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The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells. In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs. Using polymerase chain reaction–single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences. Among several mutations found, two are expected to cause amino acid substitutions. One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined. Transport of the MRP1 substrates leukotriene C[sub 4] , 17β-estradiol 17β-(d)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1. [ABSTRACT FROM AUTHOR]

We have previously shown that the doxorubicin-selected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line. Levels of immunoreactive topoisomerase II alpha are also reduced in H69AR cells. In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells. In addition, the resistant cells contained lower levels of both topoisomerase II alpha and topoisomerase II beta protein and mRNA. However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67 M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts. These data suggest that reduced levels of topoisomerase II alpha and II beta may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes. [ABSTRACT FROM AUTHOR]

H69AR is a multidrug-resistant human small-cell lung carcinoma cell line that was selected in doxorubicin and has previously been shown to be cross-resistant to a variety of natural-product-type anticancer drugs. H69AR is unlike many other multidrug-resistant cell lines in that it does not overexpress P-glycoprotein. In the present study, the drug sensitivity and cross-resistance patterns of H69AR cells were further characterized. A total of 15 drugs belonging to a number of chemical classes were screened. These compounds included anthracyclines, DNA binders (anthrapyrazoles, benzothiopyranoindazoles, and pyrazoloacridines), and lipophilic antifolates. The alkylating agent melphalan and the antimetabolite cytosine arabinofuranoside (Ara-C) were also tested. In general, the drug sensitivity and cross-resistance profiles of H69AR cells were consistent with those reported by others using other drug-resistant cell lines. However, there were several unexpected instances of cross-resistance. Thus, the H69AR cell line was more resistant than its parent cell line to the potent 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin, bisantrene, the pyrazoloacridine PD 114541, Ara-C, and melphalan. In addition, no cross-resistance to the four lipophilic antifolates tested, including trimetrexate, was found. The absence of a consistent pattern among the various drug-resistant cell lines indicates that assumptions about the efficacy of anticancer drugs in multidrug resistance should be made with caution. [ABSTRACT FROM AUTHOR]

Numerous procedures have been described which test the chemosensitivity of tumor cell lines. A major disadvantage of most of these assays is that practical limitations prevent the testing of more than a few variables. We have adapted a rapid and efficient colorimetric assay for testing the chemosensitivity of human lung tumor cells. In this assay, a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, MTT) is converted to a colored formazan product by enzymes active only in living cells. The MTT assay may be carried out entirely in 96-well microtiter plates, so that large experiments examining a number of variables can be readily performed. Thus, drug concentration, time of exposure to drug, length of assay, and cell density can be varied and tested. Moreover, the simplicity of this assay allows simultaneous testing of multiple drugs on multiple cell lines. Finally, the MTT assay is useful for monitoring the development of multidrug-resistant cells in culture. [ABSTRACT FROM AUTHOR]