The aim of this application is to study the role of hematopoietic growth factors (HGFs) in improving the antileukemic selectivity of Ara-C, and to examine the biochemical and molecular mechanisms underlying this effect. These studies will expand on the applicant's previous findings that IL-3 and/or GM-CSF selectively enhance the metabolism and cytotoxicity of Ara-C against leukemic versus normal bone marrow progenitor cells.The effect of HGFs individually or in various combinations of early (stem cell growth factor, SCF), intermediate (IL-3 and GM-CSF) and lineage specific HGFs (G-CSF) will be examined on the cell cycle status (by flow cytometry) and the percentage of clonogenic S phase normal bone marrow progenitor cells (from healthy volunteers), AML blasts and established myeloid leukemia cell lines HL-60 and KG1. Following exposure to the HGFs in conjunction with Ara-C (1-100 uM) for different schedules and intervals, the applicant will compare the colony growth inhibition of normal (CFU-GM, CFU-GEMM and multipotent blast colonies) and leukemic (L-CFU) progenitor cells in semi-solid media. HGF combinations which in conjunction with Ara-C result in improved antileukemic selectivity of Ara-C will be examined for their effects on a variety of biochemical correlates of Ara-C cytotoxicity. These will include deoxycytidine kinase and deaminase activity, Ara-CTP accumulation, Ara-CTP/dCTP pool ratios (utilizing previously described HPLC methods) Ara-C DNA incorporation including the chain terminus position of Ara-C residues in normal and leukemic bone marrow elements. In addition, as a preliminary examination, the in vivo effects of GM-CSF on the intracellular metabolism of high dose Ara-C will be determined in the context of a laboratory companion study to a currently active cooperative group (CALGB) trial. To further explore the molecular mechanisms underlying the enhanced antileukemic activity of the combinations of HGFs and Ara-C, the applicant will examine his ability to produce programmed cell death (PCD) or apoptosis. Specifically, he will examine the ability of different doses and schedules of Ara-C with or without the HGFs to produce oligonucleosomal DNA fragmentation characteristic of PCD. These studies will also be performed in the presence of known modulators of protein kinase C (PKC) activity, phosphatase inhibitors and calcium ionophores. Ara-C mediated appearance of the characteristic ladder of DNA nucleosomal oligomers in agarose gel will be correlated with the altered expression on Northern blots of those genes which are known to regulate or are associated with the induction of PCD. These include bcl-2, c-jun, c-fos, c-myc and TGF-beta-1.These studies will explore the interaction of GHFs and Ara-C in activating PCD and correlate it with Ara-C DNA incorporation as well as the clonogenic survival of AML cells. The ultimate goal of these studies is to design rational antileukemic drug combinations of HGFs and Ara-C which will be based on an understanding of the biochemical and molecular mechanisms underlying the Ara-C mediated cytotoxicity.
