mRNA Expression Levels Of MDR1 Research Articles

Correlation between cyclin A gene expression in adult patients with acute leukemia and drug resistance.

In order to investigate the relationship between the expression of cyclin A and drug resistance in adult patients with acute leukemia (AL), the mRNA expression of cyclin A, mdr1, Top II alpha, bcl-2 was detected in 64 adult patients with AL and 20 normal controls by semi-reverse transcription polymerase chain reaction (semi-RT-PCR). It was found that the cyclin A and Top II alpha mRNA expression levels in drug resistant group were significantly lower than in sensitive group (P < 0.01). Under the same experimental condition no cyclin A mRNA expression was detectable in all normal controls. The mdr1 and bcl-2 mRNA expression levels in resistant group were significantly higher than in sensitive group (P < 0.01), cyclin A and Top II alpha gene expression levels were closely correlated (rs = +0.794, P = 0.000, n = 64) in all AL patients, but cyclin A was not correlated with mdr1 and bcl-2 gene expression levels. In drug resistant group there was a negative correlation between the gene expression levels of cyclin A and mdr1 (rs = -0.337, P = 0.029). The 10 AL patients with positive lower expression of both cyclin A and Top II alpha were all resistant to drugs. Logistic regression of Binary analysis showed the correlation between the lower expression of cyclin A and drug resistance. It was concluded that lower expression of cyclin A gene might be an unfavorable prognostic factor for patients with AL, and detection of both cyclin A and Top II alpha gene expression would predict drug resistance in AL patients.

Reversal effects of nomegestrol acetate on multidrug resistance in adriamycin-resistant MCF7 breast cancer cell line.

Chemotherapy is important in the systematic treatment of breast cancer. To enhance the response of tumours to chemotherapy, attention has been focused on agents to reverse multidrug resistance (MDR) and on the sensitivity of tumour cells to chemical drugs. Hundreds of reversal drugs have been found in vitro, but their clinical application has been limited because of their toxicity. The reversal activity of progestogen compounds has been demonstrated. However, classical agents such as progesterone and megestrol (MG) also have high toxicity. Nomegestrol (NOM) belongs to a new derivation of progestogens and shows very low toxicity. We studied the reversal activity of NOM and compared it with that of verapamil (VRP), droloxifene (DRO), tamoxifen (TAM) and MG, and investigated the reversal mechanism, i.e. effects on the expression of the MDR1, glutathione S-transferase Pi (GSTpi), MDR-related protein (MRP) and topoisomerase IIalpha (TopoIIalpha) genes, as well as the intracellular drug concentration and the cell cycle. The aim of the study was to examine the reversal effects of NOM on MDR in MCF7/ADR, an MCF7 breast cancer cell line resistant to adriamycin (ADR), and its mechanism of action. MCF7/ADR cells and MCF7/WT, an MCF7 breast cancer cell line sensitive to ADR, were treated with NOM as the acetate ester. With an assay based on a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MTT], the effects of various concentrations of NOM on MDR in MCF7/ADR cells were studied. Before and after the treatment with 5 microM NOM, the expression of the MDR-related genes MDR1, GSTpi, TopoIIalpha and MRP were assayed with a reverse transcriptase polymerase chain reaction (RT-PCR) immunocytochemistry assay. By using flow cytometry (FCM), we observed the intracellular ADR concentration and the effects of combined treatment with NOM and ADR on the cell cycle. Results collected were analysed with Student's t test. NOM significantly reversed MDR in MCF7/ADR cells. After treatment NOM at 20, 10 and 5 microM, chemosensitivity to ADR increased 21-fold, 12-fold and 8-fold, respectively. The reversal activity of NOM was stronger than that of the precursor compound MG, and comparable to that of VRP. After treatment with 5 microM NOM, the expression of both the MDR1 and the GSTpi mRNA genes began to decline on the second day (P <0.05 and P <0.01, respectively), and reached the lowest level on the third day (both P <0.01); however, on the fifth day the expression levels began to increase again (both P <0.05). The expression of MRP and TopoIIalpha had no significant changes. Changes in the expression of P-glycoprotein (P-gp) and GSTpi were similar to those of their mRNA expressions, showing early declines and late increases. Two hours after treatment with 20, 10 and 5 microM NOM, the intracellular ADR concentration increased 2.7-fold, 2.3-fold and 1.5-fold respectively. However, NOM did not increase ADR accumulation in MCF7/WT cells. FCM data showed that after 48 h of combined administration of NOM (20 microM) and ADR (from low to high concentration), MCF7/ADR cells showed a gradual arrest at the G2M phase with increasing ADR dose. The arrest effect with combined drug treatment was stronger than that with the single ADR treatment. MDR is the major mechanism of drug resistance in malignant tumour cells. To overcome MDR and to increase chemosensitivity, many reversal agents have been found. Most progestogen compounds have been demonstrated to have reversal effects, but we found no data on NOM, a new progestogen compound. Our results show that NOM has strong reversal activity. The reversal effects were stronger than those of the precursor compound, MG, and were comparable to that of VRP. Because NOM has low toxicity, it might have good prospects in clinical application. Using RT-PCR and immunocytochemistry assays, we studied the effects of NOM on MDR-related genes. The results were that NOM could markedly downregulate the mRNA and protein expression levels of MDR1 and GSTpi. TopoIIalpha and MRP gene expression showed no significant changes. It is known that P-gp induces MDR in tumour cells mainly by decreasing the intracellular drug concentration. After treatment with NOM, the intracellular drug concentration in MCF7/ADR cells increased significantly. Combined treatment with NOM and ADR induced arrest at the G2M phase. It is worth noting that NOM caused an early decrease and a late increase in the expression of some MDR-related genes in a time-dependent manner. The phenomena raise a question for the continued administration of reversal agents in clinics that merits further study. We demonstrate that NOM has strong reversal effects on MDR in MCF7/ADR cells. The reversal is via different routes, namely downregulating the mRNA and protein expression levels of MDR1 and GSTpi, increasing intracellular drug concentration and arresting cells at the G2M phase (NOM in combination with ADR). The reversal mechanism needs further study.

