Selective growth-inhibition of multidrug-resistant CHO-cells by the monoclonal antibody 265/F4.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
T Efferth
Publications and source records attributed to T Efferth.
Resistance to doxorubicin in tumor cells in vitro and in vivo after pretreatment with verapamil.
Sarcoma 180 (S180) cells pretreated with verapamil increase their resistance to doxorubicin in vitro. This result could be confirmed by in vivo experiments using P388 cells. The mechanism for this resistance is different from the P-glycoprotein-mediated mechanism characteristic for multidrug-resistant cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Overexpression of P-glycoprotein in human lung carcinoma xenografts after fractionated irradiation in vivo.
In vivo exposure of a human epidermoid lung carcinoma xenograft to seven irradiation treatments of 10 Gy in consecutive passages resulted in expression of resistance to vincristine. This about threefold drug resistance was detectable with a single dose of 1 mg/kg vincristine. Characterization of the radiation-pretreated subline showed that overexpression of P-glycoprotein, as determined by immunofluorescence and Mabs C219 and 265/F4, occurred in this tumor. After six X-ray fractions, only single positive cells were observed, whereas seven fractions produced an intense immunofluorescent reaction with both antibodies. Southern blot analyses indicated that no gene amplification had occurred. This result shows that irradiation can influence expression of P-glycoprotein and in this way influences drug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Relationship of DNA ploidy to chemoresistance of tumors as measured by in vitro tests.
To examine whether patients with aneuploid tumors might derive more benefit from chemotherapy than would patients with diploid tumors, predictive tests for determining resistance in human tumors were carried out and the test results compared with the DNA ploidy of the corresponding tumors. Multidrug-resistance in 15 kidney carcinomas grown as primary cultures was determined by immunofluorescence by Mab C219, which is specific for the plasma membrane glyco-protein P-170, and by the use of tritiated nucleotide incorporation after addition of doxorubicin. Aneuploid tumors had a higher tendency to be more sensitive than diploid tumors, but the correlation was not significant. This was confirmed by reanalyzing our earlier data on ovarian and lung cancers. In conclusion, DNA measurement using flow cytometry does not appear to be a suitable tool for prediction of resistance of human tumors to chemotherapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Correlations between natural resistance to doxorubicin, proliferative activity, and expression of P-glycoprotein 170 in human kidney tumor cell lines.
The natural resistance to doxorubicin of 15 human renal carcinoma cell lines was analyzed and compared to proliferative activity and expression of P-glycoprotein. We found a significant negative correlation between proliferative activity and natural resistance to doxorubicin, as well as between proliferative activity and the expression of P-glycoprotein. A positive correlation between resistance and expression of P-glycoprotein was found.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Overexpression of P-glycoprotein in rat hepatocellular carcinomas induced with N-nitrosomorpholine.
The most frequently reported alteration of multidrug resistance (MDR) is the overexpression of the 170 kd membrane glycoprotein. An increased expression of the MDR-gene in pre-neoplastic and neoplastic liver lesions produced experimentally by different carcinogens has been reported. As shown in rat liver by stop experiments, specific carcinogen-induced alterations can be separated from non-specific, toxic changes. Therefore, we induced rat hepatocellular carcinomas with N-nitrosomorpholine and investigated whether expression of the MDR-gene also takes place in hepatocellular carcinomas after withdrawal of the carcinogen. Using mAb C219 against P-glycoprotein, both normal liver and hepatocellular carcinomas show specific immunoreactivity by means of immunofluorescence and immunohistochemistry. In hepatocellular carcinomas, however, the reaction is increased if compared to normal liver of untreated control animals. These results were confirmed by immunoblotting. Using the cDNA probe 1.5, a significant increase in MDR-gene transcripts was found in hepatocellular carcinomas as compared to normal liver.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Detection of drug resistance and P-glycoprotein in human renal cell carcinomas.
