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H J Broxterman

Publications and source records attributed to H J Broxterman.

At least 73 records · Page 4Linked to original sources
PubMed1991

Differentiation dependent expression of P-glycoprotein in the normal and neoplastic human kidney.

Adult renal cell carcinoma (RCC) is clinically resistant to chemotherapy. However, in nephroblastoma (NBL) chemotherapy has increased survival dramatically. We studied the P-glycoprotein (P-gp) expression of 18 RCC and 9 NBL as well as 1 benign renal adenoma and fetal renal tissue using three different monoclonal antibodies (MRK-16, C-219, JSB-1). P-gp was found positive with all three antibodies in 12/18 RCC, while only 2 tumors were completely negative. Staining varied with respect to intensity and number of positive cells [5%-90%]. Intense staining was seen at the apical side of malignant tubules in well differentiated parts of RCC and in tubular structures of the benign renal adenoma. Poorly differentiated parts of the tumors showed less staining. In NBL blastemal parts were negative. In 4/8 specimens showing focal epithelial differentiation, however, the luminal side of more differentiated tubular structures did stain, strongly resembling P-gp staining in the developing fetal human kidney. These results indicate that P-gp expression in normal (fetal) human kidney as well as in benign and malignant tumors derived from this organ depends on the degree of differentiation of tubules, which may have implications for chemotherapy sensitivity in both malignant tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient.

Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line.

Two different mechanisms that contribute to multidrug resistance (MDR) were found in derivatives of the human squamous lung cancer cell line SW-1573. The parental cell line has a low amount of mdr1 P-glycoprotein mRNA. In three independent selections for doxorubicin resistance, MDR variants arose in which mdr1 P-glycoprotein mRNA and protein was not detectable. Selection on higher doxorubicin concentrations gave rise to variants containing high levels of mdr1 mRNA, due to transcriptional activation of the mdr1 gene. Upon continued selection for higher levels of doxorubicin resistance, the mdr1 gene became amplified, resulting in an additional increase in the level of mdr1 mRNA. The cross-resistance pattern of the sublines that lack mdr1 P-glycoprotein expression is different from that seen in the mdr1 overexpressing cells. Both types of MDR cell lines are resistant to doxorubicin, daunorubicin, etoposide, colchicine, gramicidin D, and vincristine. However, in the non-P-glycoprotein-mediated MDR cell lines, resistance levels are lower and a preferential resistance for etoposide is seen.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Differential sensitivity of multi-drug-resistant and -sensitive cells to resistance-modifying agents and the relation with reversal of anthracycline resistance.

Calcium channel blockers, calmodulin inhibitors, and some other classes of non-related compounds reverse multi-drug resistance. In the present study, we found that several resistance modifiers are more toxic for MDR cells than for the corresponding sensitive parent cells, whereas others show the opposite effect. Several calcium channel blockers including bepridil, diltiazem, nifedipine and verapamil, as well as the calmodulin inhibitor trifluoperazine, were more toxic for several MDR cell lines than for the parent cell lines. In contrast, cross-resistance for cyclosporin A and the verapamil analogue Ro II-2933/001 was observed in the MDR/sensitive cell couple CHRC5/AUXB1, probably due to a concentration-dependent stimulation of cell growth in the range of 0-4 microM cyclosporin A and of 1-4 microM Ro II-2933/001. In partially revertant CHRC5 cells, growth inhibition by Ro II-2933/001 at concentrations below I microM, as seen in CHRC5 cells, changed into growth stimulation, and the collateral sensitivity to verapamil and bepridil disappeared almost completely. In the MDR cells CHRC5, 2780AD and DC3F/DMXX, cross-resistance to another calcium channel blocking agent, Ro II-1781/001 (tiapamil), was observed as well. This compound showed exceptional behavior: it induced marked potentiation of Dx cytotoxicity as well as stimulation of Dx accumulation in AUXB1 cells, even at low tiapamil concentrations, but not in the CHRC5 cells, even at high concentrations. It is concluded that resistance modifiers can selectively influence growth of MDR cells via more than one process, and resulting in either strong growth inhibition in MDR cells relative to the effect on sensitive cells or in growth stimulation.

Animals↗
PubMed1990

Cyclosporin A and verapamil have different effects on energy metabolism in multidrug-resistant tumour cells.

Cyclosporin A (Sandimmune) rapidly induced an increase in daunorubicin accumulation in multidrug-resistant human ovarian carcinoma cells (2780AD) and was more potent than verapamil. Steady-state 3H-cyclosporin A accumulation at 37 degrees C in 2780AD cells was 60-70% of that in the sensitive A2780 cells. A rapid increase of ATP consumption and lactate production was induced in 2780AD cells by verapamil, but not by cyclosporin A. These results suggest that the interactions of cyclosporin A and verapamil with P-glycoprotein, which leads to inhibition of drug transport, have a different mechanistic basis.

