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W T Beck

Publications and source records attributed to W T Beck.

At least 91 records · Page 5Linked to original sources
PubMed1988

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Cytogenetic and molecular characterization of tumors in nude mice derived from a multidrug-resistant human leukemia cell line.

We have evaluated whether selection of a human tumor leukemic line for resistance to vinblastine (Velban; VLB) alters its tumorigenicity. To address this question, CEM and CEM/VLB100 cells [which express the multiple drug-resistant (MDR) phenotype via amplification of the P-glycoprotein gene] were characterized by several techniques including chromosome banding, in situ hybridization, Southern blotting, RNA dot blotting, in vitro drug sensitivity, and tumorigenicity in nude mice. Analysis of the chromosome banding patterns of both drug-sensitive CEM cells and the MDR CEM/VLB100 cells revealed that the two lines differed primarily by the presence of a large metacentric marker chromosome associated with the acquisition of VLB resistance. In situ hybridization of a P-glycoprotein complementary DNA to metaphase chromosomes showed that the amplified P-glycoprotein genes in the CEM/VLB100 cell line were localized to this large marker. Tumorigenicity of both the CEM and CEM/VLB100 cell lines was measured after injection of 10(7) cells/nude mouse. The results showed that 4 of 4 drug-sensitive and 4 of 5 drug-resistant cell lines formed tumors in 5-10 wk. By comparison with the parental line, three of the four tumors arising from the CEM/VLB100 line retained their drug-resistance properties as measured by vinblastine resistance in vitro and elevated P-glycoprotein mRNA expression associated with P-glycoprotein gene amplification. In addition, tumors retaining the MDR phenotype also retained the large metacentric marker chromosome. One tumor arising from CEM/VLB100 reverted to the drug-sensitive phenotype, with a resultant decrease in P-glycoprotein mRNA expression and loss of P-glycoprotein gene amplification. This revertant was also missing the large metacentric marker present in all cells from the CEM/VLB100 parent. Our experiments show that the acquisition of the MDR phenotype resulting from overexpression of P-glycoprotein in the plasma membrane does not effect the tumorigenicity of human CEM cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Comparison of ion channels in multidrug-resistant and -sensitive human leukemic cells.

Tumor cell lines selected to grow in the presence of one "natural product" antineoplastic drug often develop cross-resistance to others. This multidrug resistance (MDR) is believed to be a major problem in cancer therapy. Organic Ca2+-channel blockers, such as verapamil, can reverse this resistance and render MDR cells in culture nearly as sensitive to the antineoplastic drugs as the drug-sensitive cells from which they were derived. It has therefore been suggested that Ca2+ channels may play a role in MDR. To determine directly whether there are electrophysiological correlates of MDR, we used whole-cell and single-channel patch-clamp techniques to survey the ion channels in a drug-sensitive human T-cell leukemia line, CCRF-CEM, and a MDR variant, CEM/VLB100. We found no evidence for a voltage-gated Ca2+ channel. However, we did identify three other current/channel types: a voltage-gated tetrodotoxin-sensitive inward current carried by Na+, a voltage-gated labile outward current carried by K+, and a nonselective cation channel reversing at 0 mV. Drug-sensitive and -resistant cells were the same with respect to the level of expression of these channels.

Antineoplastic Agents↗
PubMed1988

Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells.

Multidrug resistance (MDR), typified by resistance to Vinca alkaloids and anthracyclines, is a well characterized experimental phenomenon that may have some clinical correlates. Verapamil, chloroquine, and related drugs have been shown previously to be capable of enhancing anticancer drug cytotoxicity in multi-drug-resistant cells, but the mechanism(s) by which these agents do this is(are) unclear. Since these agents did not seem to have common features, we studied these and other compounds for their ability to "modulate" Vinca alkaloid resistance in order to determine whether they possessed any common chemical or physical features. In addition to verapamil, 24 compounds, consisting of indole alkaloids, lysosomotropic agents, and amines, were tested for their ability to enhance the cytotoxicity of vinblastine and/or vincristine in our human leukemic multidrug-resistant cell line, CEM/VLB100. Seventeen compounds that enhance the cytotoxicity of the Vinca alkaloids by more than 5-fold have been identified. These include quinolines (chloroquine, quinine, chinchonidine, and primaquine), acridines (acridine, acridine orange, and quinacrine), and indole alkaloids (yohimbine, corynanthine, reserpine, physostigmine, and the vindoline and catharanthine moieties of the Vinca alkaloids), as well as other alkaloids and amines (chlorpromazine, propranolol, atropine, and tryptamine). Vindoline, catharanthine, and quinacrine also enhanced the cytotoxicity of doxorubicin and teniposide in these cells, indicating that this "modulation" was not limited to Vinca alkaloids. We examined some well known lysosomotropic compounds (methylamine, epinephrine, suramin, and trypan blue) and found that they were not able to enhance the cytotoxicity of vincristine in the CEM/VLB100 cells, indicating that lysosomotropic activity per se is not required for modulator activity. Three-dimensional computer modeling permitted molecular comparisons of conformationally related congeners of vinblastine, vindoline, and verapamil and revealed three regions of structural homology. We measured the hydrophobicity (by oil/water partitioning) and calculated the molar refractivity (by the additive substituent constant method) of active and inactive compounds. We found that those cationic agents--verapamil, quinacrine, indole alkaloids, and quinolines--that were lipid soluble at physiologic pH and had similar molar refractivities were best able to enhance the cytotoxicity of the Vinca alkaloids in our multidrug-resistant cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibiotics, Antineoplastic↗
PubMed1987

