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

Abstract

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 (CO 2 "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

P-glycoprotein Does Not Protect Cells against Cytolysis Induced by Pore-forming Proteins

“…In agreement with these studies were those of Bomstein and Fishelson (14), who demonstrated that P-gp ϩve tumor cell lines were more sensitive to complement-mediated lysis compared with matched P-gp Ϫve parental cells. However, in contrast to these findings were two reports demonstrating a role for functional P-gp in inhibiting membrane damage by complement (15,16). The authors of these studies presented evidence suggesting that P-gp-mediated intracellular alkalinization (pH i ) and/or decreased plasma membrane potential (V max ) resulted in a reduction in the rate of formation of the "membrane attack complex" (MAC) at the cell surface.…”

“…Functional MACs consist of complement components C5b, C6, C7, C8, and multiple C9 molecules to form pores of ϳ100 Å (17). Expression of functional P-gp was shown to correlate with a reduction in the rate of MAC formation rather than the molecular stoichiometry of MAC complexes (16). It was hypothesized that altered pH i and/or V max may somehow directly or indirectly affect C9 polymerization, resulting in a net loss of functional MAC formation (16).…”

MDR1 Genotype-Related Pharmacokinetics and Pharmacodynamics.

“…[96][97][98][99] Recently, MDR1 has been demonstrated to regulate the apoptosis. [100][101][102][103][104][105][106][107][108] For example, Robinson et al reported that transfected MDR1 conferred resistance to apoptosis induced by serum starvation, which could be reversed by addition of verapamil. 104) However, little information is available concerning the role of MDR1 in the system.…”

“…107,108) Apoptosis is often preceded by intracellular acidification, 109,110) and MDR1 was suggested to delay the onset of the apoptosis via an increase of intracellular pH. 104) Some investigators pointed out that the change in intracellular pH contributed to the alteration of uptake by MDR1.…”

Mdr1b facilitates p53-mediated cell death and p53 is required for Mdr1b upregulation in vivo

“…Further studies by several groups have suggested that MDR1 plays a role in apoptosis. For example, ectopic overexpression of MDR1 diminishes the apoptotic response induced by growth factor withdrawal (Robinson et al, 1997) decreases complement mediated cytotoxicity (Weisburg et al, 1999) and impairs the activation of caspase-dependent cell death pathways (Kojima et al, 1998;Smyth et al, 1998). In contrast, it is unknown if mdr1b overexpression a ects cell viability.…”