Adenosine Monophosphate↗
PubMed1990

Distribution of multi-drug resistance-associated P-glycoprotein in normal and neoplastic human tissues. Analysis with 3 monoclonal antibodies recognizing different epitopes of the P-glycoprotein molecule.

P-glycoprotein is a molecule strongly associated with multi-drug resistance to certain cytostatic drugs, including adriamycin, vincristine, and daunorubicin. Using 3 monoclonal antibodies directed at different epitopes of this molecule (JSB-1, MRK16, C219) we investigated the tissue distribution of P-glycoprotein in normal and malignant human tissues, employing a routine immunoperoxidase technique. P-glycoprotein was found in the gastrointestinal epithelium, epithelia of the bronchi, mammary gland, pancreatic ducts, renal tubules, prostate gland, salivary gland, sweat glands of the skin, as well as in bile canaliculi and ductules, the adrenal and in endothelium of capillaries in various organs, most notably the brain. Mostly the pattern of reactivity of the 3 antibodies was similar, but some distinctly different staining reactions were seen. Reactivity was found in a variety of human tumors, arising from tissues normally expressing P-glycoprotein. Knowledge of the distribution and function of this molecule in both normal and malignant tissues may predict resistance and thus may influence choice of therapy. The role of P-glycoprotein in normal tissues and its implications for chemotherapy is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Quantitative determination of factors contributing to doxorubicin resistance in multidrug-resistant cells.

There is a large discrepancy between the changes in drug accumulation and the changes in drug cytotoxicity that accompany development of anthracycline resistance in multidrug-resistant cells. In our study, a quantitative relationship has been established between reversal of multidrug resistance by resistance modifiers and a concomitant decrease in intracellular levels of doxorubicin measured at equitoxic concentrations (IC50) in CHRC5 and 2780AD multidrug-resistant cells. (IC50 = concentration required for 50% growth inhibition.) We have demonstrated that resistance modifiers like verapamil and Ro 11-2933/001 act by increasing the effectiveness of intracellular doxorubicin, apparently by inducing redistribution of the drug from the cytoplasm to the nucleus of a multidrug-resistant cell, as shown by quantitative fluorescence microscopy. At complete reversal of resistance, as measured directly or inferred by extrapolation, the amount of intracellular doxorubicin at the IC50 as well as the ratio of nuclear doxorubicin to cytoplasmic doxorubicin were the same as those in sensitive cells. These results offer an explanation for the frequently observed discrepancies between drug accumulation and cytotoxicity and also show quantitatively that a decrease in drug accumulation and a change in intracellular drug distribution together are the only determinants of doxorubicin resistance in the multidrug-resistant cells studied.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells.

A 3H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with 3H2. 1-[3H]dMM as well as its precursor 1-[3H]2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-[3H]dMM. Thus, 1-[3H]dMM had chemical and biological properties identical with authentic dMM. Uptake of [14C]mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-[3H]dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-[3H]dMM by K562 cells was observed at 0 degrees C.

1-Deoxynojirimycin↗
PubMed1989

Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells.

Four multidrug-resistant variants of the human squamous lung cancer cell line SW-1573 with levels of doxorubicin resistance ranging from 10- to 2000-fold were characterized with respect to drug accumulation and efflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression. For all these parameters except the antioxidant defenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity (as measured for doxorubicin) progressively decreased, initial drug efflux rates (as measured for daunorubicin) progressively increased, while the subcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cytoplasmic" pattern. Our data suggest that in the present set of cell lines the same mechanism of resistance is operating at all levels of doxorubicin resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Glycolysis in P-glycoprotein-overexpressing human tumor cell lines. Effects of resistance-modifying agents.

We show that drugs, such as verapamil, which reverse multidrug resistance (MDR), in P-glycoprotein-overexpressing tumor cells, increased the rate of lactate production in four human MDR cell lines, but not in the parent, sensitive cell lines. The effect on glycolytic rate was maximal at a medium concentration of 2 microM verapamil. The glycolytic rate in sensitive (A2780) and MDR 2780AD) cells showed the same pH dependence, but the effect of verapamil was seen only in 2780AD cells at all pH values investigated (6.6, 7.4 and 8.2). A series of drugs such as nigericin, oligomycin, amiloride and monensin had similar effects in the two cells. Phorbol myristate acetate increased lactate formation in neither cell line. Verapamil induced an extra amount of ATP consumption in P-glycoprotein-expressing 2780AD cells of approx. 25 pmol/s per 10(6) cells, which was estimated to be about 10% of cellular energy turnover.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Immunohistochemical detection of P-glycoprotein in human tumor cells with a low degree of drug resistance.

