Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells Journal Article


Authors: Weisburg, J. H.; Roepe, P. D.; Dzekunov, S.; Scheinberg, D. A.
Article Title: Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells
Abstract: 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 HL6O 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 pHi 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 pHi or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pHi 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.
Keywords: cancer chemotherapy; controlled study; protein expression; human cell; flow cytometry; antineoplastic agent; animals; mice; cell survival; fluorescent dyes; kinetics; immunoglobulin g; isotope labeling; cell membrane; murinae; carboxyfluorescein; cell nucleus; antigen binding; multidrug resistance; internalization; hydrogen-ion concentration; p-glycoprotein; complement dependent cytotoxicity; drug resistance, multiple; photometry; complement; immunoglobulin m; carbon dioxide; glycoprotein p; k562 cells; radioimmunoassay; bicarbonate; hl-60 cells; cell strain hl 60; chloride ion; cell ph; oryctolagus cuniculus; complement system proteins; complement membrane attack complex; humans; human; priority journal; article; fluoresceins; vinca; 2,7 bis(carboxyethyl) 6 carboxyfluorescein; dihydro 4,4' diisothiocyano 2,2' stilbenedisulfonic acid; mycobacterium avium complex (mac)
Journal Title: Journal of Biological Chemistry
Volume: 274
Issue: 16
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1999-04-16
Start Page: 10877
End Page: 10888
Language: English
DOI: 10.1074/jbc.274.16.10877
PUBMED: 10196165
PROVIDER: scopus
DOI/URL:
Notes: Article -- Export Date: 16 August 2016 -- Source: Scopus
Altmetric
Citation Impact
MSK Authors