Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.
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PMID:Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias. 135 60

In this report the effects of single doses of ionizing radiation on the mRNA expression of several proteins involved in multiple drug resistance were analyzed. Murine NIH 3T3 cells treated with single doses of 5, 10 and 20 Gy during the time interval from 1.5 to 72 h after irradiation were compared with their corresponding controls at the same points of time. The glutathione S-transferase-pi (GST pi) level was elevated in cells treated with 10 or 20 Gy from 24 to 72 h after irradiation compared with the control. Topoisomerase II alpha and thymidylate synthase were decreased in irradiated cells 24-72 h after exposure. These down-regulations were associated with cellular proliferation, determined by mRNA expression of the proliferation marker histone 3. Irradiated cells exhibited no alteration in the P-glycoprotein or glutathione peroxidase mRNA content. The finding that GST pi mRNA was overexpressed after irradiation was validated by investigations on a human lung carcinoma cell line (LXF 289) on the mRNA and protein level. Thus, our results indicate that irradiation alters the expression of proteins involved in multidrug resistance and may, therefore, play a role in clinical drug response.
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PMID:Effects of single doses of irradiation on the expression of resistance-related proteins in murine NIH 3T3 and human lung carcinoma cells. 755 53

Recent studies have shown that high levels of the apoptosis-related proteins bcl-2 and bcl-xL increase, while over-expression of bcl-xs or bax decreases, resistance to drugs that induce apoptosis in some human cancer cells. In the present report, we investigated whether expression of these apoptosis-related proteins correlates with changes in the degree of resistance to apoptosis induced by doxorubucin, taxol, vincristine and VP-16 and contributes to the development of acquired resistance in multidrug-resistant MCF-7/Adr breast cancer cells. In this study, high levels of bcl-xL and bax proteins are detected in both MCF-7 and MCF-7/Adr cells. In contrast, bcl-2 protein is down-regulated about 10-fold in MCF-7/Adr cells compared with MCF-7 cells. RT-PCR analysis showed that MCF-7/Adr cells express approximately 2-fold less bcl-2 mRNA than MCF-7 cells. Moreover, 4-24 hr cycloheximide treatment of MCF-7 and MCF-7/Adr cells did not affect the expression of bcl-2 protein, indicating that this protein is very stable in both cell lines. Our results suggest that bcl-2 expression is modulated partly by transcriptional, but mainly by post-transcriptional, mechanisms. Despite the down-regulation of bcl-2 in MCF-7/Adr cells and equal levels of bcl-x, and bax proteins in both cell lines, cytoplasmic DNA-histone complexes induced by doxorubucin, taxol, vincristine and VP-16 indicate that MCF-7/Adr cells are highly resistant to apoptosis. Moreover, treatments of MCF-7/Adr cells with P-glycoprotein (P-gp) modulators, cyclosporin A and verapamil increased doxorubicin and vincristine-induced DNA fragmentation about 1.4- and 2.5-fold, indicating that P-gp is involved in the development of resistance to chemotherapy-induced apoptosis in this cell line.
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PMID:Down-regulation of apoptosis-related bcl-2 but not bcl-xL or bax proteins in multidrug-resistant MCF-7/Adr human breast cancer cells. 878 46

Doxorubicin- (OAW-dox, SK-OV-dox), taxol- (OAW-tax, SK-OV-tax) and cisplatin- (SK-OV-cis) resistant cells derived from the parental OAW-42 and SK-OV-3 cell lines were established. OAW-42 sublines showed high resistance, the SK-OV-3 sublines only low resistance. OAW-42 sublines showed a cross-resistance profile typical of multidrug resistance (MDR). The sublines of SK-OV-3 showed a cross-resistance profile different from the OAW-42 sublines. The mRNA expression of several resistance proteins and related factors was analyzed. An overexpression of P-glycoprotein 170 (P-170), glutathione-S-transferase-pi (GST-pi), thymidylate synthase (TS), glutathione peroxidase (GP) and c-jun was found in OAW-dox and OAW-tax cells. Additionally, OAW-tax cells expressed a higher mRNA level of protein kinase Cbeta2. DNA analysis revealed a 2-fold gene amplification of P-170, whereas the genes for GST-pi, TS and GP were not amplified. SK-OV-dox and SK-OV-tax cells showed a decreased level of histone 3 (H3) and TS mRNA. This shows that the sublines of OAW-42 developed resistance by co-expression of several resistance-related proteins and proto-oncogenes whereas the sublines of SK-OV-3 expressed resistance by decreased expression of the proliferation-dependent proteins H3 and TS.
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PMID:Messenger RNA expression of resistance proteins and related factors in human ovarian carcinoma cell lines resistant to doxorubicin, taxol and cisplatin. 907 15

