Mechanism of action of dexniguldipine-HCl (B8509-035), a new potent modulator of multidrug resistance.

It has previously been shown that dexniguldipine-HCl (B8509-035) is a potent chemosensitizer in multidrug resistant cells [Hofmann et al., J Cancer Res Clin Oncol 118: 361-366, 1992]. It is shown here that dexniguldipine-HCl causes a dose-dependent reduction of the labeling of the P-glycoprotein by azidopine, indicating a competition of dexniguldipine-HCl with the photoaffinity label for the multidrug resistance gene 1 (MDR-1) product. Exposure to dexniguldipine-HCl results in a dose-dependent accumulation of rhodamine 123 in MDR-1 overexpressing cells. In the presence of 1 microM dexniguldipine-HCl, rhodamine 123 accumulated in multidrug resistant cells to similar levels as in the sensitive parental cell lines. At this concentration, dexniguldipine-HCl enhances the cytotoxicities of Adriamycin and vincristine. The resistance modulating factors (RMF), i.e. IC50 drug/IC50 drug + modulator, were found to be proportional to the expression of MDR-1, ranging from 8 to 42 for Adriamycin and from 16 to 63 for vincristine. Transfection with the MDR-1 gene was found to be sufficient to sensitize cells to the modulation by dexniguldipine-HCl. The compound does not affect the expression of the MDR-1 gene. Dexniguldipine-HCl has no effect on a multidrug resistant phenotype caused by a mutation of topoisomerase II. It is concluded that dexniguldipine-HCl modulates multidrug resistance by direct interaction with the P-glycoprotein.

Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias.

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.

Mdr1/P-glycoprotein expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood.

The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question. The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest. We examined the expression of the transmembrane multidrug transporter mdr1/P-glycoprotein in NK-cells (CD56+) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA. As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers. Distinct, but rather inhomogeneous mdr1/P-glycoprotein expression was found in NK-cells enriched from the peripheral blood. NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine. Mdr1/P-glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin. The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A. Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.

Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.

Adrenal catecholamine release by trivalent metallic cations.

The order of effectiveness of trivalent metallic cations in releasing catecholamines from isolated perfused bovine adrenals is La greater than Ce greater than Pr greater than Nd. Other trivalent metals are essentially inactive in this system. A second exposure of the glands to trivalent metals causes little or no secretion. Omission of calcium from the perfusing medium decreases the response to La+3 or Ce+3. Trivalent metallic cations of a relatively narrow range of charge density appear to temporarily open a calcium gate which results in calcium influx and adrenomedullary catecholamine secretion.