mdm2 gene mediates the expression of mdr1 gene and P-glycoprotein in a human glioblastoma cell line.

The overexpression of the multidrug resistance (mdr1) gene and its product, P-glycoprotein (P-gp), is thought to limit the successful chemotherapy of human tumours. The mechanism by which mdr1 gene and P-gp are overexpressed in human tumours, however, is not yet clear. In this report, we show that the mdm2 (murine double minute 2) gene induced the expression of the mdr1 gene and P-gp in human glioblastoma U87-MG cells, which did not express the MDM2 protein or P-gp. The mdm2 gene, in addition, conferred the resistance of U87-MG cells to the apoptotic cell death induced by etoposide (VP-16) or doxorubicin. Furthermore, treatment with mdm2 antisense oligonucleotides inhibited the expression of P-gp in MDM2-expressing U87-MG cells. These findings suggest that the mdm2 gene may play an important role in the development of MDR phenotype in human tumours.     

Characterization and modulation of drug resistance of human paediatric rhabdomyosarcoma cell lines

The role of multidrug resistance (MDR) and p53 functional status in the treatment of paediatric rhabdomyosarcoma is unclear. We have characterized a panel of seven human rhabdomyosarcoma cell lines for MDR and p53 phenotype. None of the cell lines had P-glycoprotein (P-gp) or multidrug resistance-related protein (MRP) detectable by Western blotting, whereas immunohistochemistry suggested that very low levels of MDR proteins may be present in some of the lines. RT-PCR studies indicated that mdr-1, mrp-1 and Irp mRNA was present in 5/7, 7/7 and 5/7 lines respectively. The function of p53 is compromised in six of the lines, either through mutation of the p53 gene or by overexpression of mdm-2. The sensitivity of many of the cell lines to vincristine could be modulated above 2-fold and as high as 16-fold using two modulating agents, PSC833 and VX710 (with VX710 being a significantly more potent modulator of the rhabdomyosarcoma lines). PSC833 also increased vincristine accumulation in all of the lines from 1.2- to 2.2-fold. These results suggest that some of these cell lines have low levels of multidrug resistance. The level of MDR proteins is very low and therefore difficult to detect, but may be sufficient to confer low-level, but clinically relevant, resistance to some cytotoxic agents, especially vincristine. These cell lines will therefore provide a suitable model to test new strategies in treatment and for further understanding relationships between protein expression and drug resistance.     

Activation of MDR1 (P-glycoprotein) gene expression in human cells by protein kinase C agonists.

P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for various lipophilic compounds. MDR1 is expressed in several types of normal human tissues and in a variety of tumors, where its expression has been correlated with resistance to chemotherapy. Some P-glycoprotein-overexpressing multidrug-resistant cell lines contain elevated amounts of protein kinase C (PKC). PKC activation was shown to increase the level of drug resistance in several cell lines, but the functional association of PKC with P-glycoprotein-mediated multidrug resistance remains unclear. We have studied the effects of lymphocyte-activating agents on P-glycoprotein activity in normal human lymphocytes, and found that 12-O-tetradecanoylphorbol-13-acetate (TPA), an efficient agonist of PKC, increased the activity as well as the levels of P-glycoprotein in these cells. TPA also increased P-glycoprotein expression in several cell lines derived from different types of leukemias and solid tumors. The increase in MDR1 gene expression was observed at both the protein and RNA levels. Induction of MDR1 mRNA was apparent as early as two hours after the addition of TPA. Diacylglycerol (DAG), a physiological stimulant of PKC, also increased the expression of MDR1 mRNA and P-glycoprotein. The induction of MDR1 expression by TPA and DAG was suppressed by staurosporine, a protein kinase inhibitor. The results suggest that MDR1 gene expression in different cell types is regulated by a PKC-mediated pathway. This finding has implications for the emergence of multidrug resistance in vitro and in vivo.     

In vitro prevention of the emergence of multidrug resistance in a pediatric rhabdomyosarcoma cell line.