Expression of the multidrug-resistance gene product P-glycoprotein (P-170) was screened in 21 untreated human renal cell carcinomas using monoclonal antibodies (265/F4, C219) and immunoperoxidase staining. The inherent drug resistance of the same samples against doxorubicin was established by a short-term chemoresistance test in order to investigate the association between the expression of P-170 and intrinsic drug resistance in kidney cancers. P-glycoprotein could be demonstrated in 10 cases. Using the short-term test for predicting resistance, 17 resistant and 4 sensitive cancers were found. In vitro 10 of 17 resistant tumors revealed an increase of P-glycoprotein. On the other hand, in the sensitive tumor in vitro, an expression of P-glycoprotein could not be demonstrated. This investigation reveals that intrinsic drug resistance exists in many renal cell carcinomas and it is associated at least in part with increased expression of P-glycoprotein. The immunohistochemical results suggest that the presence of the P-glycoprotein may be useful as a marker for screening the multidrug-resistant phenotype in renal cell carcinomas and as an indicator of the therapeutic efficacy of multidrug-resistant kidney cancers.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Detection of P-glycoprotein in human leukemias using monoclonal antibodies.
Overexpression of a Mr 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines. Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein (greater than 5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Induced multidrug resistance in murine leukemia L1210 and associated changes in a surface-membrane glycoprotein.
The aim of this study was to find out whether only resistant cells of the "multidrug-resistant" phenotype show the described changes of plasma membrane glycoprotein (170 kDa) or whether resistant cells that do not express this phenotype reveal corresponding results. Doxorubicin-resistant (L1210dox) and daunorubicin-resistant L1210 ascites tumor cells (L1210dnr) (multidrug-resistant tumor cells) were therefore compared with cytosine-arabino-side-resistant (L1210AraC) and cyclophosphamide-resistant L1210 ascites tumor cells (L1210ctx) (not multi-drug-resistant tumor cells). The resistant cell lines were generated in vivo in tumor-bearing mice and the resistance to cytostatic agents was evaluated in vivo and in vitro. Using the accumulation assay with rhodamine-123, the multidrug resistance can be detected. In order to determine alterations in the plasma membranes we used the monoclonal antibodies 265/F4 and C219, which were prepared against the membrane glycoprotein P170 (170 kDa) in colchicin-resistant Chinese hamster ovary cells. The results demonstrate that L1210dox and L1210dnr tumor cells show an intense immunostaining by the streptavidin/biotinylated-peroxidase-complex method and by the streptavidin/biotin/phycoerythrin immunofluorescence method. In contrast no specific immunostaining was observed in parental (sensitive) and L1210AraC or L1210ctx tumor lines. The results were confirmed by immunoblotting. To determine whether multidrug-resistant DNA sequences were expressed in the multidrug-resistant tumor cells, Northern blots with RNA od sensitive and resistant cells were performed using the clone pcDR1.5. Elevated RNA levels were detected only in resistant cells with the multidrug-resistant phenotype. Thus, the results of this study demonstrate that only resistant cells with the multidrug-resistant phenotype show an increased expression of the membrane 170-kDa glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Detection of drug resistance in human ovarian carcinoma.
Drug-resistant cancer cells with the multidrug-resistance phenotype show overexpression of P-glycoprotein, and we therefore tested carcinoma tissue from five patients with stage III or IV ovarian cancer for P-glycoprotein using 265/F4 and C 219 monoclonal antibodies, prepared against membrane glycoproteins in colchicine-resistant CHO cells. Using immunofluorescence and immunoblotting techniques, one of the tumors showed a positive reaction. Using the pcDR 1.5 clone we found that the same cancer tissue had elevated expression of the genes responsible for multidrug resistance. The demonstration of elevated P-glycoprotein in ovarian carcinomas indicates that P-glycoprotein overexpression is not limited to experimental tumor models.
Carcinoma↗
Detection of the multidrug resistant phenotype in human tumours by monoclonal antibodies and the streptavidin-biotinylated phycoerythrin complex method.