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

Preclinical evaluation of illudins as anticancer agents.

Illudins are low molecular weight natural products which were previously evaluated as anticancer drugs using rodent tumor models. In the present studies, we used in vitro cultures of human cancer cells to reevaluate their potential as anticancer agents. Using continuous exposure, Illudins S and M were cytotoxic to human leukemia cells at concentrations of 6-100 nM, but dihydroilludin M was 3 orders of magnitude less toxic, thus identifying a ketone site as a structural feature critical for cytotoxicity. Cytokinetic studies showed that illudin S caused a complete block at the G1-S phase interface of the cell cycle. Kinetics of inhibition of radiolabeled thymidine, uridine, and leucine incorporation suggested a primary effect on DNA synthesis. In colony and liquid culture assays, cell killing was time dependent but near maximal with a 2-h exposure. Myeloid and T-lymphocyte leukemia cells were most sensitive (50% inhibitory concentration, 6-11 nM), but B-cell leukemia/lymphoma, melanoma, and ovarian carcinoma cells were at least 10 times more resistant. Bone marrow granulocyte/macrophage progenitors showed intermediate sensitivity. Illudin S was equally effective against CEM T-lymphocyte leukemia cells expressing the multidrug resistance phenotype associated with Mr 180,000 glycoprotein and the parental cell line. CEM cells resistant to doxorubicin, epipodophyllotoxins, and 1-beta-D-arabinofuranosylcytosine showed only a 2-fold increased resistance to illudin S. Illudins are novel and potent cytotoxins which may be preferentially active against human myeloid and T-cell leukemias, including cells resistant to more conventional chemotherapeutic agents. The present studies illustrate the breadth of information which can be obtained on a new agent using present in vitro screening procedures and human cells.

Antineoplastic Agents↗
PubMed1987

Atypical multiple drug resistance in a human leukemic cell line selected for resistance to teniposide (VM-26).

Resistance to the cytotoxic effects of many natural product drugs after exposure to a single agent is a common observation. The classes of drugs included in the "classic" multiple drug resistance phenotype are Vinca alkaloids, anthracyclines, epipodophyllotoxins, and antibiotics. We report here the characterization of a human leukemic cell line (CEM/VM-1) with "atypical" multiple drug resistance: despite resistance and cross-resistance to etoposide, anthracyclines, mitoxantrone, and 4'-[(9-acridinyl)amino]methanesulphon-m-anisidide (mAMSA), these cells retain sensitivity to the Vinca alkaloids. Further, even though this cell line is approximately equal to 40-fold cross-resistant to the cytotoxic effect of etoposide (VP-16), it is similar to drug-sensitive CEM cells in the cellular pharmacology of [3H]VP-16 as determined by zero time binding, initial influx rate, steady state drug concentration, and unidirectional efflux. Our studies suggest that the resistance of CEM/VM-1 cells to epipodophyllotoxins is due to an altered interaction between drug and its cellular target(s) by a mechanism unrelated to the decreased cellular concentration of drug associated with the "classic" multiple drug resistance phenotype.

Cell Line↗
PubMed1986

Chloroquine enhancement of anticancer drug cytotoxicity in multiple drug resistant human leukemic cells.

Vinblastine-sensitive (CCRF-CEM) and -resistant (CEM/VLB100) human T-cell lymphoblasts were treated with the lysosomotropic agent chloroquine. As measured by growth inhibition, this drug enhanced the cytotoxicity of vinblastine in the CEM/VLB100 cells but was less effective in the CCRF-CEM cells. Chloroquine also enhanced the cytotoxic activity of vincristine, daunorubicin and doxorubicin and, to a lesser extent, teniposide (VM-26) in the CEM/VLB100 cells. Histological examination revealed that the vinblastine-resistant cells contained more cytoplasmic vacuoles than their drug-sensitive counter-parts. When the CEM/VLB100 cells were treated with chloroquine, vinblatine, or a combination of the two, the cells displayed many more cytoplasmic vacuoles than the controls. Coincident with the increased number of vacuoles, these treated cells stained more intensely than controls for the lysosomal enzyme, acid phosphatase, but not for lipid. The vacuolization did not increase as much in the CCRF-CEM cell line when these cells were exposed to the chloroquine + vinblastine combination. Vacuolization was also associated with vincristine, doxorubicin, and daunorubicin treatments, but not with VM-26. We conclude that chloroquine is a modulator of anticancer drug action in the CEM/VLB100 cell line.

