Essential features of the P-glycoprotein pharmacophore as defined by a series of reserpine analogs that modulate multidrug resistance.

We have shown previously that reserpine is an effective "modulator" of P-glycoprotein-associated multidrug resistance (MDR). In addition to enhancing drug cytotoxicity in our multidrug-resistant human leukemia cell line, CEM/VLB100, reserpine strongly competes with a photoactivatible analog of vinblastine, N-(p-azido-3-[125I]iodosalicyl)-N'-(beta-aminoethyl)vindesine, for binding to P-glycoprotein. We also demonstrated previously that there are three substructural domains present in many compounds that modulate P-glycoprotein-associated MDR: a basic nitrogen atom and two planar aromatic rings. In the present study, we wished to test more rigorously the hypothesis that not only are these domains necessary for modulators of MDR but also they must exist in an appropriate conformation. Reserpine is a modulator of MDR in which these domains are present in a well-defined conformation. Accordingly, we prepared eight compounds that vary the spatial orientation of these domains, using either naturally occurring reserpine or yohimbine as chemical templates. When tested for their ability to enhance the cytotoxic activity of natural product antitumor drugs in CEM/VLB100 cells, five compounds that retained the pendant benzoyl function in an appropriate spatial orientation all modulated MDR. By contrast, compounds lacking this moiety failed to do so. These active modulators competed strongly with the 125I-labeled vinblastine analog for binding to P-glycoprotein in plasma membrane vesicles prepared from these cells. Conformational analysis using molecular mechanics revealed the structural similarities of the active modulators. Our results support the hypothesis that the relative disposition of aromatic rings and basic nitrogen atom is important for modulators of P-glycoprotein-associated MDR, and they suggest a ligand-receptor relationship for these agents. These results also provide direction for the definition of an MDR "pharmacophore."

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26.

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.

Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine.

Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp). Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells. We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time. These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-[3-125I]salicyl)-N'-beta-aminoethylvindesine ([125I]NASV). Both reserpine and, to a lesser extent, verapamil, compete with [125I]NASV for binding to P-gp. We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited [125I]NASV labeling of P-gp. However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet. Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

Pharmacological, molecular, and cytogenetic analysis of "atypical" multidrug-resistant human leukemic cells.

We previously described the cross-resistance patterns and cellular pharmacology of a human leukemic cell line, CEM/VM-1, selected for resistance to the epipodophyllotoxin teniposide (M. K. Danks et al., Cancer Res., 47: 1297-1301, 1987). Compared to CEM/VLB100, which is a well characterized "classic" multidrug-resistant (MDR) cell line, the CEM/VM-1 cells display "atypical" multidrug resistance (at-MDR) in that they are cross-resistant to a wide variety of natural product antitumor drugs, except the Vinca alkaloids, and they are not impaired in their ability to accumulate radiolabeled epipodophyllotoxin. We have extended our characterization of this at-MDR cell line in the present study. In comparison to CEM/VLB100 cells, we found that CEM/VM-1 cells are not cross-resistant to either actinomycin D or colchicine. Verapamil and chloroquine, which enhance the cytotoxicity of vinblastine in CEM/VLB100 cells, had little or no ability to do so in the CEM/VM-1 cells. Membrane vesicles of the two resistant sublines were examined for overexpression of the MDR-associated plasma membrane protein (P-glycoprotein, Mr 170,000 protein, or 180,000 glycoprotein) by photoaffinity labeling with the vinblastine analogue N-(p-azido[3-125I]salicyl)-N'-beta-aminoethylvindesine. We were unable to visualize the MDR-associated protein in the CEM/VM-1 membranes with this photoaffinity probe under conditions in which the P-glycoprotein was readily seen in the membranes of CEM/VLB100 cells. Furthermore, no hybridization of the pMDR1 complementary DNA was seen in slot-blot analyses of the RNA from at-MDR cells, indicating that the mdr gene coding for P-glycoprotein is not overexpressed as is the case in the classic MDR cells. However, cytogenetic analysis indicated that the CEM/VM-1 cells contained an abnormally banded region on chromosome 13q, suggesting that a gene other than mdr may be amplified in these cells. Thus, despite the two cell lines having approximately equal degrees of resistance to epipodophyllotoxins, our data indicate that the mechanism(s) responsible for at-MDR is different from that for classic, P-glycoprotein-associated MDR.