Flow cytometric evaluation of Fas expression in relation to response and resistance to anthracyclines in leukemic cells

Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis. The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport. Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways. Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry. Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated. Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist. We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin. The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11. After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis. Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line. The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis. On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway.

Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate-resistant human leukemia MOLT-3 cells developed in thymidine.

Various gene alterations are involved in the drug resistance of leukemia cells. To understand the mechanism that underlies the emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. Trimetrexate-resistant (70- and 60-fold) sublines were developed in the presence or absence of an exogenous supply of thymidine (MOLT-3/TMQ70/Th+, MOLT-3/TMQ60/Th-, respectively). Ten clonal lines were isolated by methyl cellulose cloning from each of the 2 trimetrexate-resistant MOLT-3 sublines. All the clonal lines from the 2 sublines expressed mutated DHFR mRNA, with a base change (T --> C) at the second position of codon 31, as well as the wild-type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQ70/Th+ cells. mdr1 mRNA expression in clonal lines from MOLT-3/TMQ70/Th+ cells and subclonal lines subsequently obtained from the 3 clonal lines with different mdr1 mRNA expression levels was heterogeneous, and its high expression levels were correlated with acquisition of the multidrug resistance (MDR) phenotype. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also progressively expanding among clones. Genomic instability may play a role in the establishment and clonal evolution of drug resistance in leukemia cells.

Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate‐resistant human leukemia molt‐3 cells developed in thymidine

Various gene alterations are involved in the drug resistance of leukemia cells. To understand the mechanism that underlies the emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. Trimetrexate-resistant (70- and 60-fold) sublines were developed in the presence or absence of an exogenous supply of thymidine (MOLT-3/TMQ70/Th+, MOLT-3/TMQ60/Th−, respectively). Ten clonal lines were isolated by methyl cellulose cloning from each of the 2 trimetrexate-resistant MOLT-3 sublines. All the clonal lines from the 2 sublines expressed mutated DHFR mRNA, with a base change (T → C) at the second position of codon 31, as well as the wild-type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQ70/Th+ cells. mdr1 mRNA expression in clonal lines from MOLT-3/TMQ70/Th+ cells and subclonal lines subsequently obtained from the 3 clonal lines with different mdr1 mRNA expression levels was heterogeneous, and its high expression levels were correlated with acquisition of the multidrug resistance (MDR) phenotype. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also progressively expanding among clones. Genomic instability may play a role in the establishment and clonal evolution of drug resistance in leukemia cells. Int. J. Cancer 82:63–69, 1999. © 1999 Wiley-Liss, Inc.

Clinical significance of MDR1 and MDRP gene expression in patients with non-small cell lung cancer.