We have established preclinical models for the development of drug resistance to vincristine (a major drug used in the treatment of pediatric rhabdomyosarcoma) using cell lines. The RD cell line has a mutant P53 phenotype and does not have detectable P-glycoprotein (P-gp) or multidrug resistance-related protein (MRP) despite expressing low levels of mdr-1 mRNA, which encodes P-gp and mrp1 mRNA. Resistant variants of RD were derived by exposure to increasing concentrations of vincristine. This was repeated on six occasions, resulting in three cell lines which could tolerate 64 x the IC(50) concentration. Six independent agents were tested for their ability to prevent the development of resistance in this model. Despite at least 10 attempts, resistance did not develop in the presence of the multidrug resistance (MDR) modulators PSC833, VX710, and XR9576. This strongly suggests that these agents may delay or even prevent the development of resistance to vincristine. This was also confirmed in a second rhabdomyosarcoma cell line, Rh30. In contrast, the agents indomethacin (MRP1 modulator), CGP41251 (protein kinase C inhibitor), and dexrazoxane (putative MDR prevention agent) did not affect the development of resistance in the RD model. Characterization of the resistant cell lines indicated the presence of increased mdr-1 and P-gp expression, which resulted in resistance to the agents doxorubicin, etoposide, and vincristine but not cisplatin. The resistance could be modulated using PSC833 or VX710, confirming that functional P-gp is present. No apparent differences were seen between the resistant cell lines derived in the absence and presence of the various agents. These experiments strongly suggest that the development of MDR may be preventable using modulators of MDR and merit clinical studies to test this hypothesis.     

Chemosensitisation of spontaneous multidrug resistance by a 1,4-dihydropyridine analogue and verapamil in human glioma cell lines overexpressing MRP or MDR1.

Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein. We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int. J. Cancer, 58, 860-864, 1994). In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells. The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157. Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells. Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells. The cellular accumulation of [3H]etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively. Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250. However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells. NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.     

用极量稀释法从HeLa细胞中筛选多药耐药的亚克隆细胞株

目的探讨肿瘤细胞亚克隆筛选在多药耐药研究中的应用.方法运用极量稀释法从亲代人宫颈癌HeLa细胞中筛选单个细胞的亚克隆细胞株,并采用Western Blot和MTT法检测亲代HeLa细胞株和亚克隆细胞多药耐药基因1（mdr^-1）的基因产物P-糖蛋白（P-gp）的表达及细胞的多药耐药性.结果共筛选了10个亚克隆细胞株,其中4个亚克隆细胞株有P-gp的表达,而有6个亚克隆株没有P-gp的表达.表达P-gp的克隆细胞株和亲代HeLa细胞株对柔红霉素、环磷酰胺及长春新碱具有多药耐药性,其IC50明显高于不表达P-gp的细胞株（P＜0.05）.结论HeLa细胞株有原发性耐药倾向,而且P-gp表达上具有异质性,亚克隆细胞的筛选既可以用于原发性耐药的研究,也可以筛选出细胞生物学特性一致的敏感细胞株进行继发性耐药细胞模型的建立及相关的研究.     

Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance

Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance. Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds. We therefore initiated a study of PKC and MDR. We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity. The mechanism of the increase appears to be quite different in the two cell lines. The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein. Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin. We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level. The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different. The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.     

Multidrug resistance

Multidrug resistance describes a complex phenotype whose predominant feature is resistance to a wide range of structurally unrelated cytotoxic compounds, many of which are anticancer agents. This phenotype occurs frequently in mammalian cell lines and transplantable tumours selected for resistance to a single drug. Reduced cellular accumulation of the drugs involved appears to account for the resistance. This may be a consequence of reduced drug influx, increased drug efflux, or both. A wide variety of biochemical changes have been identified in multidrug resistant cell lines, the most consistent of which is the increased expression of P-glycoprotein, a conserved, high molecular weight, plasma membrane glycoprotein. The level of P-glycoprotein expression correlates with the degree of drug resistance in a variety of different cell types. In a number of multidrug resistant cell lines, overexpression of P-glycoprotein results from gene amplification. While the function of P-glycoprotein is unknown, independent lines of evidence support the notion that P-glycoprotein is the causative molecule mediating the multidrug resistance phenotype. Significant levels of P-glycoprotein expression have been detected in some biopsy specimens from patients with ovarian and sarcoma tumours. These findings suggest that multidrug resistant tumour cells can occur in human malignancies. The presence of such cells may affect the outcome of chemotherapy.     

