The applicant's central hypothesis is that a patient's therapy selects intrinsically resistant cells that share genetic traits which confer a selective advantage enabling such cells to repopulate the tumor mass. The identification of these traits will provide targets of opportunity for improved therapeutic intervention. She has previously demonstrated that some cells selected in vitro or in vivo for resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) retained an overrepresentation of chromosomes 7 and 22. This is in contrast to the dominant cell type in the population before BCNU selection that consists of cells with overrepresentation of chromosome 7 and under representation of chromosome 22. Fluorescent in situ hybridization (FISH) and chromosome painting techniques demonstrated that many recurrent glioma cells that appear to lack 7/22 polyploidy by standard banding techniques contain additional fragments of these chromosomes in marker chromosomes. Genes known to cause BCNU resistance are not mapped to either of these chromosomes. There is also no predominant mechanism responsible for BCNU resistance in gliomas, and some tumors do not appear to use any of the classically known mechanisms of BCNU resistance suggesting that there are mechanisms of resistance yet to be defined. The applicant proposes to use matched primary/recurrent tumor samples from the same patient to expand on these observations, and to define the genetic and molecular characteristics of human malignant gliomas that lead to BCNU resistance. She will define the region(s) of chromosome 22 that is retained using bacterial artificial chromosomes (BACs) that span the length of chromosome 22 as probes for molecular and molecular cytogenetic analysis (Specific Aim 1). She will also use differential mRNA display to identify genes whose expression is altered in recurrent tumor relative to primary tumor (Specific Aim 2). Genes identified in Specific Aims 1 and 2 will be directly assessed for BCNU resistance and their cDNAs will be cloned in the forward and reverse directions into the pIRES-EGFP or pIRES-EYFP bicistronic expression vectors and transfected into sensitive and resistant glioma cells (Specific Aim 3). The clinical significance of these findings will be validated in fresh frozen and paraffin embedded samples by analyzing tissue from their extensive tumor bank (Specific Aim 4).
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