To investigate the clinical significance of MDR1 andMDRP gene expression in patients with non-small cell lung cancer (NSCLC) . Using reverse transcription polymerase chain reaction (RT-PCR) technique , the expression of MDR1 and MDRP gene was determined in cancer and paracancerous tissues from 33 NSCLC patients. Positive expression of MDR1 gene was 69.7% and 36.4% in lung cancer tissues and paracancerous tissues respectively , and a highly significant difference was observed between the two groups ( P < 0.01) . The expression levels of MDR1 mRNA were much higher than those in paracancerous tissues. The positivity rate of MDRP gene expression in cancer tissues was 63.6% , which was higher than 30.3% in paracancerous tissue. The gene expression levels in cancer tissues were also significantly higher than those in paracancerous tissues. The expression levels of MDR1 and MDRP in cancer tissues of the patients receiving pre-operative chemotherapy were remarkably higher than those of the patients not receiving chemotherapy ( P < 0.05) . NSCLC had primary and acquired multidrug resistance. It was of important significance to detect MDR1 and MDRP gene expression in directing chemotherapy of lung carcinoma and in predicting prognosis.

Selectively induced high MRP gene expression in multidrug-resistant human HL60 leukemia cells

A subclone HL60/DOX was selected from a human leukemic HL60 cell line for resistance to doxorubicin (DOX) by exposure to stepwise increasing concentrations of the drug and coexposure to a potential P-glycoprotein (P-gp) inhibitor, cepharanthine (a biscoclaurine alkaloid). Compared with the parent HL60 cells, the HL60/DOX cells were 13.0-fold more resistant to DOX and showed multidrug-resistant (MDR) phenotype characterized by 4.6-fold, 2.3-fold, and 5.7-fold cross-resistance to vincristine, pirarubicin, and etoposide, respectively, but no cross-resistance to alkylating agent, cisplatin. Immunocytochemical analyses using the specific monoclonal antibody, MRPr1, and quantitative analyses using a competitive reverse transcription-polymerase chain reaction (CRT-PCR) confirmed overexpression of MRP gene products (about 8-fold determined by CRT-PCR) in this resistant clone. The P-gp expression was not detectable by the monoclonal antibody, C219, in the HL60/DOX cells, and that was consistent with extremely low levels of mdr1 mRNA expression determined by CRT-PCR in this clone. Drug accumulation and efflux studies demonstrated the significantly increased efflux rate of DOX compared to the parent HL60 cells. This enhancement of DOX efflux was reversed by the addition of 10 μM verapamil. To investigate the additional underlying mechanisms contributing to MDR phenotype in the HL60/DOX cells, the levels of DNA topoisomerases (Topo) including Topo I, Topo IIα, and Topo IIβ, and γ-glutamylcystein synthetase (γ-GCS) expression were determined using CRT-PCR techniques. Normal expression of each enzyme at the transcriptional level was demonstrated in this resistant clone. Southern blot analysis of the gene organization in the HL60/DOX cells revealed the amplification of MRP gene. These results indicate that alteration of the drug accumulation from enhanced efflux appears to be a major mechanism(s) of MDR phenotype and attributable to high levels of MRP expression in the HL60/DOX cells. Overexpression of MRP in this clone is regulated by the genomic amplification of DNA and increased levels of the MRP mRNA, independently with the normal expression of Topo I, Topo IIα, Topo IIβ, or γ-GCS.

Evaluation of newer prognostic markers for adult soft tissue sarcomas.

In addition to tumor size, grade, location, and the presence of metastases, other factors may be useful in prognostication for adults with soft tissue sarcoma (STS). This study examines the relationship of MDR-1 mRNA, p-glycoprotein (P-gp), Ki-67 expression, and DNA content expression to clinical outcome in adults with STS. Snap-frozen STS specimens from 65 patients were analyzed and compared with clinical outcomes. Immunohistochemistry was performed for the Ki-67 antigen and P-gp. DNA content was determined using the Feulgen reaction and quantitated using image analysis. MDR-1 mRNA expression was determined using a reverse-transcriptase polymerase chain reaction (RT-PCR)-based assay. P-glycoprotein expression was found by immunohistochemistry in 48% of cases with 5-year overall (54% v 14%, P = .07) and disease-free survival rates (32% v 18%, P = .039) higher in high-grade tumors that did not express P-gp. MDR-1 mRNA was detected in 51% of cases and no patient with high levels of MDR-1 mRNA expression was a long-term survivor. Patients with diploid tumors had significantly better survival than those with nondiploid tumors (51% v 31%, P = .03). High levels of Ki-67 were associated with poorer overall survival (46% v 31%, P = .04). On multivariate analysis, American Joint Committee on Cancer (AJCC) staging, DNA content, Ki-67, and P-gp staining were significant prognostic factors for 5-year overall and disease-free survival. P-gp expression, high-level Ki-67 expression, and nondiploid DNA content are independent prognostic indicators that correlate with poor outcomes in STS patients. However, MDR-1 mRNA was not found to be predictive of survival. These newer markers are useful additions to AJCC staging for prognostication for patients with STS. Such markers may be useful in selecting high-risk STS patients who could benefit from systemic adjuvant therapy.

