EXPRESSION OF THE MULTIDRUG RESISTANCE GENE IN LEUKEMIC CELLS

P-glycoprotein(Pgp; Mr=170,000) to encoded by a family of genes-Multldrug resistance gene. The Pgp has been demonstrated to mediate resistance to multiple structurally dissimilar drugs, which fuctions as an energy- dependent efflux pump so that a cell with high level of mdr expression can more effectively eliminate cytotoxic drugs. In this report, a simplified method for analysis of clinical samples and assess the level of gene expression was set up. Furthermore, by using 32P labelled mdr- 1 cDNA as the probe and RNA dot blotting the mdr-mRNAs from 5 cases of myeloblastic leukemia cells were analysed. It was shown that the level of mdr-1 expression In different myeloleukeic cells was various and reduced In one case after remission. The established method for mRNA analysis could be generalized for evaluating the level of mRNA in clinical samples.     

mdr1基因表达与人肺腺癌多药抗药细胞系LC-3／CDDP抗药性的关系

目的:探讨多药抗药基因(mdr_1基因)表达与人肺腺癌多药抗药细胞系LC-3/CDDP抗药性产生的关系.方法:应用地高辛标记的mdr_1 cDNA探针,采用斑点杂交方法,检测该细胞系中的mdr_1mRNA含量,同时以免疫细胞化学染色方法,检测P-糖蛋白(P-gP)表达,并与亲代(LC-3)细胞比较.结果:LC-3/CDDP细胞存在mdr_1基因过表达.结论:mdr_1基因过表达是LC—3/CDDP细胞系产抗药性的机制之一.     

Multidrug resistance phenotype in the RMS-GR human rhabdomyosarcoma cell line obtained after polychemotherapy.

Classical cytotoxic treatment of rhabdomyosarcoma (RMS), the most common soft tissue malignacy in children, is often accompanied by significant morbidity and poor response. Chemotherapy may induce multidrug resistance (MDR) associated with the expression of P-glycoprotein, a drug efflux pump which modifies the sensitivity of tumoral cells to drugs. To analyze MDR in RMS we used the RMS-GR cell line, obtained from an embryonal rhabdomyosarcoma treated in vivo with polychemotherapy. The RMS-GR cells showed cross-resistance to vincristine, doxorubicin and actinomycin D, the drugs of choice in the conventional treatment of RMS. Polymerase chain reaction (PCR) analysis showed that these RMS cells overexpressed mdr1/P-glycoprotein. The pattern of resistance and the level of P-glycoprotein expression were similar to those found in the resistant RMS TE.32.7.DAC cell line obtained in vitro. Southern blot analysis showed that mdr1 overexpression was not due to amplification of the gene. Our results showed that the in vivo treatment of embryonal RMS may induce an MDR phenotype mediated by mdr1/P-glycoprotein in RMS cells.     

Multidrug Resistance Phenotype in the RMS-GR Human Rhabdomyosarcoma Cell Line Obtained after Polychemotherapy

Classical cytotoxic treatment of rhabdomyosarcoma (RMS), the most common soft tissue malignancy in children, is often accompanied by significant morbidity and poor response. Chemotherapy may induce multidrug resistance (MDR) associated with the expression of P-glycoprotein, a drug efflux pump which modifies the sensitivity of tumoral cells to drugs. To analyze MDR in RMS we used the RMS-GR cell line, obtained from an embryonal rhabdomyosarcoma treated in vivo with polychemotherapy. The RMS-GR cells showed cross-resistance to vincristine, doxorubicin and actinomycin D, the drugs of choice in the conventional treatment of RMS. Polymerase chain reaction (PCR) analysis showed that these RMS cells overexpressed mdr1 /P-glycoprotein. The pattern of resistance and the level of P-glycoprotein expression were similar to those found in the resistant RMS TE.32.7.DAC cell line obtained in vitro . Southern blot analysis showed that mdr1 overexpression was not due to amplification of the gene. Our results showed that the in vivo treatment of embryonal RMS may induce an MDR phenotype mediated by mdr1 /P-glycoprotein in RMS cells.     

腺相关病毒载体介导外源基因导入人外周血干细胞的研究

目的：观察腺相关病毒载体介导外源基因导入人外周血干细胞的转染效率及表达。方法：本文采用腺相关病毒（adeno-associated virus,AAV）载体介导的基因转移方法，将绿色荧光蛋白（green fluorescent protein,GFP）基因和人多药耐药基因（multi-drug resitance,MDR1）导入人外周造血干／祖细胞，并对转染效率和转基因造血细胞的耐药性进行了初步研究。结果：rAAV/GFP感染CD34阳性细胞后，在荧光显微镜下观察，约30％细胞中可见绿色荧光。将rAAV/MDR1重组病毒感染CD34阳性细胞，经PCR和MTT法证实，导入MDR1基因的CD34阳性细胞对秋水仙素的耐药性与未导入MDR1基因的细胞有明显差异。结论：腺相关病毒载体能有效地将外源基因导入外周血CD34阳性细胞，并可在其中有效地表达。     

Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates

The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters. Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein. This response may protect cells from the cytotoxic effects of these compounds. In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P- glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression. In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy- acetylaminofluorene. The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5- fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene. In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export. These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N- hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein. The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.      

