Multidrug Resistance 1 mRNA Research Articles

Up-regulation of MDR1 and induction of doxorubicin resistance by histone deacetylase inhibitor depsipeptide (FK228) and ATRA in acute promyelocytic leukemia cells

The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.

Exploring the mechanisms of macrolides in cystic fibrosis

Several studies have reported clinical improvements in cystic fibrosis (CF) patients on macrolide antibiotics although the mechanism of action remains unclear. We conducted an open-label study of azithromycin (500 mg daily for 2 weeks) in 9 adult CF patients to explore 3 possible mechanisms: up-regulation of the multi-drug resistance (MDR) or cystic fibrosis transmembrane regulator (CFTR) proteins, correction of epithelial ion transport and reduced bacterial adherence. End-points included nasal potential difference (PD) measurements, nasal epithelial MDR and CFTR mRNA levels and Pseudomonas aeruginosa adherence to nasal epithelium. Forced expiratory volume in the 1st second (FEV(1)) increased significantly after 2 weeks of azithromycin (pre- 41.1 [5.0]%; post- 44.6 [5.8]%; P<0.05), although improvements in forced vital capacity (FVC) did not reach significance (pre- 61.3 [4.0]%; post- 67.1 [5.4]%, NS). Before treatment all subjects had nasal PD measurements characteristic of CF. Treatment led to no significant group differences in any measures of either sodium absorption or chloride secretion. Neither CFTR nor MDR mRNA levels had altered significantly and the adherence of P. aeruginosa did not decrease. We conclude that these are unlikely to be significant contributing mechanisms accounting for the consistent beneficial results observed in clinical trials of macrolides in CF.

TEL/AML1 Overcomes Drug Resistance Through Transcriptional Repression of Multidrug Resistance-1 Gene Expression

The t(12;21)(p12;q22) chromosomal aberration, which is frequently observed in pediatric precursor B-cell acute lymphoblastic leukemia (ALL), generates the TEL/AML1 chimeric gene and protein. TEL/AML1-positive ALL has a favorable prognosis, and one possible reason is that this subtype of ALL rarely shows drug resistance. AML1/ETO, another AML1-containing chimeric protein, has been shown to transcriptionally repress the activity of the multidrug resistance-1 (MDR-1) gene promoter; thus, we examined whether TEL/AML1 also represses MDR-1 gene expression, possibly preventing the emergence of multidrug resistance. In this study, we show that the TEL/AML1 protein binds to the consensus AML1 binding site in the MDR-1 promoter and transcriptionally represses its activity. Following transient transfection of TEL/AML1 protein into Adriamycin-resistant K562/Adr cells, we also demonstrate that TEL/AML1 can down-regulate the expression of P-glycoprotein, a product of the MDR-1 gene, and restore the chemosensitivity to the cells. Furthermore, we report that MDR-1 mRNA levels in leukemic cells obtained from TEL/AML1-positive ALL patients are lower than those from TEL/AML1-negative ALL patients. Thus, TEL/AML1 protein acts as a transcriptional repressor of MDR-1 gene expression, and although TEL/AML1 has been implicated in leukemogenesis, its effects on the MDR-1 gene may contribute to the excellent prognosis of TEL/AML1-positive ALL with current therapy.

Tacrolimus therapy according to mucosal MDR1 levels in small-bowel transplant recipients.

To clarify the clinical applicability of intestinal absorptive barriers, we have quantified messenger ribonucleic acid (mRNA) expression levels of multidrug resistance 1 (MDR1) protein and cytochrome P450 (CYP) 3A4 in intestinal biopsy specimens from 2 small-bowel transplant recipients. Postoperative immunosuppressive therapy was started with intravenous and oral administrations of tacrolimus and a small amount of steroids. The daily dosage of tacrolimus was modified mainly on the basis of trough levels. After confirmation that the enterocyte MDR1 level was decreasing, tacrolimus was administered via the oral route only. The mRNA levels in the biopsy specimens varied widely throughout the period. With high-dose steroid-pulse treatment, the enterocyte mRNA expression of CYP3A4, but not of MDR1, was markedly enhanced. The mRNA levels of MDR1, but not CYP3A4, correlated well with the concentration/oral dose ratio and the oral dosage of tacrolimus. The good progress after transplantation in both cases suggested that monitoring the change in expression of MDR1 mRNA in the graft intestine might be helpful for understanding the pharmacokinetic profile and determining when to change the route of tacrolimus administration in small-bowel transplant recipients.