Quantitative analysis of multidrug resistance gene expression in human osteosarcomas.

We evaluated the MDR1 expression levels in 77 osteosarcomas and investigated whether MDR1 mRNA expression in osteosarcomas varies with location within the tumour, following chemotherapy, or after metastasis. We modified the semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to determine accurately the levels of MDR1 mRNA expression in clinical specimens. We show that specimens collected from multiple locations in six tumours revealed very little variation in MDR1 expression suggesting that the levels of MDR1 in these tumours do not vary greatly with location within the tumour mass. In a comparison of pre and post-chemotherapy specimens it was found that MDR1 levels did not change appreciably following chemotherapy in 16 of 20 cases. In addition, in eight of ten specimens obtained before and after metastasis, the amount of MDR1 mRNA was found to remain relatively constant despite metastatic spread. Thus, many osteosarcomas exhibited intrinsic expression of MDR1 mRNA before multidrug regimens which invariably included doxorubicin and, in most cases, MDR1 expression was not induced following chemotherapeutic treatment. Our results suggest that some osteosarcoma patients may have primary tumours which are resistant to doxorubicin. These individuals may benefit from different chemotherapeutic regimens, e.g. the addition of MDR reversal agents.

Drug-transport and volume-activated chloride channel functions in human erythroleukemia cells: Relation to expression level of P-glycoprotein

The characteristics of volume-activated chloride currents, drug transport function and levels of P-glycoprotein (PgP) expression were compared between two human chronic erythroleukemia cell lines: a parental (K562) cell line and a derivative obtained by vinblastine selection (K562 VBL400). Parental K562 cells showed no detectable P-glycoprotein expression, measured at the protein level (immunofluorescence labeling with monoclonal antibodies), and had very low levels of MDR-1 mRNA expression (RT-PCR analysis), when compared with levels measured in K562 VBL400. Differences in Pgp-mediated transport were estimated by comparing the rates of Fluo3 accumulation. The higher drug-transport function of K562 VBL400 cells (e.g., lower Fluo3 accumulation) correlated with their elevated levels of MDR-1. The rate of dye transport was sensitive to verapamil but was not affected by the tonicity of the extracellular medium. In contrast to the clear differences in transport function, the characteristics of chloride currents induced by cell swelling were indistinguishable between the two cell lines. Currents measured in the whole-cell configuration were outwardly rectifying, had a higher permeability to iodide than to chloride (SCN- > I- > Cl- > gluconate), were potently blocked by NPPB and were unresponsive to verapamil. The percentage of responding cells and the mean current density were nearly identical in both cell lines. In addition, activation of the volume-sensitive current was not prevented during whole-cell recordings obtained with pipettes containing high concentration of cytotoxic drugs (vincristine or vinblastine). These results do not lend support to the previously reported association between Pgp expression and volume-sensitive chloride channels, and suggest that a different protein is responsible for this type of chloride channel in K562 cells.

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line.