We report the immunohistochemical detection of the 170-180 kDa multi-drug-resistance-related P-glycoprotein in human tumor cells with a low level of resistance. A series of human squamous lung cancer cell lines with increasing levels of resistance to doxorubicin (DOX) was developed and stained for P-glycoprotein, using the JSB-IMAb. Subline SW1573/50A with a 4- to 6-fold cross-resistance to daunorubicin (DNR) and vincristine (VCR) showed rather uniform positive staining for P-glycoprotein apparently at cytoplasmic sites. Only in cells with higher degrees of resistance (greater than 10-fold) could plasma-membrane-associated P-glycoprotein be made visible. DNR efflux was increased in SW1573/50A as compared to the parent line SW1573 (52 and 70% DNR were retained during 3 min efflux respectively). Verapamil partially reversed DNR and VCR resistance in SW1573/50A. Cells obtained from a metastasized renal cell carcinoma and cultured in vitro stained in a similar way to SW1573/50A and showed some sensitivity to verapamil modulation of VCR cytotoxicity. Our results suggest that weakly resistant cancer cells obtained from patients can be routinely detected with JSB-I on cytospins, and implicate that in such weakly resistant cells P-glycoprotein may be present, while plasma membrane expression is not yet readily detectable.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: association with level of drug resistance and drug accumulation.

Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells. The level of staining intensity for P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system. Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation less than or equal to 13%). Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox6 and 8226/Dox40) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D0) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P less than 0.0005). Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D0. P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining. The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox6 and 8226/Dox40) by comparing the optical densities for individual cells. Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Monoclonal antibody JSB-1 detects a highly conserved epitope on the P-glycoprotein associated with multi-drug-resistance.

Resistance to multiple chemotherapeutic agents is a common clinical problem in the treatment of cancer. This resistance may occur before primary therapy or be acquired during treatment. We have generated a monoclonal antibody (MAb) (JSB-I), specific for a conserved epitope on the plasma membrane 170- to 180-kDa glycoprotein, the expression of which is strongly correlated with the degree of multi-drug resistance (MDR). JSB-I strongly binds to both Chinese-hamster-derived MDR cell lines and human MDR cell lines, including cell lines derived from lung and ovary. A drug-sensitive revertant line, and the corresponding drug-sensitive parent lines, showed only weak reactivity or none at all. JSB-I reacts strongly to air-dried or acetone-fixed cells and therefore has potential value for diagnostic detection of MDR cells in human tumor samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein.

An overexpression of plasma membrane 170-180 kDa P-glycoproteins is consistently found in multidrug-resistant (MDR) cell lines. In this study MRK-16, a monoclonal antibody (mAb) reacting with P-glycoprotein is used to study the putative functional role of this protein in vincristine (VCR) and daunorubicin (DNR) cellular accumulation in the MDR human ovarian carcinoma cell line 2780AD. We established that this cell line is highly cross-resistant to vincristine and daunomycin, related to a greatly reduced drug accumulation. Verapamil (Vp) (8 microM) caused a 3.6-fold increase in DNR as well as VCR accumulation. Exposition of 2780AD cells to MRK-16 led to an increase of 30% in cellular accumulation of VCR, both in normal growth medium as well as in medium without added glucose and with sodium azide, which largely depleted cellular ATP levels. No increase in DNR accumulation was found under these conditions. However, in the presence of 8 microM Vp, MRK-16 increased not only VCR but also DNR accumulation with about 30%. The relative increase of DNR accumulation was constant in a concentration range of 0.2-4 microM DNR. These data indicate that mAbs against P-glycoprotein might potentiate the action of calcium antagonists like Vp to increase cellular anthracycline accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Synthesis of cyclic oligonucleotides by a modified phosphotriester approach.

Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].

Drug Stability↗
PubMed1988

Recycling glycoproteins do not return to the cis-Golgi.

Recycling of a number of glycoproteins along the site of action of mannosidase I (the distal endoplasmic reticulum/cis-Golgi) was followed in several different cell lines. Treatment of cells with 1-deoxymannojirimycin (dMM) produced high mannose oligosaccharides at positions otherwise occupied by complex-type oligosaccharides in these glycoproteins. Conversion of high-mannose-type oligosaccharides to complex oligosaccharides of proteins initially synthesized in the presence of dMM was used as a marker for recycling of glycoproteins along the site of action of dMM. In contrast to findings reported by Snider and Rogers (Snider, M. D., and O. C. Rogers. 1986. J. Cell Biol. 103:265-275), removal of dMM did not result in reconversion of high-mannose oligosaccharides to complex-type sugars, even after prolonged periods of culture. We conclude that surface glycoproteins do not recycle through the cis-medial Golgi elements.

1-Deoxynojirimycin↗