A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells

Background: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings. We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 ( MDR1) transcripts, and applied it to clinical samples. Method: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control. Results: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression—compensated with β-actin expression—ranged from 1.4×10 2 to 2.5×10 6 (median 4.8×10 5) copies/μg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9×10 4 to 5.2×10 5 (median 2.2×10 5) copies/μg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples. Conclusions: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.

Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure. Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold). Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the U0126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of U0126-treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway. HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.

Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene

We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line.

Quantitative Analysis of a MDR1 Transcript for Prediction of Drug Resistance in Acute Leukemia

Assessing the drug resistance of leukemic cells is important for treatment of leukemia. We developed a quantitative reverse transcription (RT)-PCR method for multidrug resistance 1 (MDR1) and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. The cutoffs for copy numbers of MDR1 and MRP1 transcripts were defined based on copy numbers in healthy bone marrow mononuclear cells. To confirm that the cutoffs reflected biological resistance, we established vincristine (VCR)-resistant K562 sublines that showed various degrees of drug resistance and examined the correlation between the copy numbers of these transcripts and the biological resistance of these clones. In addition, we compared the sensitivity and specificity of quantitative RT-PCR to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric (FCM) analysis. The defined cutoff for copy numbers of MDR1 transcripts corresponded with the degree of biological resistance of VCR-resistant K562 sublines. Clinical study revealed that the concentrations of MDR1 mRNA in all relapsed patients with acute myelogenous leukemia (AML) were above the cutoff. Moreover, both AML and acute lymphoblastic leukemia patients with high MDR1 mRNA expression at diagnosis tended to show a low remission rate and short remission periods. No association was observed between the amounts of MRP1 transcripts and clinical outcomes. The specificity and sensitivity of quantitative RT-PCR for MDR1 were superior to the MTT assay and FCM analysis. These results suggest the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.

Modulation of doxorubicin sensitivity by cyclosporine A in hepatocellular carcinoma cells and their doxorubicin-resistant sublines.

Cyclosporine A (Cys) and verapamil (Ver) sensitize multidrug-resistant (MDR) cells to various anticancer drugs by interacting with membrane glycoproteins involved in the drug efflux. In the present study, we assessed the effect of Cys on the modulation of doxorubicin (DOR) sensitivity in hepatocellular carcinoma (HCC) cell lines, and their DOR-resistant sublines. The sensitivity to DOR and the chemosensitizing effects of Cys were assessed by using two human HCC cell lines, PLC/PRF/5 and Hep-3B, and their DOR-resistant sublines, PLC/DOR and 3B/DOR. The expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein (MRP) mRNA in these cells were detected by using a RT-PCR. The HCC cell lines grown in individual wells of 24-well plates were incubated with DOR that were sequentially diluted in culture medium in combination with 5 micromol/L Cys for 24 h. The cell viability in each well was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA of MDR1 and that of MRP were readily detectable in the HCC cell lines by RT-PCR. When 5 micromol/L Cys was added to the culture, the 50% inhibiting concentration (IC50) of DOR was reduced from 0.93 +/- 0.29 microg/mL to 0.32 +/- 0.10 microg/mL in PLC/PRF/5, and from 0.25 +/- 0.07 microg/mL to 0.09 +/- 0.04 microg/mL in Hep-3B. Furthermore, in the presence of 5 micromol/L Cys, the IC50 of DOR was reduced from 48.63 +/- 17.04 microg/mL to 0.49 +/- 0.14 microg/mL in PLC/DOR, and from 4.60 +/- 1.22 microg/mL to 0.15 +/- 0.06 microg/mL in 3B/DOR. The amounts of PCR products of MDR1 mRNA in PLC/DOR and 3B/DOR were greater than those in PLC/PRF/5 and Hep-3B, respectively. In HCC, the amplification of MDR1 mRNA is probably the main mechanism underlying acquired DOR resistance. Cyclosporine is also indicated to be highly active in potentiating the anticancer activity of DOR in HCC cells and their DOR-resistant sublines.