Differentiation and the multiple drug resistance phenotype in human leukemic cells.

A common feature of mammalian cell lines selected for multiple drug resistance is the overexpression of a high-molecular-weight surface membrane glycoprotein(s). While its amount has been shown to be related to the degree of resistance of such cells, its function in this phenomenon remains obscure. Because there are some biochemical and functional similarities between drug-resistant cells and differentiated cells, we asked if resistance-associated glycoproteins were also associated with cellular differentiation. Using three monoclonal antibodies against antigens known to be associated with differentiation and three monoclonal antibodies that distinguish our multiple drug-resistant human leukemic CEM/VLB100 cells from their drug-sensitive counterparts, we found that the resistant cells were neither altered in their apparent state of differentiation nor were they altered in their ability to respond to a differentiation stimulus with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. We did find, however, that one antibody that recognizes the resistance-associated glycoprotein, Mr 180,000 glycoprotein (gp180), was only minimally altered in amount bound after treatment with the phorbol ester, but two other resistance-associated glycoproteins, Mr 155,000 glycoprotein (gp155) and, to a lesser extent, Mr 130,000 glycoprotein (gp130), were reduced in expression after this treatment. We suggest that the function of the previously described "marker" glycoprotein associated with multiple drug resistance remains unknown, but that the expression of two other resistance-associated glycoproteins also appears to be related to cellular differentiation or maturation in these cells.

Reversal of Vinca alkaloid resistance but not multiple drug resistance in human leukemic cells by verapamil.

We examined the ability of verapamil, a Ca2+ channel blocker, to overcome Vinca alkaloid and multiple drug resistance in our CEM/VLB100 and CEM/DOX human leukemic lymphoblasts. Compared with the parent CCRF-CEM cells, CEM/VLB100 cells are approximately equal to 200- to 800-fold resistant to vinblastine and express cross-resistance to vincristine, doxorubicin, and other "natural product" drugs, as determined by comparing 50% inhibitory concentrations in a 48-h growth inhibition assay. Verapamil (10 microM) decreased the 50% inhibitory concentrations for Vinca alkaloids in CEM/VLB100 cells by approximately equal to 75- to 85-fold but caused only slight (approximately equal to 2- to 5-fold) decreases in 50% inhibitory concentrations for anthracyclines, epipodophyllotoxins, and other tubulin-binding drugs (colchicine and podophyllotoxin). Qualitatively similar results were obtained with doxorubicin-resistant cells, termed CEM/DOX; verapamil caused a 19-fold increase in doxorubicin toxicity but 67- and 3500-fold increases in the toxicities of vinblastine and vincristine, respectively. These results indicate that the effect of verapamil is relatively greater for Vinca alkaloids, with less pronounced effects for the other natural product drugs against which these cells express multiple drug resistance. In flow cytometric studies, individually nontoxic or minimally toxic concentrations of vinblastine plus verapamil caused measurable accumulation in the G2 + M phase as early as 4 h after the drug combination was added to cultures of CEM/VLB100 cells; this finding correlated with a comparable increase in the number of cells in mitosis and measurable decreases in the total number of cells. Since similar effects on cell cycle distribution, percentage of cells in mitosis, and cell number were seen when CEM/VLB100 cells were treated with toxic concentrations of vinblastine alone, we conclude that the primary toxicity of the vinblastine-verapamil combination stems from the alkaloid. Further, the rapid lytic effect of the drug combination was associated with cellular vacuolization. The vacuoles did not stain for lipids, and increases in their number were evident within 2 to 4 h after drug treatment. We suggest that verapamil enhances Vinca alkaloid cytotoxicity by altering "cryptic" cytotoxic targets, possibly related to these vacuoles, through some as yet undefined membrane effect.

Cross-resistance patterns and antigen expression in Vinca alkaloid- and other multiple drug-resistant human leukemic cell lines.

The studies presented in this report demonstrate that Vinca alkaloid-resistant human leukemic lymphoblasts display patterns of cross-resistance to other drugs that differ from those of cell lines selected for primary resistance to anthracyclines or epipodophyllotoxins. These various drug-resistant cell lines also showed differential expression of an antigen recognized by an antibody that distinguishes VLB-resistant from VLB-sensitive cells. Furthermore, comparable levels of resistance or cross-resistance to one drug are not predictive of cross-resistance to other drugs. Our data suggest, then, that the MDR phenotype is complex and may be the result of many and different biochemical lesions. Thus, in order to predict MDR, it may be necessary to document more than one of these changes with specific reagents.

