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A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents. The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems. Glutathione S-transferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. There was no difference in overall GST activity between the sensitive and resistant cells. Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line. When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity. Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 +/- 0.1 nmol NPSH/10(6) cells) as compared to the drug-sensitive parental subline 8226/S (11.6 +/- 1.9 nmol NPSH/10(6) cells) (P less than 0.001). In addition, when the 8226/DOX40 cells were cultured in medium without doxorubicin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells. However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays. Depletion of glutathione by D,L-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline. Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.

Incubation of human carcinoma cells with mitoxantrone resulted in an intracellular distribution of the drug into cytoplasmic, nuclear and cytoskeletal compartments occurring within 1 min of drug treatment. Incubation of the cells in drug-free medium resulted in an efflux of the drug such that 80% of the intracellular drug was eliminated from the cells by 72 hr. Approximately 20% of the initial intracellular drug concentration remained in the cells after the drug had been removed from the medium. The majority of the persistent intracellular drug was associated with soluble cytoplasmic proteins and fractions enriched in nucleic acid. Approximately 10% of the persistent drug binding was associated with cellular structures that had been depleted of soluble cytoplasmic protein and nucleic acid. During the persistent drug binding, the cells enlarged at least 2-fold as determined by microscopic examination. An increasing percentage of the cells was also observed to contain a DNA content consistent with a G2 cell cycle arrest. Taken together, these data suggest that the persistent intracellular binding of mitoxantrone results in a G2 cell cycle arrest and cellular damage.

A mitomycin C-resistant (MMCR) strain of L1210 mouse leukemia was developed by continuous drug exposure in vitro. MMC concentrations were increased in a stepwise fashion beginning at 0.033 microM and ending at 0.34 microM. This produced a 10-fold resistant cell line over the parental line. Resistance simultaneously developed to anthracene and anthracycline DNA intercalators, to vinca alkaloids and epipodophyllotoxins but not to cisplatin, bleomycin, fluorouracil or ionizing X-rays. MMC resistance was reversed using the membrane-active agent verapamil. The level of non-protein sulfhydryls was increased 2-fold in the MMCR cells. Intracellular uptake of unchanged MMC was reduced by 40% in the MMCR cells. Cytogenetic analyses demonstrated no recognizable clonal chromosomal alterations unique to the resistant subline and no evidence of double minutes or homogeneously staining regions in the DNA. Gel renaturation analysis failed to document the presence of an amplified DNA domain. Southern blotting of parental and MMCR DNA using a cDNA probe (CHP1) for the P-glycoprotein gene also failed to demonstrate amplification or rearrangement of P-glycoprotein-related homologous sequences. However, an Mr 180,000 glycoprotein was detected in the plasma membranes from MMCR cells. This protein also specifically reacted with a monoclonal antibody (C219) to the P-glycoprotein of Ling and co-workers [Kartner et al., Nature, Lond. 316, 820 (1985)]. These results suggest a pleiotropic drug resistance pattern in the MMCR cells, associated with membrane glycoprotein alterations, enhanced non-protein sulfhydryl levels, and reduced MMC accumulation. This is a novel observation for a resistant cell line selected with an alkylating agent.