Overexpression Of Multidrug Resistance 1 Research Articles

Metformin alleviates adriamycin resistance of osteosarcoma by declining YY1 to inhibit MDR1 transcriptional activity

Chemotherapy resistance hinders the successful treatment of osteosarcoma (OS) to some extent. Previous studies have confirmed that metformin (Met) enhances apoptosis induced by chemotherapeutic drugs, but the underlying mechanism remains unclear. To establish adriamycin (ADM)-resistant MG-63 (MG-63/ADM) cells, the dosage of ADM was progressively increased. The results of qRT-PCR and Western blotting demonstrated that the expression level of Yin Yang 1 (YY1) and multi-drug resistance-1 (MDR1) in MG-63/ADM cells were remarkably increased compared with those in MG-63 cells. Met dramatically enhanced ADM cytotoxicity and accelerated apoptosis of MG-63/ADM cells. Moreover, Met suppressed the expressions of YY1 and MDR1 in MG-63/ADM cells. YY1 promoted its transcriptional expression by directly binding to the MDR1 promoter. Furthermore, the effects of Met on ADM sensitivity in MG-63/ADM cells was reversed due to overexpression of YY1 or MDR1. Collectively, these findings suggested that Met inhibited YY1/MDR1 pathway to reverse ADM resistance in OS, providing a new insight into the mechanism of Met in ADM resistance of OS.

MiR-381 overcomes cisplatin resistance in breast cancer by targeting MDR1.

Increasing evidence suggests the involvement of microRNA-381 (miR-381) in chemoresistance of cancer treatment. However, its function and molecular mechanisms in breast cancer chemoresistance are still not well elucidated. In the present study, we aimed to investigate the functional role of miR-381 in cisplatin (DDP) resistance of breast cancer and discover the underlying molecular mechanism. The expression levels of miR-381 and MDR1 were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis in breast cancer tissues and cell lines. The DDP sensitivity and cell apoptosis of breast cancer cells were determined by MTT assay and flow cytometric analysis, respectively. The relationship between miR-381 and MDR1 was explored by target prediction and luciferase reporter analysis. miR-381 was decreased in DDP-resistant breast cancer tissues and cell lines. Low miR-381 expression was correlated with poor prognosis of breast cancer patients. miR-381 overexpression improved DDP sensitivity of MCF-7/DDP and MDA-MB-231/DDP cells. Conversely, miR-381 inhibition lowered the response of MCF-7 and MDA-MB-231 to DPP. Moreover, miR-381 could directly suppress multidrug resistance 1 (MDR1) expression. MDR1 knockdown could overcome DDP resistance in MCF-7/DDP and MDA-MB-231/DDP cells, while MDR1 overexpression led to DDP resistance in MCF-7 and MDA-MB-231 cells. Notably, MDR1 overexpression counteracted the inductive effect of miR-381 mimics on DDP sensitivity of MCF-7/DDP and MDA-MB-231/DDP cells. On the contrary, miR-381 inhibition-mediated DDP resistance in MCF-7 and MDA-MB-231 cells was reversed by MDR1 knockdown. In summary, miR-381 could overcome DDP resistance of breast cancer by directly targeting MDR1, providing a novel therapeutic target for breast cancer chemoresistance.

Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant.

PurposePregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant.Experimental DesignArginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin.ResultsAMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts.ConclusionsPRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with chemotherapy drugs may be a new strategy for overcoming tumor MDR.

F2A sequence linking MGMTP140K and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of haematopoietic stem cells

Chemoprotection of haematopoietic stem cells (HSCs) by gene therapeutic transfer of drug-resistance genes represents the encouraging approach to prevent myelosuppression, which is one of the most severe side effects in tumor therapy. Thus, we cloned and evaluated six different bicistronic lentiviral SIN vectors encoding two transgenes, MGMT(P140K) (an O(6)-benzylguanine-resistant mutant of methylguanine-DNA methyltransferase) and MDR1 (multidrug resistance 1), using various linker sequences (IRESEMCV, IRESFMDV and 2A-element of FMDV (F2A)). Expression of both transgenes in HL-60 and in K562 cells was assayed by quantitative real-time PCR. Combination therapy with ACNU plus paclitaxel in HL-60 cells and with carmustin (BCNU) plus doxorubicin in K562 cells resulted in the most significant survival advantage of cells transduced with the lentiviral vector HR'SIN-MGMT(P140K)-F2A-MDR1 compared with untransduced cells. In human HSCs, overexpression of both transgenes by this vector also caused significantly increased survival and enrichment of transduced cells after treatment with BCNU plus doxorubicin or temozolomide plus paclitaxel. In summary, we could show significant chemoprotection by overexpression of MDR1 and MGMT(P140K) with a lentiviral vector using the F2A linker element in two different haematopoietic cell lines and in human primary HSCs with various combination regimens. Consequently, we are convinced that these in vitro investigations will help to improve combination chemotherapy regimens by reducing myelotoxic side effects and increasing the therapeutic efficiency.

MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice

The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.

Molecular Predictors for the Response to Nilotinib Treatment After Acquired Imatinib Failure In Ph+ Chronic Myeloid Leukemia

AbstractAbstract 2283 Introduction:A switch to the 2nd generation tyrosine kinase inhibitor nilotinib has been proven to be effective in case of resistance or intolerance to imatinib for the majority of patients with Ph+ chronic myeloid leukemia (CML) in chronic phase (CP). Besides mutations in the BCR-ABL kinase domain and various BCR-ABL-independent mechanisms, e.g. clonal evolution and activation of pathways bypassing BCR-ABL, efficacy of imatinib and nilotinib are dependent on intracellular drug levels, which are influenced by the activity of the efflux transporter protein multidrug resistance 1 (MDR1). Cell culture data suggest overexpression of MDR1 as a cause of resistance to nilotinib (Mahon et al., Cancer Res 2008). Moreover, Dulucq et al. (Blood 2008) report the association of MDR1 single nucleotide polymorphisms (SNPs) with the chance to achieve major molecular response (MMR) on first-line imatinib therapy. Aim:In order to allow risk stratification, we sought to elucidate molecular markers, e.g. MDR1 gene expression, BCR-ABL transcript burden, BCR-ABL mutation status and common SNPs in the MDR1 gene regarding their potency to predict therapy-related endpoints, such as MMR, complete cytogenetic response (CCyR), and progression-free survival (PFS) during second line therapy with nilotinib in imatinib-resistant CML-CP patients. Patients and Methods:A cohort of 83 imatinib-resistant patients in chronic phase CML treated with nilotinib was investigated within the AMN107A2101 phase I/II trial. Baseline BCR-ABL mutations were detected by D-HPLC and direct sequencing. MDR1 and BCR-ABL mRNA expression levels were determined by qRT-PCR using LightCycler™ technology, normalized against beta-glucuronidase (GUS) expression. BCR-ABL levels were standardized according to the international scale (IS). MDR1 SNPs (1236C>T, 2677G>T) were investigated using conventional sequencing. Log-rank tests were performed to compare the time to MMR (BCR-ABL IS ≤0.1%), CCyR, and PFS. Results:By 12 or 24 months, patients with MDR1/GUS ratios ≥2 achieved MMR in an estimated rate of 34%, CCyR was attained in rates of 53% and 58%, and PFS rates were 88% and 75%, whereas those with initial MDR1/GUS ratios <2 showed MMR in 13% (p=0.026), CCyR in 25% and 35% (p=0.028), and PFS in 71% and 50% (p=0.027), respectively (Table). Further, combining BCR-ABL load and MDR1 expression prior to nilotinib produced a significant association with subsequent molecular and cytogenetic response and PFS. By 12 or 24 months, patients presenting with BCR-ABL IS <13% and MDR1/GUS ≥2 showed MMR in 56%, CCyR in 75% and 81% and longer PFS in 93% and 85% compared to those with BCR-ABL IS ≥13% and MDR1/GUS <2 achieving MMR rates of 5% (p<0.001), CCyR rates of 26% and 38% (p=0.002) and PFS rates of 72% and 41%, respectively (p=0.013). Also, BCR-ABL kinase domain mutation status prior to the onset of nilotinib was significantly associated with PFS under nilotinib therapy: Patients without any mutation showed PFS rates of 90% and 71% at 12 or 24 months, respectively; whereas those harboring less sensitive or resistant mutations (Hughes et al., JCO 2009) attained only 26% PFS (p=0.028). Except for patients harboring less sensitive or resistant mutations there was no significant difference between unmutated and mutated patients. 31 of 39 patients revealed SNPs at the 1236 position and 33 of the 39 showed SNPs at the 2677 position. Nilotinib efficacy was not affected by MDR1 SNPs. Neither an association with the molecular outcome nor MDR1 gene expression under nilotinib therapy was found. Conclusions:The rates of MMR, CCyR, and PFS on nilotinib 2nd line therapy are significantly associated with baseline levels of MDR1 gene expression and tumor burden of imatinib-resistant CML-CP patients. Further, baseline BCR-ABL mutations predict the probability of PFS rates but do not seem to do so for MMR and CCyR. These findings might deliver new molecular tools for the clinical risk stratification for treatment with nilotinib in case of resistance under imatinib front line therapy and suggest the need for prospective validation.Table:MDR1/GUS ratio predictive for MMR, CCyR and PFSRatio MDR1/GUS dichotomized at 2pCutoff<2≥2MMR by 12 months13%34%0.026MMR by 24 months13%34%CCyR by 12 months25%53%0.028CCyR by 24 months35%58%PFS by 12 months71%88%0.027PFS by 24 months50%75% Disclosures:Reynolds: Novartis Oncology: Employment, Equity Ownership. Woodman: Novartis Oncology: Employment. Robin: Novartis Oncology: Employment. Hofmann: Novartis Oncology: Honoraria, Research Funding. Hochhaus: Novartis Oncology: Honoraria, Research Funding. Müller: Novartis Oncology: Honoraria, Research Funding.