Overexpression of the mdr1 gene in blast cells from patients with acute myelocytic leukemia is associated with decreased anthracycline accumulation that can be restored by cyclosporin-A

Typical multi-drug resistance (MDR) in human and animal cell lines is caused by overactivity of a unidirectional drug efflux pump. This pump is composed of a 170-kDa transmembrane glycoprotein (P-glycoprotein) that is encoded by the so-called mdr1 gene. The functionally relevant characteristic of MDR cells is a defect in drug accumulation that can be restored by agents which inhibit the P-glycoprotein pump. The purpose of our study was to find out whether P-glycoprotein inhibitors could increase the daunorubicin (DNR) accumulation in acute myelocytic leukemia (AML) cells, overexpressing the mdr1 gene. Using dot blot analysis with an mdr1-specific cDNA probe, we identified leukemic cell samples, obtained from chemotherapy-resistant AML patients, that had relatively high levels of mdr1 expression. These leukemic cells showed a reduced ability to accumulate DNR in vitro, as quantitated by flow cytometry. Addition of cyclosporin-A (Cy-A), a drug known to inhibit the P-glycoprotein pump, to the incubation medium resulted in an increase (up to 60%) in steady-state drug uptake by the leukemic cells. The degree of Cy-A-induced increase in drug accumulation in the leukemic cells correlated approximately with the level of overexpression of the mdr1 gene. Our data indicate that Cy-A is a good candidate for combination chemotherapy with cytotoxic drugs in clinical trials, aimed at the treatment of drug resistance in AML.     

The development and characterization of vinca alkaloid-resistant Caco-2 human colorectal cell lines expressing mdr-1

Several novel cell lines with variable resistance to Vinca alkaloids have been derived from the Caco-2 human colorectal carcinoma cell line. Parental Caco-2 cells were found by PCR analysis and immunofluorescence studies to produce a low amount of the mdr-1 gene product (P-glycoprotein) that may well be clinically significant. These cells, which were initially highly sensitive to desacetylvinblastine sulfate (DAVLB sulfate) were selected, without mutagenesis, through continuous culture with increasing concentrations of DAVLB sulfate over a 335-day period. This selection resulted in cell lines that displayed an mdr (multiple-drug-resistance) cross-resistance profile that could be reversed with agents such as verapamil and vindoline. During the selection process the amount of mdr-I mRNA present, the extent of gene amplification and the amount of gp 170 expressed all correlated well with the level of drug resistance. However, this correlation does not hold in the absence of selective pressure for the more resistant cell lines where gene amplification and the amount of P-glycoprotein present remained constant while the level of drug resistance and the amount of mdr-l mRNA present declined. These cell lines are potential models for studying mdr-1 gene expression and drug resistance in human epithelial malignancies.     

Chemosensitization of murine fibrosarcoma cells to drugs affected by the multidrug resistance phenotype by the antidepressant trazodone - an experimental-model for the reversal of intrinsic drug-resistance

A variety of resistance phenotypes to cytotoxic agents in bacteria, protozoa parasites and mammalian cells are mediated by evolutionarily conserved proteins of the mdr family. The finding that chloroquine resistance in the malarial parasite, Plasmodium falciparum, that is mediated by an mdr-1 gene product can be circumvented by tricyclic antidepressant drugs has stimulated the present study to assess whether this class of agents might also modulate the multidrug resistance (MDR) phenotype(s) in mammalian tumor cells. The possible chemosensitizing effects of nine antidepressant drugs have been tested against the UV-2237M murine fibrosarcoma line and its MDR variant. At nontoxic concentrations all nine antidepressants markedly enhanced the cytotoxicity of ADR against the parental cells but were much less effective against the MDR cells. The most active antidepressant, trazodone, also enhanced the cytotoxicities of vinblastine and vincristine, but not those of actinomycin D, mitomycin C, or 5-fluorouracil. The parental cells treated with trazodone exhibited an increased accumulation of intracellular ADR, but lacked detectable alterations in the expression and drug-binding activity of plasma membrane P-glycoprotein, and trazodone did not affect the activities of isolated protein kinase C and calmodulin. These data suggest that the antidepressant drug trazodone may be useful in the reversal of the intrinsic drug resistance of tumor cells that express low levels of P-glycoprotein.     

人多药耐药基因mdr1野生型mRNA转导脐血干细胞提高其对化疗耐药性的体外研究

目的:将人类多药耐药基因(mdr1)全长野生型mRNA 转导至人脐带血干细胞,分析其P糖蛋白的表达及干细胞的耐药性。方法: 通过体外转录方法经人野生型mdr1 基因全长cDNA制备相应的mRNA, 经脂质体介导转染人造血干细胞,采用RT- PCR 和Western blot 检测转染效果,流式细胞仪检测蛋白表达、体外培养及细胞活率计数观察耐药性。结果: 转染后P糖蛋白表达丰度为9 .05%,对照组为1. 01%; 转染后具有Rhodamin 123 排泌功能的细胞占98 .72%, 对照组为66. 83%;转染后细胞耐药性明显增强,P=0 .002。结论: 通过脂质体介导,将人类mdr1基因野生型全长mRNA转导入脐血干细胞,增加了P糖蛋白的表达,提高了细胞的耐药性。该系细胞可用于缓解和治疗恶性肿瘤化疗带来的骨髓抑制。     

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.     

Detection of multidrug resistance markers, P-glycoprotein and mdr1 mRNA, in human leukemia cells

We have examined the expression of P-glycoprotein in clinical leukemic cell samples by using a monoclonal antibody (MRK16) against P-glycoprotein. We found that leukemia cells isolated from 3 out of 6 patients with blast crisis of chronic myelogenous leukemia were reactive to MRK16. These 3 cell lines expressed high levels of mdr1 mRNA, which codes for P-glycoprotein. The present result indicates that the clinically refractory state of the tumor may be predicted in part by determining P-glycoprotein expression using the monoclonal antibody against P-glycoprotein, and the mdr1 probe.     

Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.