Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.
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PMID:A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells. 167 47

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ200, MOLT-3/TMQ800, and MOLT-3/TMQ2500 based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention. These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug. Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with 125I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the Mr 170,000 protein of the TMQ-resistant cells. These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.
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PMID:Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate. 257 16

Exposure of MOLT-3 human leukemic cells in culture to a lipophilic antifolate, trimetrexate (TMQ), resulted in the development of sublines resistant to antifolates as well as to drugs related to multidrug resistance. The TMQ-resistant sublines had an increase in dihydrofolate reductase (DHFR) activity and overexpression of P-glycoprotein. In these sublines, neither the DHFR gene nor the MDR1 gene were amplified. In these cells, DHFR transcripts were also not overexpressed but DHFR protein was increased, indicative of translational or post-translational control of DHFR activity. In contrast, MDR1 transcripts were found to be overexpressed, in parallel with P-glycoprotein production. Therefore, increases in P-glycoprotein appear controlled at the transcriptional level. These data support evidence that TMQ produced two phenotypic changes independently: the former probably from folate deficiency and the latter from the lipophilic nature of the compound.
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PMID:Expression of dihydrofolate reductase and multidrug resistance genes in trimetrexate-resistant human leukemia cell lines. 809 75

In order to reverse P-glycoprotein-mediated drug resistance in a specific manner, we designed two hammerhead ribozymes which can cleave the GUC sequence in codon 179 and 196 of MDR1 (PGY1) mRNA. The ribozymes were directly synthesized using a set of primers, one containing a bacteriophage T7 RNA polymerase promoter. A target MDR1 RNA was created by a reverse transcription polymerase chain reaction using a MOLT-3 human acute leukemia cell line resistant to trimetrexate (TMQ) (MOLT-3/TMQ800), which displayed MDR1 overexpression. In a cell-free system, both ribozymes cleaved a target piece of MDR1 RNA into 2 fragments at the specific sites at a physiological pH and temperature. The cleavage reaction was dependent on time, ribozyme:substrate ratio, and magnesium concentration. The 196 MDR1 ribozyme was more active than the 179 MDR1 ribozyme. The 196 MDR1 ribozyme was then cloned into a human expression vector, and MOLT-3/TMQ800 cells were transfected. The original MOLT-3/TMQ800 cells were nearly 700-fold resistant to vincristine, whereas the transfectant cells selected in G418 became only 20- to 30-fold resistant. The level of resistance and the amount of MDR1 RNA expressed appeared to correlate inversely with the amount of ribozyme expression. A disabled 196 MDR1 ribozyme was capable of neither specific cleavage in vitro nor decreasing MDR1 expression in transfectant cells. These results indicate that it was the ribozyme activity and not antisense activity which was responsible for decreased MDR1 RNA. This approach may be applicable to cancer patients as a specific means to reverse tumors with P-glycoprotein-mediated MDR phenotype back to a drug-sensitive one.
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PMID:Reversal of drug sensitivity in multidrug-resistant tumor cells by an MDR1 (PGY1) ribozyme. 811 16

Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.
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PMID:Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs. 1100 May 80