Co-suppression of MDR1 (multidrug resistance 1) and GCS（glucosylceramide synthase）restores sensitivity to multidrug resistance breast cancer cells by RNA interference (RNAi)

Multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle to successful treatment in patients with breast cancer. Besides overexpression of MDR1, the member of the ABC transporter family, glucosylceramide synthase (GCS) which allows cellular escape from ceramide-induced cellular apoptosis by mediating ceramide glycosylation was considered to be related with multidrug resistance (MDR) in breast cancer. To specifically and efficiently reverse MDR of breast carcinoma cells, two small interference RNA (siRNA) targeted multidrug resistance 1 (MDR1) and GCS mediated by plasmids were constructed and co-transfected into the MDR breast cancer cell line MCF-7/ADM. The results showed that both transient transfection and stable transfection of the two siRNA-expression plasmids could greatly decrease the relative expression of GCS mRNA and MDR1 mRNA, significantly lower than the controls (p < 0.01). Evaluation of chemosensitivity displayed a 96-fold reduction in drug resistance for Adriamycin. And the reversing effects could keep for a long period (at least 3 weeks). Our results demonstrated that co-inhibition of MDR1 and GCS could more effectively reverse MDR in drug resistant breast carcinoma cells in vitro.

Phospholipid/deoxycytidine analogue prodrugs for the treatment of cancer

We synthesized thioether phospholipid carrier molecules, conjugated each of them to 1-b-D-arabinofuranosylcytosine (ara-C), and synthesized amido containing phospholipid carriers conjugated to gemcitabine. Changing the alkyl chain at the C1- and C2-positions of the phospholipid increased the conjugates’ cytotoxicity over previous conjugates. Dipyridamole increased ara-C’s and gemcitabine’s IC50 value while the IC50 values for the phospholipid conjugates were relatively unchanged suggesting that phospholipid conjugates do not require a transporter for entry into the cell. The phospholipid conjugates were cytotoxic to MCF-7 cells and its multidrug resistance-1 (MDR-1) overexpressing cell line derivative (BC-19). Ara-C had no effect on either cell line. Therefore, these novel phospholipid/nucleoside analogue conjugates could be used for the treatment of tumor cells that express certain resistance phenotypes such as a loss of transporter activity and/or MDR-1 overexpression. In vivo the gemcitabine-phospholipid conjugate was well tolerated and prolonged the survival of tumor bearing mice compared to control mice.

Melanoma differentiation associated gene-7/interleukin-24 reverses multidrug resistance in human colorectal cancer cells

