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. In most 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 to other members of the ABC transporter family. 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 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 in the NCI-60 cell lines with resistance to specific cytotoxic drugs. Furthermore, this analysis has revealed that some drugs are more toxic to P-gp-expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazides, have been identified. One compound, NSC73306, has been studied in detail and shown to kill P-gp-expressing cells with high specificity by blocking them in S phase. Treatment with NSC73306 and related drugs also results in increased turnover of ABCB1 mRNA. Cells that survive NSC73306 treatment do not express P-gp and are sensitive to chemotherapy with natural product drugs such as anthracyclines, paclitaxel and Vinca alkaloids. A quantitative structure activity analysis of NSC73306 analogs and a further correlation analysis in the NCI-60 cell lines has yielded many additional compounds with improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. In addition, the compound tiopronin, which is a sulfhydryl donor used clinically to treat cystinosis, and some of its derivatives have been shown to be powerful selective agents for killing MDR cells. Technology enabling a high-throughput screen for new agents that are substrates, inhibitors or specifically kill P-gp-expressing cells has been developed. In cell lines derived from patients with Kaposi sarcoma, exposure to HIV protease inhibitors results in overexpression of ABCB1, for which the protease inhibitors are important substrates. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethoxy-loperamide has been developed in collaboration with Robert Innis in NIMH to PET image distribution of this specific P-gp substrate in cancers and in the brain, with and without treatment with potent inhibitors of P-gp such as tariquidar. PET ligands that are weak bases are trapped in lysosomes, amplifying the uptake signal in the brain, in some normal tissues, and in cancers. We have shown that among three most prominent transporters at the blood-brain barrier (ABCB1, ABCC1, ABCG2), this compound is specific for ABCB1 (P-gp). Common polymorphic variants of P-gp have also been detected, but coding polymorphisms do not appear to alter the drug transport functions of P-gp. However, a synonymous polymorphism (C3435T, no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping by altering the rhythm of protein folding and changing substrate and inhibitor interactions with P-gp. This haplotype appears to change mRNA folding, and cause a major translational delay which results in altered conformation of P-gp. Stable transfectants of porcine LLC-PK1 cells with the haplotype form of P-gp show altered drug resistance and inhibitory sensitivity compared to wild-type P-gp transfectants. We have created a highly sensitive, quantitative assay for ABC transporter mRNAs in human cancer samples using TaqMan Low Density Arrays. In initial studies of several cancers whose cell lines are represented in the NCI-60 cell lines we have found that patterns of expression of ABC transporter genes in clinical cancers differ substantially from those of the established cell lines, irrespective of the manner in which these cells are cultured (monolayer, 3D culture or xenografts). This result indicates that current in vitro cancer cell models may not be adequate to study drug resistance in cancer and efforts are underway to develop novel ex vivo cancer cell growth conditions that maintain in vivo patterns of expression of MDR genes. 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. Using recombinant SV40 capsid proteins, it is possible to package DNA and RNA in vitro. In particular, siRNA and chemically modified siRNAs such as peptide nucleic acids (PNAs) can be delivered with high efficiency and at much lower concentrations than are needed for lipofection. Delivery of toxic DNAs, such as Pseudomonas exotoxin cDNA, can be used to target cancers in vitro and in mouse xenoplant models.
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