Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The nucleotide sequence of the mdr1 gene encoding a putative drug efflux pump (P-glycoprotein) is homologous to a class of bacterial membrane-associated transport proteins. These bacterial proteins are part of a multicomponent system that includes soluble periplasmic proteins that bind substrates, channeling them through the membrane in an energy-dependent manner. We have investigated the possibility that a similar multicomponent transport system exists in a multidrug-resistant human MCF-7 breast cancer cell line that was initially selected for resistance to doxorubicin (AdrR MCF-7). AdrR MCF-7 cells overexpress both the mdr1 gene and the pi class isozyme of glutathione S-transferase (GST-pi) (EC 2.5.1.18). The latter is one of several isozymes known to have a ligand-binding function in addition to drug-metabolizing capabilities. Although we have recently shown that transfection of a functional GST-pi expression vector is insufficient to confer resistance to doxorubicin in cells that lack P-glycoprotein expression [Mol. Pharmacol. 36:22-28 (1989)], we examined the possibility that GST-pi interacts with P-glycoprotein to alter multidrug resistance. To do this, we have cloned cDNAs encoding these proteins from AdrR MCF-7 cells, constructed expression vectors containing these two genes, and transfected these vectors sequentially into drug-sensitive MCF-7 cells. The human mdr1 cDNA isolated from AdrR MCF-7 is a variant gene whose sequence differs from that isolated previously from vinblastine-resistant KB cells [Cell 53:519-529 (1989)], resulting in an amino acid substitution of alanine to serine at position 893 (mdr1/893ala). Transfection of eukaryotic expression vectors containing the mdr1 gene isolated from AdrR MCF-7 cells produced a multidrug-resistant phenotype in recipient cells, with a cross-resistance pattern similar to that in the AdrR MCF-7 cells. To determine whether GST-pi expression could augment resistance provided by mdr1, two clones transfected with mdr1, one with high levels (153% of mdr1 RNA in AdR MCF-7 cells) and one with low levels (10% of mdr1 RNA in AdrR MCF-7 cells), were subsequently cotransfected with a GST-pi expression vector and pSVNeo and selected for resistance to G418. Six of these clones contained levels of GST-pi that were 8- to 18-fold greater than GST levels found in mdr1-expressing clones transfected with nonspecific DNA. We found no difference in the degree of resistance to doxorubicin, actinomycin D, and vinblastine between the clones expressing mdr1 only and the clones expressing both mdr1 and GST-pi.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 Jun
PMID:Multidrug resistance in cells transfected with human genes encoding a variant P-glycoprotein and glutathione S-transferase-pi. 197 72

The relationship between terminal cell differentiation and changes in the subcellular levels of the HER-2/neu antigen was investigated in cultured human breast cancer cells: AU-565 cells (which overexpress the HER-2/neu gene) and MCF-7 cells (which do not overexpress this gene). Differentiation was achieved by treating the cells with mycophenolic acid (MPA), phorbol 12-myristate 13-acetate (PMA), retinoic acid (RA), or the TA-1 monoclonal antibody to the extracellular domain of the HER-2/neu protein. Ten to twenty percent of the cells in untreated, sparsely growing AU-565 cultures exhibited morphological maturation characterized by large lacy nuclei surrounded by sizable flat cytoplasms. A fraction of these cells harbored milk factors such as casein and large lipid droplets. Treatment of the AU-565 cells for 4 d with 9 microM MPA, 1.6 nM PMA, 2.5 microM RA, or 1 microgram/mL TA-1 antibody resulted in cell growth inhibition and an increase in the percentage of cells (48-97%) that exhibit a mature phenotype. MCF-7 cells were also susceptible to differentiation by MPA and RA, but to a lesser degree than the AU-565 cells. Differentiation in the AU-565 and MCF-7 cells was associated with reduced immunostaining for the HER-2/neu protein at the cell surface membrane and with a transient increased diffuse immunostaining for this protein in the cytoplasm.
Mol Carcinog 1990
PMID:Differentiation of cultured human breast cancer cells (AU-565 and MCF-7) associated with loss of cell surface HER-2/neu antigen. 198 May 88