The aim of this study was to find out whether the membrane glycoprotein P-170 can be detected in human tumours with both acquired and intrinsic resistance to chemotherapeutic agents using monoclonal antibodies (265/F4 and C219) and the streptavidin-biotinylated phycoerythrin complex method. Pretreated leukaemia cells and untreated lung and ovarian carcinomas were analysed. Two plasmacytomas and one leukaemia expressed high levels of P-glycoprotein, whereas two leukaemias showed moderate, and three leukaemias no expression of this protein. The intrinsic resistance was analysed with a panel of four human epidermoid lung cancer xenografts grown in nude mice. The expression of P-glycoprotein could be correlated with the degree of resistance. In addition, one out of five ovarian carcinomas revealed a high level of P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein expression in treated and untreated human breast cancer.
The expression of P-glycoprotein in primary and recurrent human breast cancer was investigated by means of immunohistochemistry, using a monoclonal antibody (C219) and the streptavidin-biotin-peroxidase method. Twelve patients received no chemotherapeutic treatment. The other 11 patients were treated with chemotherapy, and all developed clinical resistance to it. No or only minimal reactivity was found in specimens coming from the untreated patients (12 cases) or from patients treated with substances not involved in the multidrug resistance phenomenon (four cases). In contrast, three out of seven tumours from patients treated with multidrug resistance related substances showed clear reactivity (positive staining in more than 20% of the tumour cells). In one of these cases, where specimens of the tumour could be studied before and after treatment, an association between the latter and expression of P-glycoprotein was suggested. Finally, this marked expression of P-glycoprotein only took place in tumours treated over a longer space of time (five courses or more of multidrug resistance related chemotherapy).
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Acquired drug resistance in human lung carcinoma xenografts.
The development of resistance to vincristine and dactinomycin has been investigated in a human epidermoid lung xenograft line grown in nude mice. With 1 mg/kg BW vincristine and 0.5 mg/kg BW dactinomycin per passage, resistance of the solid tumors was visible already at the second transplantation. The vincristine-resistant subline showed cross-resistance to dactinomycin and doxorubicin, and the dactinomycin-resistant subline only to vincristine. To determine whether multidrug resistance genes were overexpressed in the resistant sublines, slot blots were performed using the cDNA probe pcDR 1.5. Slightly elevated RNA levels could be detected in the vincristine-resistant subline and in the dactinomycin-resistant subline.
Animals↗
Rapid detection assays for multidrug resistance.
In attempts to develop simple and rapid assays for detection of multidrug resistance (MDR) doxorubicin-resistant S180 (S180DOX), colchicine-resistant CHO (CHOCOL) and cytarabine (cytosine-arabinoside)-resistant L1210 cell lines (L1210AraC) were investigated. S180DOX and CHOCOL cells express the MDR-phenotype while L1210AraC does not. Using a previously described short-term assay firstly, inhibition of incorporation of radioactive nucleic acid precursors into tumour cells after addition of doxorubicin was measured. Secondly, the accumulation of the fluorescent dye rhodamine 123 (R123) in the different cell lines were analyzed. It could be observed that the resistant S180DOX and CHOCOL cells needed significantly more time to accumulate R123 than their sensitive parental cell lines or L1210AraC cells. Therefore, both assays are adequate tools for the rapid detection of MDR.
Animals↗
Detection of murine S180 cells expressing a multidrug resistance phenotype using different in vitro test systems and a monoclonal antibody.
Doxorubicin (adriamycin) preconditioned S180 cells were more resistant to doxorubicin. The resistance was detected by three different methods (short-term test, colony assay, tissue culture assay). The doxorubicin-resistant S180 cells express the pleitrop drug resistance phenotype. There exists a multidrug resistance to doxorubicin, dactinomycin (actinomycin D), vincristine and colchicine. In addition, collateral sensitivity was found to fluorouracil (5-fluorouracil) and methotrexate. This multidrug phenotype is in accordance with the pleiotropic phenotype of colchicine-resistant CHO cells. Resistant S180 cells express a glycoprotein of Mr 170 kd determined by indirect immunofluorescence using a monoclonal antibody (Mab 265/F4) to the P-glycoprotein of colchicine-resistant CHO-cells. The P-glycoprotein could be an important prognostic factors for tumors with multidrug resistance.