Overexpression of the multidrug resistance 1 (MDR1) gene, encoding P-glycoprotein (P-gp), facilitates resistance to diverse chemotherapeutic drugs and current P-gp inhibitors display high toxicity. We studied the effects of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), which exhibits cancer-specific apoptosis-inducing properties, in drug-sensitive (SW620) and drug-resistant (SW620/Dox) colorectal carcinoma cells. Adenovirus administered mda-7/IL-24, Ad.mda-7, effectively reversed resistance to doxorubicin-induced apoptosis in SW620/Dox cells by increased intracellular accumulation and decreased efflux of doxorubicin. Unexpectedly, P-gp-overexpressing cells (SW620/Dox) displayed increased apoptosis following Ad.mda-7 infection compared with parental SW620 cells, which correlated with more MDA-7/IL-24 protein in SW620/Dox than SW620 cell and potentially explains the increased sensitivity of P-gp-overexpressing cells to mda-7/IL-24. Transient overexpression of MDR1 in SW620 cells significantly increased apoptosis, decreased anchorage-independent growth, and increased MDA-7/IL-24 protein following Ad.mda-7 infection, whereas down-modulation of MDR1 in SW620/Dox cells by small interfering RNA decreased apoptosis following Ad.mda-7 infection. The increased mda-7/IL-24 sensitivity observed in SW620/Dox cells was partly due to increased reactive oxygen species generation and lower mitochondrial membrane potential. These findings confirm that mda-7/IL-24 is a potent MDR reversal agent, preferentially causing apoptosis in P-gp-overexpressing MDR cells, suggesting significant expanded clinical implications for the use of mda-7/IL-24 in treating neoplasms that have failed chemotherapy mediated by the P-gp MDR mechanism.

Inhibitory effect of curcumin onMDR1 gene expression in patient leukemic cells

When patients with cancers are treated with chemotherapeutic agents a long time, some of the cancer cells develop the multidrug resistance (MDR) phenotype. MDR cancer cells are characterized by the overexpression of multidrug resistance1(MDR1) gene which encodes P-glycoprotein (Pgp), a surface protein of tumor cells that functions to produce an excessive efflux and thereby an insufficient intracellular concentration of chemotherapeutic agents. A variety of studies have sought potent MDR modulators to decrease MDR1 gene expression in cancer cells. Our previous study has shown that curcumin exhibits characteristics of a MDR modulator in KB-V1 multidrug-resistant cells. The aim of this study was to further investigate the effect of curcumin on MDR1 gene expression in patient leukemic cells. The leukemic cells were collected from 78 childhood leukemia patients admitted at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, in the period from July 2003 to February 2005. There were 61 cases of acute lymphoblastic leukemia (ALL), 14 cases of acute myeloblastic leukemia (AML), and 3 cases of chronic myelocytic leukemia (CML). There were 47 males and 31 females ranging from 1 to 15 years old. Bone marrows were collected. The leukemic cells were separated and cultured in the presence or absence of 10 microM curcumin for 48 hours. MDR1 mRNA levels were determined by RT-PCR. It was found that curcumin reduced MDR1 gene expression in the cells from 33 patients (42%). Curcumin affected the MDR1 gene expression in 5 of 11 relapsed cases (45%), 10 of 26 cases of drug maintenance (38%), 7 of 18 cases of completed treatment (39%), and 11 of 23 cases of new patients (48%). The expression levels of MDR1 gene in leukemic patient cells as compared to that of KB-V1 cells were classified as low level (1-20%) in 5 of 20 cases (25%), medium level (21-60%) in 14 of 32 cases (44%), and high level (61-100%) in 14 of 20 cases (70%). In summary, curcumin decreased MDR1 mRNA level in patient leukemic cells, especially in high level of MDR1 gene groups. Thus, curcumin treatment may provide a lead for clinical treatment of leukemia patients in the future.

Overexpression ofMDR1Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection

Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P < 10(-4)) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed approximately 1.4- 4-fold increases in transcript levels (e.g. ALDH1A, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment.

Microarray Analysis of Differentially Expressed Genes in Response to Retrovirally Mediated Overexpression of MDR1.

An important strategy for improving cancer therapy is the protection of healthy cells against therapy-induced damage. In this context gene therapy can complement radio- and chemotherapy in the treatment of tumours. Retroviral overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, can deliver such a protective effect. P-gp possesses two relevant functions: its detoxifying effect acts as an efflux pump and could be important in high-dose chemotherapy, the anti-apoptotic effect of overexpressed P-gp could protect normal cells in tumour radio- or chemotherapy. The protection of haematopoietic stem cells in lymphoma, myeloma or solid tumor therapy is a promising candidate for this approach. However, little is known which other gene expressions might be influenced by MDR1-overexpression. Therefore we analyzed differentially expressed genes in the human lymphoblastoid cell line TK6 transduced with onco-retrovirus SF91m3 encoding MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). 61 annotated genes showed a significant change in expression (p<10−4) in MDR1 overexpressing compared to control transduced (overexpression the neomycin resistance gene) and untransduced cells. We found that genes coding for detoxifying and exocytotis proteins (e.g. ABCB4, ALDH1A, unc13) were up-regulated. Furthermore several proapoptotic genes were down-regulated (e.g. Casp1, Casp4) with concomitant increased expression of antiapoptotic genes (e.g. Akt3). The differential expressions for several genes were verified with real-time PCR. The results confirmed the dependence of these gene regulations on MDR1 overexpression. The influence of MDR1/P-gp overexpression on apoptotic signalling and cell survival was further corroborated by reduced apoptosis rates in response to irradiation in SF91m3 transduced cells compared to the control cell lines. Our genomics results support the findings from other studies that overexpression of MDR1 confers protection against apoptotic stimuli. The multidrug resistance phenotype does not seem to be only due to high P-gp expression but also to other metabolic proteins. Our results could have important implications for MDR1-gene therapy in patients receiving chemotherapy regimens in combination with radiotherapy.

