Genetic Analysis of the Multidrug Resistance Phenotype i

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Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol, and to the synthetic drug cisplatin. In both cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene or other members of the ABC transporter family. For cisplatin, cross-resistance to methotrexate, some nucleoside analogs, heavy metals, and toxins is due to a reduction in drug influx resulting from a pleiotropic defect in uptake systems. Recent evidence suggests a global defect in endocytosis in these cisplatin resistant cells and defects in intracellular protein trafficking, the cytoskeleton, and in glucose metabolism. Single-step cisplatin resistant mutants show a defect in protein trafficking which results in accumulation of cell surface receptors/transporters/channels in the cytoplasm; a putative cisplatin carrier/channel is presumed to be among these mislocalized proteins resulting in decreased cisplatin uptake. At higher levels of resistance, after multiple steps of selection in cisplatin, there is increased methylation of genes for binding proteins (e.g., folate binding protein), altered regulation of cytoskeletal proteins including filamin, actin, tubulin and g-catenin, and genes involved in regulation of glucose metabolism. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. We have developed a tet-repressible P-gp cell line and demonstrated that although reduced drug accumulation is a direct consequence of P-gp expression, many other phenotypes frequently attributed to P-gp expression, including altered membrane fluidity and membrane potential, are not due directly to P-gp. Common polymorphic variants of P-gp have been detected, but coding polymorphisms do not appear to alter the drug transport functions of P-gp. However, a synonymous polymorphism (no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping for reasons still under investigation. To explore the possibility that other members of the ABC family of transporters may be involved in drug resistance in cancer, we have developed real-time PCR and microarray technology for detection of most of the 48 known ABC transporters; these techniques have been used to correlate expression of novel ABC transporters in cancer cell lines of known drug resistance. Expression of approximately 30 ABC transporters has been shown to correlate with resistance to specific cytotoxic drugs. Furthermore, some drugs are more toxic to P-gp expressing cells than to non-expressors. Exposure of multidrug-resistant cells to these compounds results in survivors that are no longer multidrug-resistant, suggesting a novel approach to treatment of multidrug-resistant cancers. In addition, an ABC ToxiChip has been created in collaboration with the NIEHS microarray center and used to analyze various cell lines selected for multidrug resistance. New resistance phenotypes associated with expression of ABCB6, ABCA12 and ABCC2 have been identified with this chip. We have also found a unique signature of ABC transporters in melanoma cells. One of these transporters, ABCB5, is closely related to P-gp (MDR1) and appears to contribute to multidrug resistance in melanoma cells. Use of the MDR1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of MDR1.