The long-term objective of this proposal is to clone and identify new genes which engender drug resistance in human tumors. Human tumors often display resistance to chemotherapeutic drugs in the clinic. In vivo tumor cell drug resistance can result from increased levels of drug resistance gene products via increased gene transcription or gene amplification. Unfortunately, clinical drug resistance cannot always be attributed to overexpression of known drug resistance genes. Therefore, it is probable that other, unidentified drug resistance genes also exist. Identification of new drug resistance mechanisms by finding new drug resistance genes can improve antitumor drug design, drug therapy, and understanding of normal cellular defenses. Recently, it has been demonstrated that a significant portion of amplified genes in human tumors are carried on acentric, circular extrachromosomal DNA molecules. Extrachromosomal DNAs lack centromeres, and are retained within a cell population via selective advantage of genes expressed on those structures. for these reasons, it is a logical strategy to isolate circular extrachromosomal DNa molecules from drug resistant human tumors and search them for drug resistance genes.
Specific Aim 1) of this proposal is to develop techniques for the cloning and sequencing of genes amplified and expressed on extrachromosomal DNAs in tumor cells. five human tumor cell lines which carry amplified genes on extrachromosomal DNAs will be used to develop strategies for electrophoretic isolation, PCR amplification, and sequencing of extrachromosomal DNA genes. Isolation strategies will exploit the physical/chemical differences between circular and linear DNAs.
Specific Aim 2) is to employ these techniques to clone and sequence genes expressed on extrachromosomal DNAs in drug-resistant human tumor tissues. Gene sequences will be compared with known gene sequences to identify new genes. Other drug resistant tumor tissues will be assayed for overexpression of new genes to identify relationships between gene expression and drug resistance.
