Cranberry juice extract, a mild prooxidant with cytotoxic properties independent of reactive oxygen species.

A cranberry juice extract (CJE), rich in proanthocyanidins, had weak prooxidant properties, generating low levels of hydrogen peroxide (H2O2) and superoxide. Generation of H2O2 was pH dependent, increasing at alkaline pH, and was lowered in the presence of catalase and, to a lesser extent, of superoxide dismutase (SOD). Growth inhibition and cytotoxicity were noted towards human oral carcinoma HSC-2 cells, with midpoint cytotoxicity at 200 µg/mL CJE, but not towards human gingival HF-1 fibroblasts. Being a mild prooxidant, CJE toxicity was unaffected by exogenous catalase and pyruvate, scavengers of H2O2, but triggered intracellular synthesis of reduced glutathione, as confirmed by cell staining with Cell Tracker™ Green. The presence of exogenous SOD potentiated the toxicity of CJE, possibly by stabilizing the CJE phenols and hindering their degradative autooxidation. Conversely, 'spent' CJE, i.e. CJE added to cell culture medium and incubated for 24 h at 37 °C prior to use, was much less toxic to HSC-2 cells than was freshly prepared CJE. These differences in toxicity between SOD-stabilized CJE, freshly prepared CJE, and 'spent' CJE were confirmed in HSC-2 cells stained with aceto-orcein, which also indicated that the mode of cell death was by the induction of apoptosis.

Regulation of IFN-gamma signaling is essential for the cytotoxic activity of CD8(+) T cells.

Previous studies have demonstrated that, as naive murine CD4(+) cells differentiate into Th1 cells, they lose expression of the second chain of IFN-gammaR (IFN-gammaR2). Hence, the IFN-gamma-producing subset of Th cells is unresponsive to IFN-gamma. Analysis of IFN-gamma-producing CD8(+) T cells demonstrates that, like Th1 cells, these cells do not express IFN-gammaR2. To define the importance of IFN-gamma signaling for the development of functional CD8(+) T cells, mice either lacking IFN-gammaR2 or overexpressing this protein were examined. While CD8(+) T cell development and function appear normal in IFN-gammaR2(-/-) mice, CD8(+) T cell function in IFN-gammaR2 transgenic is altered. IFN-gammaR2 transgenic CD8(+) T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8(+) T cells proliferate normally and produce cytokines, such as IFN-gamma in response to antigenic stimulation. Therefore, although IFN-gamma signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-gamma signaling can selectively impair the cytotoxic function of CD8(+) T cells.

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

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 (pHi) 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 pHi 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 pHi. 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 pHi. 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.

The multidrug resistance phenotype confers immunological resistance.

Multidrug resistance (MDR), which is due, in part, to the overexpression of P-glycoprotein, confers resistance to a variety of natural product chemotherapeutic agents such as daunorubicin, vincristine, and colchicine. RV+ cells are a P-glycoprotein overexpressing variant of the HL60 myeloid leukemia cell line. In addition to classic MDR, RV+ cells displayed relative resistance to complement-mediated cytotoxicity with both immunoglobulin G and M antibodies against different cell surface antigens, but not to antibody-dependent cellular cytotoxicity and lymphokine-activated killing. Complement resistance was reversed both by treatment with verapamil and with specific monoclonal antibodies (mAbs) capable of binding to P-glycoprotein and blocking its function. To further confirm that the resistance of RV+ cells was not a consequence of the selection of the cells on vincristine, a second system involving P-glycoprotein infectants was also investigated. K562 cells infected with the MDR1 gene, which were never selected on chemotherapeutic drugs, also displayed relative resistance to complement-mediated cytotoxicity. This MDR1 infection-induced resistance was also reversed by mAbs that bind to P-glycoprotein. Therefore, the MDR phenotype as mediated by P-glycoprotein provides resistance to complement-mediated cytotoxicity. The increased intracellular pH and the decreased membrane potential due to the MDR phenotype may result in abnormal membrane attack complex function. This observation may have implications for the possible mechanisms of action of P-glycoprotein and for a possible physiologic role for P-glycoprotein in protection against complement-mediated autolysis.

Novel Cl(-)-dependent intracellular pH regulation in murine MDR 1 transfectants and potential implications.

Previously [Luz et al. (1994) Biochemistry 33, 7239-7249], we determined that Cl(-)- and -HCO3-dependent pHi homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991). Via single-cell photometry experiments performed under various conditions, we are now able to separate Na(+)-dependent and Na(+)-independent components of Cl-/-HCO3 exchange in the MDR transfectants and the parental LR73 cells. Cl(-)-dependent, Na(+)-independent reacidification of pHi, mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein. Analysis of H+ flux at different pHi shows that Cl(-)-dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pHi = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells). MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl(-)-dependent pHi regulation are more severe. Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pHi homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein. Verapamil reverses the anomalous behavior.(ABSTRACT TRUNCATED AT 250 WORDS)