Antibiotics, Antineoplastic↗
PubMed1986

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.

Antibodies, Monoclonal↗
PubMed1986

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.

Cell Cycle↗
PubMed1986

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.

Antibodies, Monoclonal↗
PubMed1985

Monoclonal antibodies to glycoproteins of Vinca alkaloid-resistant human leukemic cells.

Drug resistance is a major problem in the successful treatment of cancer. Resistance to one drug is often associated with cross-resistance to other anticancer agents. This is commonly seen with the "natural product" anticancer drugs such as the Vinca alkaloids and anthracyclines. In experimental systems, specific changes in plasma membranes characterize this "multiple drug resistance." The most prominent of these is the enhanced expression in several systems of high-molecular-weight glycoproteins ranging from Mr approximately equal to 150,000 to approximately equal to 180,000, the amount of which has been shown to be related to the degree of drug resistance. We report here the production of three monoclonal antibodies that bind preferentially to the surfaces of cultured human leukemic lymphoblasts resistant to the Vinca alkaloid vinblastine. Each antibody recognizes a surface membrane glycoprotein with molecular weights of 180,000 to 210,000. Additionally, two of the antibodies also recognize a second surface glycoprotein with molecular weights of either approximately equal to 155,000 or approximately equal to 130,000. All of these glycoproteins are overexpressed in the alkaloid-resistant cells. While a Mr approximately equal to 180,000 protein has been shown to be associated with multiple drug resistance, the other two glycoproteins have not been described previously in these cells. These antibodies may be useful in studies of the mechanisms of drug resistance, as well as in screening cells from drug-resistant patients for these resistance-associated glycoproteins.

Antibodies, Monoclonal↗
PubMed1984

Cellular pharmacology of Vinca alkaloid resistance and its circumvention.

Vinca alkaloid-resistant human leukemic cells express the multiple drug-resistant phenotype, characterized by cross resistance to natural product compounds of unrelated structure and action. While there are also distinct biochemical lesions associated with this phenotype, their role(s) in the expressions of resistance is not known at this time. More clear, however, is our understanding of the pharmacologic determinants of this resistance--apparent decreased drug uptake and decreased drug retention. This latter phenomenon has been attributed to the workings of an active efflux pump, but data presented here and elsewhere permit an alternative explanation--that of altered drug binding to an as yet unidentified target(s). Elucidation of the mechanism of multiple drug resistance is important in the design of new chemotherapeutic strategies to overcome it. In this regard, calcium channel blocking agents and calmodulin inhibitors can cause an apparent reversal of resistance by enhancing the cytotoxic effectiveness of the anticancer drugs, possibly by increasing the amount of drug retained by the tumor cells. The basis for this enhanced retention and cytotoxicity is not presently known, but it may be related to cellular calcium fluxes, calmodulin content or membrane fluidity and permeability. The meaning of these findings is unclear at the present time, but they may provide new insights into the mechanism of action and ultimate cellular target(s) for Vinca alkaloids. Whether these modifying drugs sensitize the cells to the action of the alkaloids or potentiate the oncolytic drug effect in the cell remains to be determined. Regardless of the mechanism, calcium channel blocking agents may have a role in the combination chemotherapy of the leukemias with Vinca alkaloids.

Biological Transport, Active↗
PubMed1984

Acquisition of multiple drug resistance by CCRF-CEM cells selected for different degrees of resistance to vincristine.

We studied the acquisition of multiple drug resistance to several "natural product" drugs by cultured human leukemic lymphoblasts selected for increasing resistance to vincristine (VCR). Three apparent types of cross-resistance patterns could be distinguished: specific, pleiotropic, and mixed. Cross-resistance to vindesine developed in parallel with VCR resistance, appearing at the lowest levels of VCR resistance (approximately fivefold). Vinblastine resistance did not become noticeable until VCR resistance was higher (approximately equal to 50-100-fold). Cross-resistance resistance to three other tubulin-binding agents developed in a different pattern, however. While cross-resistance to maytansine was seen in cells of intermediate (approximately equal to 50-fold) resistance to VCR, colchicine cross-resistance occurred only in cells that were highly resistant to VCR (approximately equal to 500-fold). Even at the highest level of VCR resistance (approximately equal to 600-fold), complete sensitivity to podophyllotoxin was retained. Conversely, cross-resistance to the epipodophyllotoxins , teniposide and etoposide, semisynthetic derivatives of podophyllotoxin, was seen in cells that were greater than 50-fold resistant to VCR. The same pattern obtained for the anthracyclines, doxorubicin and daunorubicin. We conclude that resistance to low concentrations of VCR does not uniformly confer cross-resistance to other classes of natural product drugs.

Animals↗