Previous studies on heterogeneity of human gliomas have increased our understanding of the natural evolution of such tumors and how therapy changes that evolution. They have also defined for the first time in a human tumor regional heterogeneity and its associated regional chemosensitivity. We now propose to test the hypothesis that contained within the heterogeneous tumor are regionally distributed foci of chemotherapy-resistant cells (BCNU and cisplatin) with specific chromosomal abnormalities. Cytogenetic studies indicate that the resistant cells are near-diploid and have over-representation of chromosomes 7 and 22. These chromosomes contain the proto-oncogenes c-erb-B and c-sis, respectively coding for epidermal growth factor receptor (EGFR) and platelet derived growth factor (PDGF). It is proposed that as therapy kills off the sensitive cells, these resistant cells re-populate the tumor, using to their selective advantage the gene-products (growth factors) of their specific chromosomal complements. We shall dissociate low- and high-grade gliomas, karyotype the cells to determine the Reference Set, and treat the primary and early passage cells with BCNU using colony forming assay (CFA). The resistant (surviving) cells are cloned and their cytogenetics are compared with those of the Reference Set of karyotypes. Regions of tumors removed in toto will undergo the same experiments, comparing the results from region to region. To define the behavior of the resistant cells, the plan is to determine if polyploidy of chromosomes 7 and 22 is a form of gene amplification, to assess mRNA encoded by the genes to determine if they are active, and to determine the receptor concentration for EGF and PDGF and the amount of PDGF secreted by the cells. To test the hypothesis that continued and increasing dosage of BCNU causes the amplification of specific intracellular proteins associated with drug resistance, we shall determine by 2-dimensional gel electrophoresis (2-D), is such proteins are amplified in patients with tumors recurrent after BCNU therapy. Using glioma clones made resistant to BCNU, probes will be developed to these proteins to determine if they are present in tumors from untreated patients and if they are involved in intrinsic resistance.
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