326. Gene Therapeutic Overexpression of MDR1 Affects Transcriptional Regulations of Proteins Involved in Apoptosis and Detoxification

An important strategy for improving cancer therapy is the protection of healthy cells against therapy-induced damages. In this context gene therapy can complement radio- and chemotherapy in the treatment of tumours. Retroviral overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, can deliver such a protective effect. P-gp possesses two relevant functions: its detoxifying effect acts as an efflux pump and could be important in high-dose chemotherapy; the anti-apoptotic effect of overexpressed P-gp could protect normal cells in tumour radio- or chemotherapy. The protection of haematopoietic stem cells in lymphoma, myeloma or solid tumor therapy is a promising candidate for this kind of MDR1 gene therapy. However, little is known which other gene expressions might be influenced by MDR1-overexpression. Therefore we analyzed differentially expressed genes in the lymphoblastoid cell line TK6 onco-retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). 204 annotated genes of the >54000 probe sets per microarray showed a significant change in expression (P<10|[minus]|3) in MDR1 overexpressing compared to untransduced cells. We found that genes coding for detoxifying and exocytotis proteins (e.g. ALDH1A, unc13) were up-regulated. Furthermore several proapoptotic genes were down-regulated (e.g. Casp1, Casp4) with concomitant increased expression of antiapoptotic genes (e.g. Akt3). The differential expressions for seven selected genes were verified with real-time PCR comparing cDNA from MDR1 transduced versus untransduced and versus Neomycin resistance gene or hrGFP (humanized renilla green fluorescent protein) transduced TK6 cells as controls. The results confirmed the dependence of these gene regulations on MDR1 overexpression. Thus we could demonstrate that MDR1 overexpression influences the transcriptome of TK6 cells. Our genomics results support the findings from other studies that overexpression of MDR1 confers protection against apoptotic stimuli. The multidrug resistance phenotype does not seem to be only due to high P-gp expression but also to other metabolic proteins. These results could have important implications for MDR1-gene therapy in patients receiving chemotherapy regimens in combination with radiotherapy.

Caveolin-1 gene is coordinately regulated with the multidrug resistance 1 gene in normal and leukemic bone marrow

Caveolin-1 is a structural protein that may function as a scaffold for plasma membrane proteins, one of which is P-glycoprotein (P-gp), product of the multidrug resistance-1 ( MDR-1) gene. We tested the hypothesis that if P-gp and caveolin-1 interacted physically, caveolin-1 and MDR-1 genes might be coordinately regulated; by quantifiying their gene expression with quantitative-polymerase chain reaction. MDR-1 and caveolin-1 gene expressions were normalized to an internal control and related to a fixed calibrator by a comparative cycle-threshold ( C T ) method. In four different groups of marrow samples (20 normal, 56 acute myeloid leukemias (AML) at diagnosis, 48 AMLs at relapse, and 51 regenerating marrows), caveolin-1 and MDR-1 gene expressions were positively correlated. In 65 samples with MDR-1 over-expression, caveolin-1 and MDR-1 expressions were also correlated. The coordinate expression of caveolin-1 and MDR-1 suggests that they may either interact physically, or are involved in the same aberrant pathway(s) activated during MDR-1 up-regulation.

