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

Abstract

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR). In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response. There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation. Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different poreforming proteins. Testing a variety of different P-gpexpressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp ؉ve or P-gp ؊ve cells to the pore-forming proteins complement, perforin, or pneumolysin. Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.P-glycoprotein (P-gp), 1 a member of the ATP-binding cassette (ABC) superfamily, is encoded by the MDR1 gene in humans and mdr1a and mdr1b in mice and has been demonstrated to act as a very efficient toxin efflux molecule (1, 2). In the clinical setting, expression of P-gp on tumor cells confers resistance to a wide range of different chemotherapeutic agents constituting a multidrug-resistant (MDR) phenotype and a poor prognosis. The current working model maintains that P-gp removes xenotoxins in an energy (ATP)-dependent manner by intercepting the drug as it moves through the lipid membrane and flips the drug from the inner leaflet to the outer leaflet and into the extracellular media (3). Consistent with its toxin clearance role, P-gp is expressed on the surface of normal human cells found in the gut, liver, and kidney tubules, and at blood-tissue barriers (4). However, P-gp is also expressed in the adrenal gland, hemopoietic stem cells, natural killer (NK) cells, antigen-presenting dendritic cells, and T and B lymphocytes (5, 6), and a role for P-gp in removing xenotoxins from these cells is not immediately apparent.Recent studies by our group and others have indicated that, in addition to its role as an efflux pump, P-gp also regulates programmed cell death mediated by some chemotherapeutic drugs, serum starvation, ultraviolet (UV) irradiation, and ligation of the cell surface death receptors Fas and tumor necrosis factor receptor (7-9). The ability of P-gp to inhibit cell death mediated by these diverse apoptotic stimuli appears to be due to P-gp-mediated inhibition of caspase activation. In contrast, other stimuli such as the chemotherapeutic agent hexamethylene bisacetamide (10), the prote...

Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux

“…We have recently reported that P-gp was unable to confer resistance to cell death by pore-forming proteins such as complement, perforin and pneumolysin. 38 CEM-GFP, CEM-P-gp WT , CEM-P-gp MM-3 and CEM-P-gp MM-32 cells were treated with normal rabbit serum as a source of complement and cell lysis was assessed by 51 Cr release (Figure 6d). Endogenous CEM cell-reactive antibodies within the rabbit serum were shown by flow cytometry to bind equivalently to all four transduced cell lines (data not shown) and enabled activation of the classical complement pathway and the rapid induction of cell lysis.…”

“…Complement treatment of cells and 51 chromium release assays were performed as previously described. 38 P-gp reversal assays were performed by preincubating cells in anti-P-gp monoclonal antibodies (16 mg/ml UIC2 (Immunotech, Marseille, France) or 100 mg/ml MRK16) for 1 h prior to addition of 50 ng/ml vincristine. Samples were then incubated for 24 h at 371C and trypan counted as above.…”

Mammalian ABC Transporters in Health and Disease

“…Five other physiological functions have been attributed to the drug-transporting Pgps (151): transport of cytokines, particularly interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-␥) (160, 161); increased self-renewal and decreased differentiation of hematopoietic stem cells (162); migration of antigen-presenting dendritic cells and T lymphocytes (163); inhibition of alloantigen-dependent, but not mitogen-dependent, T-cell activation (164); and inhibition of apoptosis [reviewed in (151)], but not cytolysis by pore-forming proteins (165,166). The evidence for these physiological functions rests mainly on the effects of Pgp inhibitors, gross overexpression of a Pgp gene, or the effects of anti-Pgp antibodies, and remains the subject of lively discussions (167).…”

P-glycoprotein as a therapeutic target: good news

“…8 This effect can be achieved via generation of membranotropic intracellular metabolites such as ROS. The toxicity of ROS is not confined to the break of biological membranes (necrosis); oxygen burst-induced damage of macromolecules can trigger apoptotic cascades.…”