Multidrug Resistance in the Laboratory and Clinic

Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases. Studies have demonstrated that this form of drug resistance occurs in cultured tumor cell lines as well as in human cancers. P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural-product drugs from the cell before they have a chance to exert their cytotoxic effects. Using the tools of molecular biology, studies are beginning to reveal the true incidence of multidrug resistance, as mediated by the MDR1 gene, in the clinical setting. It has been demonstrated, at least in the laboratory, that resistance mediated by P-glycoprotein may be modulated by a wide variety of compounds, including verapamil and cyclosporine A. These are compounds which, by themselves, generally have little or no effect on the tumor cells, but when used in conjunction with antineoplastic agents act to decrease, and in some instances eliminate, drug resistance. The mechanism(s) by which these agents act to reverse resistance is not fully understood. Clinical trials to modulate P-glycoprotein activity are now under way to determine whether such strategies will be feasible. The detection of the P-glycoprotein in patient samples is very important in the design of these studies, as it appears that drug-resistant cells lacking P-glycoprotein will be unaffected by agents such as verapamil. Clinical studies are needed in which patients are stratified into chemotherapy protocols based on levels of MDR1 mRNA or P-glycoprotein expression in the primary tumors. Several research areas have been identified that are important to the transfer of the discovery of the MDR1 gene and its protein product from the research laboratory to the clinical environment. There is an immediate need for comprehensive information on the prevalence and levels of expression of the human MDR genes and their protein products in human organs and tissues. Data are needed on P-glycoprotein levels in specific subpopulations (e.g., according to age, sex, race, and diet), and the study of the heterogeneity and variability of expression of P-glycoprotein in normal human tissues should be given high priority. Since early studies have indicated some successes in identifying patients with classic multidrug resistance who might be responsive to chemosensitization, it can be anticipated that clinical research will accelerate in this area. The next wave of clinical studies will provide clinical investigators with opportunities to develop and evaluate P-glycoprotein tests and correlate test results with clinical outcomes.

MDR1 gene expression and treatment outcome in small cell lung cancer: MDR1 gene expression as an independent prognostic factor.

We report a preliminary study to determine whether MDR1 gene expression level in small cell lung cancer (SCLC) tumors is a useful predictor of tumor response to chemotherapy and patient survival in association with myc amplification in the tumor. We analyzed 18 patients with SCLC receiving adriamycin and etoposide combination chemotherapy between August 1989 and November 1991; 16 males and 2 females, median age of 68 years, and 7 with limited disease and 11 with extensive disease. MDR1 mRNA expression level and myc family gene amplification were simultaneously determined by polymerase chain reaction using transbronchial biopsy specimens which were obtained at diagnosis. Patients with tumors expressing low MDR1 mRNA responded more favorably to chemotherapy than those with tumors expressing high MDRI mRNA, however, the difference in tumor response was statistically not significant (84.6% versus 40%). The overall survival was significantly shorter in the latter than in the former (7.2 months versus 11.7 months; p = 0.023). The survival of the 4 patients with tumor showing myc family gene amplification was almost identical to that of patients with tumors showing no amplification of the gene (8.2 months versus 8.8 months; p = 0.73). Multivariate Cox's regression analysis supports the notion that MDR1 may be a useful independent prognostic factor.

Expression of MDR1 and glutatione S transferase-π genes and chemosensitivities in human gastrointestinal cancer

The relationship was analyzed between drug resistance and MDR1 (with MDR signifying multiple drug resistance) and glutathione S transferase-pi (GST-pi) gene expression in four stomach and four colon cancer cell lines. Northern blot analysis by pmdr1 probe showed that stomach cancer cell lines had no detectable level of MDR1 mRNA expression. By contrast, some levels of MDR1 mRNA expression were found in two colon cancer cell lines, indicating doxorubicin resistance. To examine the MDR1 mRNA in each cell level, in situ hybridization was used. It was found that all colon cell lines and two stomach cell lines had more silver grains per cell than KB cells (a human KB kidney epidermoid carcinoma cell line). However, the number of silver grains in each cell was heterogeneous in the colon and stomach cell lines. Low-level MDR1 mRNA expression could be detected even in cell lines without MDR1 mRNA expression by northern blot hybridization. These results suggest the possibility that all gastrointestinal cell lines can acquire multiple drug resistance. In addition, all examined gastrointestinal cell lines had high GST-pi mRNA expression. This GST-pi gene expression shows cisplatin resistance in the examined cell lines. Heterogeneity of GST-pi mRNA expression also was shown at the cellular level.