Expression of multidrug resistance-1 and multidrug resistance-associated protein genes in pediatric rhabdomyosarcoma

Overexpression of multidrug resistance-1 (MDR-1), and multidrug resistance-associated protein (MRP) genes has been linked with resistance to chemotherapy in vitro and in vivo. Their role in chemotherapy resistance in pediatric rhabdomyosarcoma is unclear. The study was undertaken to analyze the expression of MDR-1 and MRP genes in the embryonal and the alveolar subtypes of rhabdomyosarcoma and to elucidate its clinical relevance. Twenty-three rhabdomyosarcoma samples were analyzed for the expression of MDR-1 and MRP genes using a semi-quantitative competitive RT-PCR assay. MRP gene expression was associated with a reduction in survival (p=0.02). The overall survival of patients with tumors positive or negative for MRP expression were 50% (95% confidence interval, 30-70%) and 93% (95% confidence interval, 76-100%) respectively. In contrast, the expression of MDR-1 gene was not predictive of survival. These findings suggest that MRP expression could be a prognostic factor in patients with rhabdomyosarcoma.

Evidence for multidrug resistance-1 P-glycoprotein-dependent regulation of cellular ATP permeability.

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release approximately 3-fold. The MDR1 inhibitors cyclosporine A (10 microm) and verapramil (10 microm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl- current density from -33.1 +/- 12.5 pA/pF to -2.0 +/- 0.3 pA/pF (-80 mV, p < or = 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.

Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines.

Previous studies have shown that gene re-arrangements play a significant role in tumorigenesis. Gene re-arrangements involving the human multidrug resistance-1 (MDR1) gene have been identified as a mechanism for MDR1 over-expression in human malignant cells. In 2 multidrug-resistant human cancer sublines with high levels of MDR1 and P-glycoprotein (MCF7/TX400 and S48-3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified. To characterize and determine the site of the re-arrangements resulting in generation of hybrid mRNAs, we first constructed a lambda phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1. In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra-Alu deletion flanked by chi-like sequences at the re-arrangement junction. By contrast, non-homologous recombination was observed in S48-3s/Adr10 cells with Alu repeats near the junction sequence. While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re-arrangements. The gene re-arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1.

A Pilot Study of Low Dose Hydroxyurea as a Novel Resistance Modulator in Metastatic Renal Cell Cancer

Mechanisms of chemoresistance in renal cell carcinoma include P-glycopro-tein, overexpression of multidrug resistance-1 (mdr1) gene, and unstable chromosomal aberrations. In vitro exposure of resistant tumor cells to low dose hydroxyurea causes loss of chromosomal aberrations, decrease in the mdr1 gene copies, and increased sensitivity to vinblastine.Patients received continuous hydroxyurea 500 mg every Monday, Wednesday and Friday. Vinblastine 5 mg/m2 was given intravenously on days 1 and 8 every 21 days. Seventeen patients with a median age of 63 (range 40-80) received a median of 3 courses of vinblastine (range 1−14). Toxicities included: > grade 3 non-hematologic toxicity (1) and febrile neutropenia (2). No treatment related mortality occurred. Three patients (17.6%) had partial responses. The median survival was 38.0 weeks (95% CI= 26.9−49.1 weeks).The addition of hydroxyurea given at the dose of 500 mg orally three times weekly had no major impact on the expected antitumor effect of vinblastine.

Overexpression of multidrug resistance protein gene in human cancer cell lines selected for drug resistance to epipodophyllotoxins.

Overexpression of either the multidrug resistance 1 (MDR1) gene or multidrug resistance protein (MRP) gene is involved in acquisition of multidrug‐resistant phenotypes in human cancer cells. In this study we examined whether selection for resistance to the epipodophyllotoxins, etoposide/teniposide (VP16/VM26), could induce overexpression of MDR1 or MRP. We have previously isolated two VP16/VM26‐resistant KB cell lines. Two VP16/VM26‐resistant KB cell lines, KB/VM‐1 and KB/VM‐4, which were selected by stepwise exposure to VM26 had decreased accumulation of [3H]VP16 and increased levels of MRP, but no apparent expression of MDR1 gene was observed. Another VP16/VM26‐resistant KB cell line, KB/VP‐4, which was further isolated from a VP16‐resistant KB cell line, KB/VP‐2, had decreased accumulation of [3H]VP16 and showed overexpression of MRP gene, but not that of MDR1 gene. We also isolated a VP16‐resistant cell line, IN157/VP‐1, from a human glioma cell line IN157. IN157/VP‐1 cells showed decreased accumulation of [3H]VP16 and overexpression of MRP gene, but not of MDR1. These findings suggest that selection for resistance to VP16/VM26, preferentially induces overexpression of MRP gene.