The overall goals of this proposal are to elucidate the mechanisms of cytotoxicity and resistance to taxol in human myeloid leukemia versus normal bone marrow progenitor cells (NBMPC). These studies are based on our recent findings that taxol induces the gene directed and active form of apoptotic death in human leukemic cells, which is significantly retarded in the leukemic cells over-expressing BCL-2 oncogene. In addition, leukemic cells over-expressing MDR-1 gene encoded membrane P-glycoprotein (PGP) are resistant to intracellular taxol accumulation and apoptosis. In the proposed studies we will examine whether clinically achievable concentrations of taxol produce the internucleosomal DNA fragmentation and morphologic features of apoptosis in patient derived AML blasts. Taxol induced apoptosis along with the intracellular microtubular bundling and cell cycle G2/M arrest will be correlated with taxol mediated inhibition of the clonogenic survival of AML blasts. Modulation of apoptosis by previously described alterations in protein phosphorylations will also be tested to determine their role in the molecular signalling of taxol induced apoptosis. This may also indicate whether a specific modulation of protein phosphorylation would augment taxol induced apoptosis in AML blasts. BCL-2 and c-myc expression in AML blasts will be determined and correlated with the degree of taxol induced apoptosis. Also, in a BCL-2 transfected and over-expressing pre-B leukemia cell line in which taxol induced apoptosis is significantly retarded, we will determine whether taxol produces intracellular microtubular bundling and cell cycle G2M arrest, and if so whether the growth arrested cells eventually die by apoptosis with declining levels of BCL-2 expression. To examine mechanisms of resistance to intracellular taxol accumulation and taxol induced apoptosis, we will sequentially examine patient derived AML blasts at diagnosis and relapse for MDR-1 and BCL-2 expressions. These will be correlated with taxol induced intracellular microtubule bungling, cell cycle G2/Marrest, apoptosis and the inhibition of clonogenic survival of AML blasts. In purified CD34+ NBMPC as well, we will examine taxol induced microtubule and cytokinetic changes and features of apoptosis. These will be correlated with BCL-2 and MDR-1 expression in NBMPC. Finally, we will compare the effects of hemopoietic growth factors (HGFs) G-CSF and pIXY 321 on taxol mediated inhibition of the clonogenic survival of normal CFU-GM and CFU- GEMM versus AML blasts (L-CFU). These studies will help elucidate the mechanisms of action and resistance to taxol induced apoptotic cell death in AML blasts versus NBMPC and establish the role of HGFs in reducing host bone marrow toxicity of taxol.
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