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.
J Steroid Biochem Mol Biol 1991 Jan
PMID:Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase. 199 20

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.
J Steroid Biochem Mol Biol 1991 Feb
PMID:Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts. 200 34

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylaminoethoxy-phenyl)-1,2-diphenylbut-1-ene] is an effective anticancer agent against estrogen receptor (ER)-positive breast cancer. The alkylaminoethane side chain is essential for antiestrogenic activity, but the potency of the antiestrogen can be increased by para hydroxylation of the phenyl ring on carbon 1 of but-1-ene. This compound, 4-hydroxytamoxifen, is a metabolite of tamoxifen and has a very high binding affinity for ER [J. Endocrinol. 75:305-316 (1977)] because the hydroxyl is located in the equivalent position as the 3-phenolic hydroxyl of 17 beta-estradiol. In this study, we have examined the relationship between the relative positions of the hydroxyl and the alkyl-aminoethane side chain and the pharmacological activity of the ligand. A fixed seven-membered ring derivative of the triphenylethylene was used to prevent isomerization. All compounds were tested, with and without 17 beta-estradiol, for their effects on the growth of estrogen-responsive T47D and MCF-7 human breast cancer cells in vitro. The growth of MDA-MB-231 ER-negative breast cancer cells was not affected by any of the compounds tested, at a concentration (1 microM) that had a profound estrogenic or antiestrogenic action in ER-positive cell lines. The relative binding affinity of the compounds was determined using rat uterine ER and was found to be consistent with the observed potencies in vitro. The compounds found to be antiestrogens in vitro were antiestrogenic against estradiol (0.08 micrograms daily) in the 3-day immature rat uterine weight test. All compounds were partial agonists in vivo. In general, the estrogenic and antiestrogenic results obtained in vivo were consistent with the potency estimates obtained with the breast cancer cells in vitro. The results of this extensive structure-activity relationship study demonstrate that the substitution for 4-hydroxytamoxifen appears to be optimal to produce a potent antiestrogen; all other substitutions produced either estrogenic compounds or less potent antiestrogens. The hydroxyl group appears to be critical to locate the alkyl aminoethoxy side chain in the correct position in the steroid-binding site to block estrogen action. Novel antiestrogens were identified that could have been predicted based upon earlier drug-receptor models for the ER.
Mol Pharmacol 1991 Mar
PMID:Structure-activity relationships of nonisomerizable derivatives of tamoxifen: importance of hydroxyl group and side chain positioning for biological activity. 200 79

To determine whether the c-Ha-ras oncogene plays a role in the initiation of mammary carcinogenesis, an immortalized human breast epithelial cell line, MCF-10A, was transfected with the plasmid vector pHo6T1 containing the T24 Ha-ras oncogene and the aminoglycoside phosphotransferase gene, which confers resistance to geneticin. Transfected cells exhibited an altered pattern of growth and tridimensional morphology in collagen gel. They also exhibited anchorage-independent growth and loss of requirement for hormones and epidermal growth factor; in addition, they expressed invasiveness and increased collagenolytic activity in an in vitro system and became tumorigenic in irradiated nude mice, all properties indicative of malignant transformation. Transformed cells contained the mutated c-Ha-ras oncogene and expressed the p21 mutated protein. These data indicate that the c-Ha-ras oncogene is capable of inducing malignant phenotypes in immortalized human breast epithelial cells.
Mol Carcinog 1991
PMID:Transformation of human breast epithelial cells by c-Ha-ras oncogene. 200 32