Energy-dependent reduced drug binding as a mechanism of Vinca alkaloid resistance in human leukemic lymphoblasts.

We studied the accumulation of [3H]vinblastine (VLB) by lines of CCRF-CEM cultured human leukemic lymphoblasts that were either sensitive or resistant to the drug. Neither cell line metabolized VLB, nor selectively retained any radioactive impurities. There was an apparent "instantaneous" accumulation of VLB by cells of both lines, resulting in cell to medium ratios greater than 1.0 within 1 sec after drug addition. Experiments between 0 and 60 sec revealed that the presumed undirectional initial rate of VLB accumulation by resistant cells, termed CEM/VLB100, was about one-half that of sensitive CEM cells. In experiments carried out over 60 min, the VLB-resistant cells accumulated considerably less [3H]VLB than did the sensitive cells. Drug accumulation by both cell lines was temperature-sensitive, since incubation of cells at 4 degrees resulted in only minimal uptake beyond that observed at zero time. CEM/VLB100 cells retained less drug than did CEM cells, apparently because of a larger fraction of readily releasable VLB compared with CEM cells. The accumulation of VLB by either cell line was related in part to cellular levels of ATP. Although depletion of ATP was associated with decreased accumulation of VLB by CEM cells, it was related to enhanced drug accumulation by CEM/VLB100 cells. Restoration of ATP levels to near control values by addition of glucose also had opposite effects on the two cell lines, causing further accumulation of VLB by the sensitive line but leading to apparent drug efflux from the resistant line. Potentially competing substrates (VM-26, colchicine, daunorubicin, and doxorubicin) failed to block this glucose-mediated release of VLB from the CEM/VLB100 cells. In experiments with energy-depleted CEM/VLB100 cells preloaded with VLB and then incubated in drug-free medium, initial drug loss was shown to be independent of cellular metabolism, being roughly the same for both metabolically intact and metabolically depleted cells. Glucose (energy) was required only for subsequent release of what appeared to be a more tightly bound cell-associated fraction of VLB. Results of zero-time binding studies tended to confirm that VLB binding by resistant cells has two components, one requiring and the other not requiring metabolic energy. Differences in the proportions of these two components between the sensitive and resistant cells suggest a mechanism for resistance to VLB and similar compounds.

Continued expression of vinca alkaloid resistance by CCRF-CEM cells after treatment with tunicamycin or pronase.

We have shown that a glycoprotein(s) with a molecular weight of approximately 180,000 exists on the surface of cultured lymphoblasts selected for resistance to vinblastine (VLB) (CEM/VLB100). The amount of this glycoprotein, which is barely detectable on the VLB-sensitive cells (CEM), appears to be related in part to the degree of resistance, up to approximately 270-fold. Exposure of the cells to Pronase for 45 to 60 min or growth of the cells for 2 days in tunicamycin, an inhibitor of glycoprotein synthesis, resulted in the absence of the resistance-associated glycoproteins, as determined by polyacrylamide gel electrophoresis of these treated cells after labeling te cells either with sodium [3H]-borohydride or with [14C]- or [3H]glucosamine. Uptake studies with [3H]VLB revealed that CEM cells normally accumulated and retained more drug than did the CEM/VLB100 cells. While the tunicamycin or Pronase treatments slightly increased the uptake of drug by CEM cells, there was no enhanced uptake of [3H]VLB by the tunicamycin- or pronase-treated CEM/VLB100 cells, when compared with untreated controls, indicating that the loss of external surface glycoproteins did not render the resistant cells more "leaky" to drug influx. Additionally, diminished drug retention by the CEM/VLB100 cells was unaffected by these treatments. Moreover, when CEM/VLB100 cells were grown grown for 2 days in the presence of tunicamycin and several concentrations of VLB, no enhanced toxicity of VLB was noted. Other experiments with [14C]glucosamine and [3H]leucine revealed that treatment with tunicamycin did not affect the distribution of proteins in these cells. Taken together, these results permit the suggestion that the carbohydrate moiety of the cell surface resistance-associated glycoproteins does not mediate resistance to the alkaloid per se; however, a role for plasma membrane proteins cannot be ruled out at this time.