Animals↗
5-Azacytidine modulates the response of sensitive and multidrug-resistant K562 leukemic cells to cytostatic drugs.
In an endeavor to improve responsiveness of tumor cells to drug combination treatments, we analyzed the effect of 5-azacytidine (5AC) as a model compound for a new class of drugs, DNA-demethylating agents. We used parental K562/WT chronic myelogenous leukemia cells and a multidrug-resistant subline thereof, K562/ADM. Multidrug-resistant cells were more resistant to daunorubicin, but more sensitive to cisplatin than parental K562 cells as measured by growth inhibition and apoptosis assays. Resistance to daunorubicin can be explained by amplification of the MDR1 drug transporter gene. Cisplatin induced more DNA damage in specific genes and in the entire genome of K562/ADM cells compared to K562/WT cells using PCR stop assays and atomic absorption spectroscopy. Pretreatment with 5AC modulated the response of K562/ADM cells toward MDR-type drugs (daunorubicin, vincristine, etoposide) and reduced function and expression of MDR1 as analyzed by flow cytometry and RT-PCR. Analysis of CpG island methylation in the promotor region of the MDR1 gene by bisulfite sequencing and a methylation-sensitive HpaII-digestion/PCR approach revealed that methylation of the MDR1 promotor of K562/ADM cells was greater than in K562/WT cells. 5AC treatment completely abolished MDR1 promotor methylation. The unexpected observation that DNA demethylation by 5AC rather decreases than increases MDR1 expression in K5612/ADM cells points to still unexplored sequences in the MDR1 promotor whose transcriptional activity may be affected by the methylation status. 5AC pretreatment also modulated K562/WT and K562/ADM cells to non-MDR-type drugs such as cisplatin and increased cisplatin-induced DNA damage.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Modulation of P-glycoprotein-mediated multidrug resistance by monoclonal antibodies, immunotoxins or antisense oligodeoxynucleotides in kidney carcinoma and normal kidney cells.
In the present investigation we show that both monoclonal antibodies against P-170 (265/F4, MRK 16, and HYB 612) and 265/F4-ricin alpha-chain immunotoxin are useful tools for the eradication of kidney carcinoma and normal kidney primary cell cultures with high P-170 expression, whereas kidney tumor and normal kidney cell lines with low amounts of P-170 are less affected. Furthermore, we found that the expression of P-170 in both kidney tumor and normal kidney cell cultures with high amounts of P-170 was inhibited by antisense oligodeoxynucleotides complementary to base pairs -9 to +6 of the MDR1 gene. These data indicate that these approaches for the eradication of P-170 expressing multidrug-resistant cells are not limited to tumors but also affect P-170 expressing normal cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Intrinsic overexpression of two different mechanisms of resistance to chemotherapy (P-glycoprotein and GST-pi) in human endometrial carcinoma.
Endometrial carcinoma is considered a tumor which does not respond well to chemotherapeutic treatment. Among the various mechanisms of resistance to chemotherapy which are under investigation, two of them (multidrug resistance, mediated by P-glycoprotein, and glutathione-S-transferase-pi [GST-pi] overexpression) are of great interest for gynecologic oncologists, because they involve several drugs commonly used in practice, among which Adriamycin and cisplatin are probably the most important ones. We have studied 23 human endometrial carcinomas of different histological varieties and 3 normal endometrial samples for the overexpression of both P-glycoprotein and GST-pi by means of immunohistochemistry. Both resistance markers were detectable in all tumor samples, and in normal endometrial tissue as well. The concomitant intrinsic overexpression of these two resistance mechanisms may in part explain why these tumors tend to be extremely resistant to chemotherapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