An irradiation-reduced somatic cell hybrid mapping panel was constructed of BALB/c mouse Chromosome 1. Nineteen hybrids were selected from a pool of 292 clones to generate a fine structure physical map of the distal 40 cM of the chromosome. The hybrids contain mouse DNA fragments only from Chromosome 1, ranging from approximately 5 cM to approximately 20 cM. Utilizing a viral infectibility assay, a cellular receptor gene, Rmc-1, for the MCF class of murine retroviruses was found to be linked to Lamb2, in the region between the Lamb2 and Bxv-1 loci. In addition, analysis of the hybrid mapping panel resulted in the remapping of three loci, Atpb, Ly-5, and Pmv-24, as compared to the mouse linkage map. Two previously unmapped endogenous proviruses are also putatively assigned positions on the chromosome.
Somat Cell Mol Genet 1991 Mar
PMID:Isolation and characterization of irradiation fusion hybrids from mouse chromosome 1 for mapping Rmc-1, a gene encoding a cellular receptor for MCF class murine retroviruses. 201 95

To begin analysis of the DNA sequences necessary for luteinizing hormone (LH) gene transcription, fusion genes containing the 5' flanking region of the rat LH beta or the human alpha-subunit gene linked to luciferase were transfected into primary cultures of rat pituitary cells. The LH beta-luciferase construct was expressed in the primary cultures at a level 50 times greater than a promoterless luciferase control plasmid. Little or no expression of the LH beta-luciferase construct was detected following transfection of MCF-7, JAR or GH3 tumor cell lines. Treatment of transfected cells with gonadotropin-releasing hormone resulted in a modest induction of LH beta-luciferase activity. Considerably higher levels of LH beta-luciferase activity were obtained with cultures from ovariectomized rats than were obtained with cultures from intact female rats. Analysis of 5' deletions of the LH beta-luciferase construct demonstrated that activity was well maintained even after substantial deletions. The shortest construct, which contained 75 base pairs of 5' flanking sequence had 38% of the activity of the longest which contained 1.7 kilobase pairs of flanking sequence. These findings demonstrate that transfection of primary cultures of rat pituitary cells may provide a useful system for analysis of the cis-acting sequences and trans-acting factors required for LH gene expression.
Mol Cell Endocrinol 1990 Dec 03
PMID:DNA sequences required for expression of the LH beta promoter in primary cultures of rat pituitary cells. 209 May 14

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.
Mol Cell Biochem 1990 Aug 10
PMID:Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines. 212 95

Estrogen is a mitogen for the rat uterus, where it induces transient activation of c-fos and c-myc protooncogene expression, followed by increases in DNA synthesis and cell proliferation. JUN-C, the product of the c-jun protooncogene, is a nuclear protein that can interact with FOS to modulate the activity of AP-1-responsive promoters. To test whether c-jun is a target for estrogen regulation, we measured the effects of 17 beta-estradiol on the expression of this gene in rat uterus. A human c-jun cDNA probe detects in rat uterus two mRNA species of 2.5 and 3.2 kilobases. Treatment of the animals with estrogen results in a rapid transient increase in the concentrations of these mRNAs; a 4- to 5-fold increase over the prestimulation level was detected starting 30 min after estrogen injection and lasting for 2 h, with a return to the prestimulation level after 4 h. In accordance with the results obtained by analysis of the mRNA, we found that estrogen increases 3- to 4-fold c-jun gene transcription in the uterus, at the same time it induces its mRNA accumulation. The ability of estrogen to induce c-jun gene expression was not abolished by the protein synthesis inhibitor cycloheximide, suggesting that transcriptional activation of this protooncogene is a primary response to the hormone. Furthermore, we found that in the estrogen-responsive MCF-7 human mammary carcinoma cells, estrogen stimulates transcription of a reporter gene containing four copies of a jun/AP-1 response element. These data demonstrate that c-jun gene expression is regulated by estrogen and suggest that JUN-C could play a role in the activation of cell proliferation by estrogen.
Mol Endocrinol 1990 Jul
PMID:Estrogen stimulates transcription of c-jun protooncogene. 212 98


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