Expression of the multidrug-resistance 1 (MDR1) gene and prognosis in human pancreatic cancer.

Multidrug-resistance 1 (MDR1) encodes a 170 kDa transmembrane glycoprotein (P-glycoprotein), which acts as a drug-efflux pump. In the present study, we analyzed the expression of MDR1/P-glycoprotein in human pancreatic cancer and correlated the results with clinical parameters. Pancreatic cancer tissue samples were obtained from 67 patients (30 female, 37 male) who underwent surgery. Normal pancreatic tissues obtained from 15 previously healthy organ donors (4 female, 11 male) served as controls. MDR1 mRNA levels were analyzed by Northern blotting, and the exact site of MDR1 mRNA expression was determined by in situ hybridization and immunohistochemistry. Northern blot analysis indicated that in comparison with the normal pancreas, MDR1 mRNA levels were only increased 1.4-fold (p = 0.03) in the pancreatic cancer samples. However, there was a 2.9-fold (p < 0.01) increase in MDR1 mRNA levels when only the samples that exhibited increased expression (38%) were analyzed. In situ hybridization and immunohistochemical analysis showed that MDR1 was highly expressed in the cancer cells of these samples. Statistical analysis revealed that patients with high MDR1/P-glycoprotein expression had a shorter postoperative survival time compared with patients with weak to moderate expression of MDR1. On the basis of in situ hybridization, survival in the intense group was 11.6 (n = 12) versus 14.2 months (n = 42) in the mild to moderate group. On the basis of immunohistochemistry, survival in the intense group was 7.5 months (n = 10) versus 14.1 months (n = 40) in the mild to moderate group. Surprisingly, survival of patients with high expression of MDR1/P-glycoprotein was not significantly different from that of patients without detectable MDR1/P-glycoprotein expression. These findings suggest that both strong expression of MDR1/P-glycoprotein and lack of expression seem to influence tumor growth via known and yet unknown mechanisms.

Establishment of new multidrug-resistant human osteosarcoma cell lines.

Multidrug-resistant clones of human osteosarcoma MNNG/HOS and MG63 cells were isolated by stepwise selection on exposure to increasing doses of doxorubicin (DXR). The final clones MNNG/HOS/DXR1000 and MG63/DXR1000, established after ethylmethane sulfonate mutagenesis, showed 96-fold and 121-fold higer resistance to DXR than their parental cell lines. They were also cross-resistant to vincristine, but not to cisplatinum or methotrexate. The levels of multidrug-resistance-1 (MDR1) mRNA expression increased gradually according to the concentration of DXR in both cell lines. Although the parental MNNG/HOS cells expressed a low level of MDR1 mRNA, the parental MG63 cells showed no MDR1 expression. The IC50 values of MNNG/HOS and its resistant variant to DXR were higher than those of MG63 and its resistant clone. Multidrug-resistant associated protein (MRP) mRNA expression was detected in MNNG/HOS or MG63 parental cell lines, and in their resistant variants. MG63 and its resistant variants revealed stable expression of MRP, whereas the resistant phenotype of MNNG/HOS showed decreased MRP expression, compared to its parental cell line. No alteration in the levels of hepatocyte growth factor (HGF) or its receptor c-MET was recognized between parental lines and their resistant variants. The results indicate that our DXR-resistant variants of MNNG/HOS and MG63 reveal a classical MDR phenotype and can offer a model with which to investigate the mechanisms of multidrug resistance in osteosarcoma.

Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay

We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1. The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments. The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/μg total RNA in MOLT-3/TMQ70, MOLT-3/TMQ800, and MOLT-3/VCR1,000, respectively. The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis. In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detect P-gp. This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples. J. Clin. Lab. Anal. 11:258–266, 1997. © 1997 Wiley-Liss, Inc. No abstract.

Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay

We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1. The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments. The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/microgram total RNA in MOLT-3/TMQ70, MOLT-3/ TMQ800, and MOLT-3/VCR1,000, respectively. The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis. In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detet P-gp. This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples.

Expression of MDR1 (multidrug resistance) gene and its protein in normal human kidney.

P-glycoprotein (Pgp), the product of the multidrug resistance (MDR) gene overexpressed in cancer cells, is present also in normal tissues. In the kidney, MDR1 Pgp has been found in the proximal tubule and in cultured mesangial cells. In situ hybridization and immunohistochemistry were used to determine the complete nephronal localization of MDR mRNA and its product, Pgp, in the human kidney. MDR mRNA expression was studied with the use of nonradioactive in situ MDR RNA probes. MDR1 Pgp was immunolocalized using the specific monoclonal antibody MRK16. The presence of MDR mRNA was confirmed in proximal tubules and mesangium, and demonstrated as well in thick limb of Henle's loops and in collecting ducts. MDR1 Pgp colocalized in the same nephronal segments. This suggests that, in addition to secreting xenobiotics, Pgp may play a role in the transport of endogenous substrates or in the regulation of Cl- channels.

Immunohistochemical Localization of P‐Glycoprotein and Expression of the Multidrug Resistance‐1 Gene in Human Pancreatic Cancer: Relevance to Indicator of Better Prognosis

We investigated immunohistochemical localization of P‐glycoprotein (F‐gp) on paraffin‐embedded sections from 103 cases of previously untreated pancreatic tumors and also analyzed multidrug resistance‐1 (MDR1) gene expression by polymerase chain reaction after reverse transcription in 35 cases. High positive staining for P‐gp was observed in 72.8% of pancreatic tumors and in 73.2% of ductal adenocarcinoma. In ductal adenocarcinoma, immunoreactivity of P‐gp was inversely correlated with biological aggressiveness of tumors determined by histologic grading (P<0.01), tumor size (P<0.01), retroperitoneal invasion (P<0.01) and portal invasion (P<0.05). Expression of the MDR1 gene was detected in all the pancreatic tumors examined and was significantly higher than that in normal pancreas (P<0.05). The levels of MDR1 mRNA showed a moderate correlation with those of P‐gp (r=0.62, P<0.0001). Higher expression levels of MDR1/P‐gp significantly correlated with better prognosis of patients with ductal carcinoma (P<0.05). Among patients with ductal carcinoma, the high staining group for P‐gp revealed a 3.5‐fold better prognosis compared with the low staining group (HR=3.47, 95% CI=1.62, 7.45; P=0.0016). In conclusion, MDR1 gene/P‐gp expression in pancreatic cancer without chemotherapy inversely correlates with biological aggressiveness and is an independent indicator of favorable prognosis.

Expression of Retroviral Vectors Containing the Human Multidrug Resistance 1 cDNA in Hematopoietic Cells of Transplanted Mice

Transfer of the human multidrug resistance 1 (MDR1) gene to hematopoietic stem cells offers an approach to overcome the myelosuppression caused by a number of antineoplastic drugs. This study was designed to determine the effect of MDR1 gene transfer on overall P-glycoprotein (P-gp) expression in murine hematopoietic cells. Mice were transplanted with bone marrow cells infected with either of two different MDR1 retroviral vectors. A reverse-transcriptase polymerase chain reaction-based assay was used to quantify expression levels of both endogenous and vector-derived P-gp encoding transcripts in hematopoietic cells of transplanted mice. Expression of both a truncated and full-length MDR1 mRNA species was noted in bone marrow and spleen colony cells. The truncated message resulted from cryptic mRNA splice sites within the MDR1 cDNA and was detected with both vectors. Full-length message levels exceeded those from the endogenous genes in all but one case and roughly approximated that seen in the modestly drug-resistant cell line SW620. We conclude that transfer of MDR1 retroviral vectors resulted in a significant increase in P-gp expression in most cases; however, aberrant splicing of MDR1 transcripts can result in reduced expression of vector-derived P-gp.

