The overall objective is to determine the molecular basis for selective antitumor activity of the dimeric Catharanthus alkaloids, vincristine (VCR) and vinblastine (VLB). Experiments will be designed to elucidate the biochemical basis for selective retention of VCR in human rhabdomyosarcomas (RMS) sensitive in vivo, but not in resistant lines, or host (mouse) limiting tissues. Cytosols will be prepared from RMS growing as xenografts in mice. The pool of unpolymerized tubulin will be determined, as will the association constants for VLB and VCR using Scatchard analysis. Isoforms of Alpha and Beta-tubulin will be identified using gel electrophoresis and monoclonal antibodies. Similar analyses will be carried out using normal tissues, where stable VCR-protein complexes are formed. Human RMS growing as xenografts, have been selected for resistance to VCR under clinically relevant conditions. One line (Rh18/VCR-3) characterized exhibits quantitative and qualitative changes in Beta-tubulin isoforms, and decreased retention of VCR. In order to determine whether these changes are a consequence of altered gene expression, mRNAs for Beta-tubulin will be isolated and characterized using 32p-cDNA homologous to Beta-tubulin mRNA. Beta-Tubulin will be hybridized to cDNA bound to filters, eluted and translated in vitro. Translation products from parent and resistant lines will be compared to soluble forms of Beta-tubulins present in these tumors. Rh12/VCR, also selected for resistance in vivo, will be characterized with respect to VCR retention, the expression of tubulins and compared to its parent line, which is exquisitely sensitive to VCR. In cytosols prepared from mouse intestine, liver, spleen and kidney, failure to retain VCR may be due to proteolytic cleavage of tubulin dimer, causing release of the ligand. This 'protease' activity will be characterized with respect to substrate specificity (using 125I-iodinated tubulin, -intermediate filaments and -neurofilaments), Ca++ dependence, pH optimum, and tissue distribution. Its relevance to the rapid efflux of VCR from tissues, and regulation of tubulin pools will be examined. Vinca alkaloids form the backbone of chemotherapy of childhood RMS, yet the cellular pharmacology, basis for selectivity, and mechanisms of cellular resistance are poorly understood. The RMS xenograft model is unique in allowing the study of changes associated with acquired resistance to VCR in human cancers, selected under controlled conditions in vivo. The phenotypes of such resistant lines may be of relevance to resistance in human cancers.
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