Resistance of tumor cells to chemotherapeutic drugs can not only be caused by treatment with antineoplastic agents but also by radiotherapy. The aim of this study was to analyze whether ionizing radiation can influence the mRNA expression of proteins which have been found to be involved in drug resistance of tumor cells. Human tumor cell lines (MCF-7, LXF and Sk-Mel) were treated with single doses of irradiation (5, 10 and 20 Gy). The expression of the resistance related proteins glutathione S-transferase-pi (GST-pi), topoisomerase II alpha (Topo II), thymidylate synthase (TS), O6-methylguanine-DNA-methyltransferase (MGMT), P-glycoprotein (Pgp), glutathione peroxidase (GPX) multidrug resistance-associated protein (MRP) and also of the heat-shock protein 70 (HSP 70) were determined at the mRNA level during the time interval from 1.5 to 72 h post-irradiation and compared with their corresponding controls. We also examined whether a relationship exists between these proteins and the proliferative activity (histone 3, Ki-67, statin) of the cells. We found that exposure of MCF-7, LXF and Sk-Mel cells to ionizing radiation increases the expression of the mRNA of GST-pi. Topo II, TS, HSP 70 and proliferation markers were also altered by exposure to ionizing radiation, but there was no common response of the three cell lines. No significant changes were observed in the expression of MGMT, Pgp, GPX and MRP after radiation treatment. Drug resistance tests revealed that irradiated MCF 7 cells were less sensitive to doxorubicin than non-irradiated control cells. Our results indicate that ionizing irradiation modifies the expression of some proteins involved in drug resistance and the response of MCF 7 cells to doxorubicin and may, therefore, play a role in clinical drug response.
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PMID:Messenger RNA expression of resistance factors in human tumor cell lines after single exposure to radiation. 941 87

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.
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PMID:Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y. 963 21

The multidrug resistance (MDR1) gene product, P-glycoprotein (Pgp), is a 170-kDa ATP-dependent pump that expels a variety of anticancer drugs out of malignant cells, reducing drug accumulation and thus antitumor activity. In recent years, considerable data has been presented that indicates the need to standardize detection methods for Pgp and MDR1. Reverse transcription (RT)-PCR is one of the most sensitive and specific techniques used to detect MDR1. Nevertheless, there is the need to address working criteria for quantitation by RT-PCR. In this study, we describe a flexible assay used to quantify MDR1 gene expression using heterologous (nonhomologous) standards for use in competitive RT-PCR (CRT-PCR). Our guidelines were to use a RT-PCR quantitation method that was independent of exponential phase kinetics, sensitive (detect low levels of gene measurement in clinical samples) and did not require radiolabel. Furthermore, the method would need to be flexible enough for the user to express quantitation as either the number of cells or amount of cDNA used in CRT-PCR. Using low-stringency amplification, heterologous DNA competitors were constructed for MDR1 and as an internal reference, the ubiquitously expressed human histone variant 3.3 (H3.3). The benefits of this approach are threefold: (i) amplification kinetics of target and competitor molecules are identical, (ii) low-stringency PCR is a simple way of constructing heterologous DNA competitors that do not require special storage conditions and (iii) heterologous competitors avoid the formation of heteroduplex molecules. We conclude that CRT-PCR is an extremely flexible and sensitive assay that can quantify MDR1 based on competitive amplification of a heterologous competitor. This might complement future efforts to standardize MDR1 detection methods using RT-PCR.
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PMID:Absolute quantitation of MDR1 transcripts using heterologous DNA standards--validation of the competitive RT-PCR (CRT-PCR) approach. 1037 51

Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. A. hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined. Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria. ORFs encoded by the remaining 46 fragments were identified as new proteins of A. hydrophila, showing no significant homology to any known proteins. Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A. hydrophila. The PPD134/91-specific genes included five known virulence factors of A. hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads. Some of these fragments encoded virulence determinants. These included genes for the synthesis of O-antigen and type II restriction/modification system. The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A. hydrophila.
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PMID:Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. 1078 58

Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) were used to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter. We have established that methyl-CpG binding protein 2 (MeCP2) is involved in methylation-dependent silencing of human MDR1 in cells that lack the known transcriptional repressors MBD2 and MBD3. In the repressed state the MDR1 promoter is methylated and assembled into chromatin enriched with MeCP2 and deacetylated histone. TSA induced significant acetylation of histones H3 and H4 but did not activate transcription. 5aC induced DNA demethylation, leading to the release of MeCP2, promoter acetylation, and partial relief of repression. MDR1 expression was significantly increased following combined 5aC and TSA treatments. Inhibition of histone deacetylase is not an overriding mechanism in the reactivation of methylated MDR1. Our results provide us with a clearer understanding of the molecular mechanism necessary for repression of MDR1.
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PMID:Precipitous release of methyl-CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene (MDR1) on activation. 1186 62

Depsipeptide (FK228) is a novel histone deacetylase inhibitor currently in clinical trials and the first to demonstrate clinical activity in patients. Responses have been observed in patients with T-cell lymphomas, despite prior treatment with multiple chemotherapeutic agents. To better understand the effects of histone deacetylase inhibitors on T-cell lymphoma, the human T-cell lymphoma cell line HUT78 was tested for sensitivity and molecular response to depsipeptide. Treatment with depsipeptide, as well as other histone deacetylase inhibitors, caused induction of histone acetylation, induction of p21 expression, and substantial apoptosis without significant cell cycle arrest. Treatment with the caspase inhibitor z-VAD-fmk significantly inhibited depsipeptide-induced apoptosis, enabling detection of cell cycle arrest. Treatment with depsipeptide increased expression of the interleukin-2 (IL-2) receptor, and combination with the IL-2 toxin conjugate denileukin diftitox resulted in more than additive toxicity. Cells selected for resistance to depsipeptide overexpressed the multidrug resistance pump, P-glycoprotein (Pgp). However, cells selected for resistance to depsipeptide in the presence of a Pgp inhibitor had a Pgp-independent mechanism of resistance. These studies confirm the activity of depsipeptide in a T-cell lymphoma model and suggest a general sensitivity of T-cell lymphoma to histone deacetylase inhibitors, an emerging new class of anticancer agents.
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PMID:T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance. 1499 4


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