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J Biol Chem, Vol. 274, Issue 16, 10877-10888, April 16, 1999

Intracellular pH and Multidrug Resistance Regulate Complement-mediated Cytotoxicity of Nucleated Human Cells

Jeffrey H. Weisburg, Paul D. Roepe^§, Sergey Dzekunov^§, and David A. Scheinberg

From the  Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York 10021 and the ^§ Department of Chemistry and Lombardi Cancer Center Program in Tumor Biology Georgetown University, Washington, D. C. 20057

In previous work (Weisburg, J. H., Curcio, M., Caron, P. C., Raghi, G., Mechetner, E. B., Roepe, P. D., and Scheinberg, D. A. (1996) J. Exp. Med. 183, 2699-2704), we showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV⁺) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pH_i) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pH_i by two independent methods (CO₂ "conditioning" or isotonic Cl substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pH_i by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl/HCO₃ exchange upon Cl substitution is blocked by treatment with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate. Continuous photometric monitoring of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), to assess changes in pH_i or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pH_i upon CO₂ conditioning and Cl substitution and release of BCECF upon formation of MAC pores. Antibody binding and internalization kinetics are similar in both the parental and resistant cell lines as measured by radioimmunoassay, but flow cytometric data showed that net complement deposition in the cell membrane is both delayed and reduced in magnitude in the MDR cells and in the cells with increased pH_i. This interpretation is supported by comparison of BCECF release data for the different cells. Dual isotopic labeling of key complement components shows no significant change in molecular stoichiometry of the MACs formed at different pH_i. The results are relevant to understanding clinical implications of MDR, the physiology of P-GP, and the biochemistry of the complement cascade and further suggest that the "drug pump" model of P-GP action cannot account for all of its effects.

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