Expression of the mdr1 gene in human colorectal carcinomas: relationship with multidrug resistance inferred from analysis of human colorectal carcinoma cell lines

To investigate whether mdr1 gene products are involved in conferring the chemoresistant phenotype to human colorectal carcinomas (HCCs), we determined the mdr1 mRNA expression level (mdr1 EL) in surgical specimens from 29 pharmacologically untreated patients and analyzed the relationship between mdr1 EL and drug resistance in an in vitro experimental model. This consisted of 7 HCC cell lines chosen to cover the range of mdr1 ELs detected in the neoplastic specimens. No relationship was observed between the mdr1 EL of the HCC cell lines and the degree of chemosensitivity found for each drug tested, regardless of whether mdr1 gene products may [doxorubicin (DOX), vincristine (VCR), and actinomycin-D (ACT-D)] or may not affect [cis-diamminedichloroplatinum (CDDP)] drug-transmembrane equilibria. Conversely, a direct relationship was found between the mdr1 EL of HCC cell lines and the number of drug-resistant (DR) colonies arising from each parent cell line treated in continuous culture with high DOX concentrations. In addition, the chemoresistance index and mdr1 EL of the DR cell variants were roughly proportional to the mdr1 EL of the parent cell line. Our findings suggest that primary HCCs derive multidrug resistance from biochemical mechanism(s) other than mdr1 gene products. However, the mdr1 EL might be indicative of a predisposition to develop DR cell variants after chemotherapeutic treatment.

Pleiotropic-resistant phenotype is a multifactorial phenomenon in human colon carcinoma cell lines.

The biochemical basis of multidrug-resistant (MDR) phenotype has been investigated in drug-resistant sublines independently obtained in our laboratories by single step doxorubicin (DOX) selection of LoVo, DLD1, and SW948 human colon carcinoma (HCC) cell lines. All the chemoresistant sublines have been found to be cross-resistant to DOX, actinomycin-D (ACT-D) and vincristine (VCR) but not to cis-diamminedichloroplatinum (CDDP), and have exhibited an increased expression level of mdr1 mRNA and gp170 glycoprotein. Comparative analyses in drug-resistant and sensitive cells of resistance index, extracellular and intracellular equitoxic DOX concentrations, and mdr1 gene products expression have indicated that MDR phenotype is a multifactorial phenomenon due to different and possibly independent biochemical mechanisms which cooperate, in varying degrees from cell line to cell line, in conferring cellular chemoresistance.

Expression of the multidrug resistance, MDR1, gene in neuroblastomas.

Metastatic neuroblastoma is a childhood malignancy that is frequently responsive to chemotherapy with doxorubicin, vincristine, and teniposide (VM26), among other drugs, but in the majority of treated patients, the tumor recurs during or after chemotherapy. In this work, we have examined the hypothesis that the development of resistance to chemotherapy in neuroblastoma might be related to the expression of the human MDR1 gene, which encodes a multidrug transporter that functions as an energy-dependent drug efflux pump. RNA samples from 49 neuroblastomas were analyzed, including 31 from untreated and 18 from treated patients. MDR1 RNA was detectable in the majority of treated and untreated tumors using a sensitive, semiquantitative slot blot assay. Of the samples from treated patients, five of 18 were found to have high MDR1 RNA levels, whereas only three of 31 from untreated patients had high MDR1 levels, a statistically significant difference (P less than .01). These results show that high levels of MDR1 RNA are often associated with resistance to chemotherapy in neuroblastoma and suggest that they may contribute to this resistance. Many of the neuroblastoma samples were also evaluated for N-myc amplification but there was no correlation between N-myc copy number and the level of MDR1 mRNA expression.

The direct activation of human multidrug resistance gene (MDR1) by anticancer agents

Enhanced expression of a multidrug-resistance gene (MDR1) is observed in some cancer patient, but any regulatory mechanism of MDR1 gene expression in this phenomenon is not yet known. In this study, the regulation of MDR1 gene was analysed by transient expression assays in the presence of anticancer agents. We found that MDR1 promoter could be activated directly on the addition of anticancer agents including vincristine, daunomycin, adriamycin and colchicine. The results suggest that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype.

Correlation of MDR1 gene expression with chemotherapy in neuroblastoma.

Forty-one neuroblastoma tumor specimens have been analyzed by Northern and slot blot hybridization techniques with human MDR1 gene probes. Only one of 15 (6%) tumors from patients who had not received chemotherapy exhibited high levels of MDR1 transcripts, while 11 of 26 (42%) treated tumors showed high levels of MDR1 expression (Fisher exact test: P = .03). The results indicate that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype in vitro regardless of neuroblastoma tissue origin or N-myc content in the genome. For the 26 treated neuroblastomas, the number of nonresponsive tumors was found to be significantly higher among those with high levels of MDR1 mRNA.