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 due to expression of an energy-dependent drug efflux system known as P-glycoprotein (P-gp), the product of the MDR1 gene. 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, including both receptor-mediated and fluid phase endocytosis. Studies on mechanism of action of P-gp have focused on the manner in which many different substrates and inhibitors are recognized by the transporter, how substrate interaction results in activation of ATPase, and how ATPase results in drug translocation and efflux. We have made molecular alterations in various domains of the 12 transmembrane (TM), 2 ATP-site MDR1 transporter, including chimeras with MDR2, and characterized the effects of these mutations on transport function after high-level transient expression of P-gp in a vaccinia virus-based system in cultured cells. Mutational and biochemical analysis of the two ATP sites demonstrates that both are essential, but their ATP binding and catalytic activities differ. These studies and others have led to the following major conclusions: (1) there are multiple, probably overlapping sites for interaction of substrates and inhibitors primarily formed by TM segments from both the amino-terminal (TM5,6) and carboxy-terminal (TM11,12) halves of P-gp; (2) substrate interaction sites include a high affinity """"""""on"""""""" site, a lower affinity """"""""off"""""""" site, and an allosteric site which affects ability of substrates to bind to the """"""""on"""""""" and""""""""off"""""""" sites; (3) both amino- and carboxy-terminal ATP sites are essential for function of P-gp and the sites are partially interchangeable, but not identical; (4) both ATP sites are not utilized simultaneously, supporting a model of alternating activation of ATPase during substrate transport; and (5) activation of ATPase results in a reduction of substrate binding to P-gp, consistent with translocation of substrate from the """"""""on"""""""" site to the """"""""off"""""""" site. A second molecule of ATP may need to be hydrolyzed to return the transporter to its native high affinity state. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs, and that its expression reduces infectivity of CD4 positive cells by HIV. 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. MDR1 can be efficiently packaged into SV40 vectors both in vivo and in vitro and delivered into hematopoietic cells and many other cell types.
| Kannan, Pavitra; Pike, Victor W; Halldin, Christer et al. (2013) Factors that limit positron emission tomography imaging of p-glycoprotein density at the blood-brain barrier. Mol Pharm 10:2222-9 |
| Kannan, Pavitra; Brimacombe, Kyle R; Kreisl, William C et al. (2011) Lysosomal trapping of a radiolabeled substrate of P-glycoprotein as a mechanism for signal amplification in PET. Proc Natl Acad Sci U S A 108:2593-8 |
| Shomron, Noam; Hamasaki-Katagiri, Nobuko; Hunt, Ryan et al. (2010) A splice variant of ADAMTS13 is expressed in human hepatic stellate cells and cancerous tissues. Thromb Haemost 104:531-5 |
| Kannan, Pavitra; Brimacombe, Kyle R; Zoghbi, Sami S et al. (2010) N-desmethyl-loperamide is selective for P-glycoprotein among three ATP-binding cassette transporters at the blood-brain barrier. Drug Metab Dispos 38:917-22 |
| Gillet, Jean-Pierre; Macadangdang, Benjamin; Fathke, Robert L et al. (2009) The development of gene therapy: from monogenic recessive disorders to complex diseases such as cancer. Methods Mol Biol 542:5-54 |
| Paterson, Jill K; Gottesman, Michael M (2007) P-Glycoprotein is not present in mitochondrial membranes. Exp Cell Res 313:3100-5 |
| Kimchi-Sarfaty, Chava; Marple, Andrew H; Shinar, Shiri et al. (2007) Ethnicity-related polymorphisms and haplotypes in the human ABCB1 gene. Pharmacogenomics 8:29-39 |
| Sauna, Zuben E; Kimchi-Sarfaty, Chava; Ambudkar, Suresh V et al. (2007) The sounds of silence: synonymous mutations affect function. Pharmacogenomics 8:527-32 |
| Gottesman, Michael M; Ling, Victor (2006) The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research. FEBS Lett 580:998-1009 |
| Szakacs, Gergely; Paterson, Jill K; Ludwig, Joseph A et al. (2006) Targeting multidrug resistance in cancer. Nat Rev Drug Discov 5:219-34 |
Showing the most recent 10 out of 48 publications