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.

Low external pH and osmotic shock increase the expression of human MDR protein.

We have studied the effects of extracellular pH (pHo) and osmotic strength on the expression of the human multidrug resistance (MDR) protein. Both lowered pHo and hypertonic shock increase the level of hu MDR protein 5-10-fold in membranes isolated from the human colon carcinoma cell lines SW620 and HCT15 and the human kidney carcinoma line SKRC-39. Increased protein expression is dependent on the duration of acid or osmotic shock and is reversed within several days when normal growth conditions are restored. Quantitative northern blot analysis with a hu MDR 1 specific probe reveals increased MDR mRNA in the acid and hypertonically shocked cells. Interestingly, we find a greater increase in mRNA levels for hypotonically shocked colon cells, without an apparent increase in protein levels. Overexpressing cells are found to retain less [3H]vinblastine relative to cells cultured under normal conditions, and they are resistant to the cytotoxic effects of doxorubicin, vinblastine, and colchicine, but not methotrexate. This resistance appears to be reversed by treatment with verapamil. In contrast, SW620 cells previously induced to overexpress MDR protein via the administration of differentiation agents [Mickley et al. (1989) J. Biol. Chem. 264, 18031-18040] did not exhibit decreased retention of [3H]vinblastine; thus low-pHo-induced overexpression of MDR protein in these cells may provide additional factors that promote the full expression of the MDR phenotype. These data may help to explain why many solid tumors (e.g., of colon and kidney origin) develop MDR prior to chemotherapy, since they usually grow under similar acidic conditions. These data also support the contention that MDR protein may play a role in intracellular pH and volume homeostasis.

Inhibition of MDR1 gene expression by H-87, a selective inhibitor of cAMP-dependent protein kinase

N-[2-( p-bromocinnamylmethylamino) ethyl]-5-isoquinolinesulfonamide (H-87), a newly synthesized inhibitor of protein kinase A, significantly decreased the drug resistance of multidrug resistant human U937 M cells, mouse FM3A M and P388 M cells. Northern blot analysis showed MDR1 gene expression was decreased in H-87-treated P388 M cells. H-87 inhibited the activity of MDR1 (multidrug resistance-1) promoter in a dose-dependent manner in transient expression assay. In contrast, the expression of c- raf-1 gene significantly enhanced the activity of MDR1 promoter. We therefore examined the effect of H-87 on MDR1 gene expression in c- raf-1 transfected CV-1 cells (CV-1/raf). A significant decrease in the level of MDR1 mRNA was observed after H-87 treatmfent of the CV-1/ raf cells. These results suggest that inhibition of MDR1 gene expression by H-87 is associated with circumvention of drug resistance in MDR cells.

P-glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture.

P-Glycoprotein (P-gp), the multidrug resistance (MDR) gene product, has previously been shown to be functional and overexpressed in adult rat hepatocytes during primary culture. In the present study, we examined P-gp expression in human, mouse and hamster hepatocytes cultured in conditions similar to those used for rat liver cells. Northern blotting and doxorubicin P-gp-mediated efflux analyses revealed that liver parenchymal cells from these three species exhibited only limited increased MDR mRNA levels with no intracellular decreased drug retention, thus suggesting that the marked functional P-gp induction observed in rat hepatocytes could reflect a species-specific response of these cells to an unfamiliar environment. An 8 h exposure to cycloheximide, which strongly induced MDR mRNAs in rat liver cells, also increased, although to a far lesser extent, MDR mRNA levels in human, mouse and hamster hepatocytes, indicating that P-gp expression in normal liver parenchymal cells could be at least partly negatively regulated by a labile protein factor.