediated Bcl-2 phosphorylation in bryostatin 1 and UCN-01 actions will be explored, as will be the contribution of p21WAF1/CIP1 to radiation-induced lethality using newly generated p21 antisense-expressing mutants. Finally, the ability of bryostatin 1 to enhance the in vivo therapeutic efficacy of radiation will be assessed using a murine leukemia model. It is hoped that information emanating form these studies will lead to the development of entirely novel therapeutic strategies designed to improve the efficacy of TBI in patients with leukemia undergoing bone marrow transplantation.