Molecular analysis of oncogenes, ras family genes (N-ras, K-ras, H-ras), myc family genes (c-myc, N-myc) and mdm2 in natural killer cell neoplasms.

Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p15INK4B, p16INK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N-ras, K-ras, H-ras, c-myc, N-myc and mdm2 by Southern blot, PCR-SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c-myc and N-myc genes. No overexpression of c-Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to carcinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.     

Molecular Analysis of Oncogenes, ras Family Genes (N-ras, K-ras, H-ras), myc Family Genes (c-myc, N-myc) and mdm2 in Natural Killer Cell Neoplasms

Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p151NK4B, p161NK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N-ras, K-ras, H-ras, c-myc, N-myc and mdm2 by Southern blot, PCR-SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c-myc and N-myc genes. No overexpression of c-Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to car-cinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.     

Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein

Fumitremorgin C (FTC) is a potent and specific chemosensitizing agent in cell lines selected for resistance to mitoxantrone that do not overexpress P-glycoprotein or multidrug resistance protein. The gene encoding a novel transporter, the breast cancer resistance protein (BCRP), was recently found to be overexpressed in a mitoxantrone-selected human colon cell line, S1-M1-3.2, which was used to identify FTC. Because the drug-selected cell line may contain multiple alterations contributing to the multidrug resistance phenotype, we examined the effect of FTC on MCF-7 cells transfected with the BCRP gene. We report that FTC almost completely reverses resistance mediated by BCRP in vitro and is a pharmacological probe for the expression and molecular action of this transporter.     

P-glycoprotein expression in multidrug-resistant human ovarian carcinoma cell lines

Multiple selections with either vinblastine or vincristine in the human ovarian carcinoma cell line SKOV3 resulted in variants with increasing degrees of multidrug resistance. SKOV3 derivatives that span a wide range in resistance (4- to 2000-fold) were obtained and analyzed for P-glycoprotein expression. In general, we observed a progressive increase in P-glycoprotein level (detected by Western blot) that paralleled the increase in multidrug resistance. However, a more detailed analysis of the P-glycoprotein mRNA and gene level indicated that the amount of P-glycoprotein expressed may be under complex control. At low levels of resistance, only an increase in P-glycoprotein mRNA and protein was observed. At intermediate to high levels of resistance P-glycoprotein gene amplification became evident. At the high level of resistance, an example was observed where only the amount of P-glycoprotein was increased without a concomitant increase in mRNA or gene copy. The mechanisms through which the content of P-glycoprotein in the plasma membrane is mediated are not understood; it is possible that the resistant variants identified here represent perturbations at different levels of regulation.     

High levels of MDM2 are not correlated with the presence of wild-type p53 in human malignant mesothelioma cell lines.

Prior analysis of 20 human mesothelioma cell lines for p53 status revealed only two mutations and one p53 null cell line, although p53 expression was detected in most cell lines. In addition, mRNA and protein expression of the retinoblastoma gene product in human mesothelioma cell lines is similar to normal controls. We have tested for p53 induction after exposure to ionising radiation and demonstrate this induction and, to a lesser extent, p21(WAF1) induction, in both normal mesothelial cells and p53-positive mesothelioma cell lines. We postulated that high levels of MDM2 might alter p53 and retinoblastoma tumour-suppressor function in mesothelioma. However, Southern blot analysis for mdm2 indicated that no amplification had occurred in 18 mesothelioma cell lines tested. Steady-state mRNA and protein levels also did not indicate overexpression. These results indicate that high levels of MDM2 are not responsible for inactivating the functions of wild-type p53 or the retinoblastoma gene product during the pathogenesis of malignant mesothelioma.     

Expression of multidrug resistance-associated protein (MRP), MDR1 and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines.

The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene. Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy. In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively. Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide. T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin. On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11. Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells. The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.     

大肠癌多药耐药机制及其逆转剂

大肠癌耐药机制主要与P-糖蛋白、多药耐药性相关蛋白、肺耐药相关蛋白、拓扑异构酶Ⅱ、蛋白激酶C、谷胱甘肽氧化还原系统等因素相关,目前针对大肠癌多药耐药逆转的研究主要有化学药物逆转、中药逆转、基因逆转,并且都取得了一定进展,这将有助于提高大肠癌患者的化疗疗效.