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

---

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

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.
...
PMID:Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line. 751 53

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.
...
PMID:Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells. 764 Feb 9

The human melanoma cell line FEM-X was selected in multiple steps with VP-16 (etoposide) and an inhibitor of P-glycoprotein (Campain et al., 1993). The resulting clones, FVP1b and FVP3, are highly resistant to the nonintercalative epipodophyllotoxins and exhibit moderate levels of resistance to doxorubicin. The topoisomerase II activity present in crude nuclear extracts from mutant and wild-type cells is similar in amount and equally sensitive to VP-16. However, in live cells, the topoisomerase II from FVP1b and FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. Using reverse transcription followed by the polymerase chain reaction (RT-PCR), we have cloned and sequenced the entire cDNA for topoisomerase II alpha from FEM-X and FVP3. The only sequence change unique to the cDNA from drug-resistant cells is a 3 bp deletion of nucleotide 1320-1322, resulting in a deletion of Ala429. Three FEM-X sublines of increasing resistance were tested, and the prevalence of the mutant RNA over wild-type increases in these cells in parallel with their resistance to VP-16. In FVP3, the most highly resistant line, expression of the wild-type allele is barely detectable. Analysis of genomic DNA shows that FEM-X is homozygous for the wild-type topoisomerase II alpha sequence and that each of the drug-resistant clones possesses both wild-type and mutant alleles. Although not definitive, these genetic results suggest that the deletion of Ala429 from topoisomerase II alpha makes the enzyme less susceptible to drug-induced cleavable complex formation and confers a growth advantage upon cells in the presence of VP-16.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:A novel mutant topoisomerase II alpha present in VP-16-resistant human melanoma cell lines has a deletion of alanine 429. 772 83

In a series of 60 ALL samples drawn during different stages of the disease we used a cDNA-PCR approach to analyse the relative mRNA levels of the MDR-associated genes encoding mdr1/P-glycoprotein, mrp, and the topoisomerase II isozymes alpha and beta. Expression analysis of the cyclin A gene was included to examine cellular proliferation activity. The expression of gapdh served as an internal standard. Calculating the mean values we found: (i) a distinctly lower mdr1 gene expression in primary ALL and first relapses compared to bone marrow from healthy donors, (ii) no change in mdr1 and mrp, but a decreased topoisomerase II alpha gene expression in first relapses of ALL compared to the primary leukaemia, and (iii) increased mdr1 and mrp levels combined to decreased topoisomerase II alpha levels in recurrent relapses of ALL showing significant correlations (mdr1/mrp: rs = +0.6833, P < 0.05; mdr1/topoII alpha: rs = -0.6727, P < 0.05). The expression of the topoisomerase II alpha gene was correlated to that of cyclin A, indicating a link of its expression to cellular proliferation. Our findings suggest that a multifactorial MDR including mrp appears particularly in recurrent relapses of ALL, which often do not respond to chemotherapy. Nonetheless, some individual samples showed gene expression levels very different from the mean values calculated for a particular state of the leukaemia, indicating the need of an individual expression analysis of MDR-associated genes.
...
PMID:Expression of mdr1, mrp, topoisomerase II alpha/beta, and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias. 787 86

The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (< 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to-cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony-stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.
...
PMID:Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia. 790 87

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.
...
PMID:Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias. 791 48

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.
...
PMID:Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection. 791 96

Fifteen samples from 11 patients suffering from chronic lymphocytic leukaemia (CLL; 5 untreated, 6 chemotherapeutically treated) were analysed for their individual gene expression of the multidrug resistance (MDR) associated genes encoding mdr1/P-glycoprotein, mrp, and topoisomerase II alpha/beta-isoenzymes by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. The expression of glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as standard. Thereby, we generally found high mdr1- and mrp-, but low topoisomerase II alpha-mRNA levels. While mdr1 levels of the CLL samples were mostly found to be in the range of values measured in the T-lymphoblastoid, P-glycoprotein MDR cell line CCRF VCR 100, mrp levels were usually found to be 2-4-fold higher compared therewith. This might represent a multifactorial MDR in CLL. In contrast to the low or even absent topoisomerase II alpha gene expression, however, the expression of the topoisomerase II beta gene was generally high in the CLL lymphocytes exceeding the value observed in the cell line CCRF VCR 100 up to 5-fold. mdr1 gene expression correlated significantly with mrp gene expression in samples from patients having received chemotherapy (rs = 0.5833, P < 0.05, n = 10). In two patients the follow-up analysis revealed combined increases in mdr1- and mrp-gene expression levels in the course of the disease.
...
PMID:High mdr1- and mrp-, but low topoisomerase II alpha-gene expression in B-cell chronic lymphocytic leukaemias. 795 50

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II alpha are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II alpha-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M(r) of 160,000. The levels of the topoisomerase II alpha 6.1-kilobase mRNA in H209/V6 cells are reduced > 10-fold. In addition, a second topoisomerase II alpha-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M(r) 180,000 topoisomerase II beta protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase II alpha-related M(r) 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M(r) 170,000 topoisomerase II alpha in H209 cells and the M(r) 180,000 topoisomerase II beta in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II alpha have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.
...
PMID:Altered topoisomerase II alpha in a drug-resistant small cell lung cancer cell line selected in VP-16. 810 87

K562 leukaemia cells were selected for resistance using 0.5 microM etoposide (VP-16). Cloned K/VP.5 cells were 30-fold resistant to growth inhibition by VP-16 and 5- to 13-fold resistant to m-AMSA, adriamycin and mitoxantrone. K/VP.5 cells did not overexpress P-glycoprotein; VP-16 accumulation was similar to that in K562 cells. VP-16-induced DNA damage was reduced in cells and nuclei from K/VP.5 cells compared with K562 cells. Topoisomerase II protein was reduced 3- to 7-fold and topoisomerase II alpha and topoisomerase II beta mRNAs were each reduced 3-fold in resistant cells. After drug removal, VP-16-induced DNA damage disappeared 1.7 times more rapidly and VP-16-induced DNA-topoisomerase II adducts dissociated 1.5 times more rapidly in K/VP.5 cells than in K562 cells. ATP (1 mM) was more effective in enhancing VP-16-induced DNA damage in nuclei isolated from sensitive cells than in nuclei from resistant cells. In addition, ATP (0.3-5 mM) stimulated VP-16-induced DNA-topoisomerase II adducts to a greater extent in K562 nuclei than in K/VP.5 nuclei. Taken together, these results indicate that resistance to VP-16 in a K562 subline is associated with a quantitative reduction in topoisomerase II protein and, in addition, a distinct qualitative alteration in topoisomerase II affecting the stability of drug-induced DNA-topoisomerase II complexes.
...
PMID:Altered stability of etoposide-induced topoisomerase II-DNA complexes in resistant human leukaemia K562 cells. 814 56


1 2 3 Next >>

---