Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 Jul
PMID:Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins. 274 27

The regulation of the human cytochrome Cyp1A2 gene by 3-methylcholanthrene was studied through the transfection of 5'-flanking sequences into human cells. The Cyp1A2 promoter sequence and 3700 bases 5' to the cap site were linked to the procaryotic chloramphenicol acetyltransferase gene. Transfection of this construct into HepG2 cells generated a 2-3-fold increase in Cyp1A2-directed chloramphenicol acetyltransferase activity when the cells were treated with 3-methylcholanthrene. Deletion of flanking sequence to -1079 resulted in a loss of 3-methylcholanthrene-induced chloramphenicol acetyltransferase activity. When 5'-flanking sequences of the Cyp1A2 gene were inserted into a plasmid containing the chloramphenicol acetyltransferase gene under control of the simian virus 40 promoter, 3-methylcholanthrene-enhanced chloramphenicol acetyltransferase activity was observed. The strongest 3-methylcholanthrene-induced chloramphenicol acetyltransferase activity, a 4-fold increase, was observed for a DNA fragment located at -3202 to -1595. When this Cyp1A2 responsive element was transfected into human breast carcinoma MCF-7 cells, 3-methylcholanthrene did not stimulate chloramphenicol acetyltransferase activity. In comparison, when a DNA fragment that contained a copy of the human Cyp1A1 xenobiotic-responsive element was analyzed for enhancer activity, 3-methylcholanthrene initiated chloramphenicol acetyltransferase activity in both HepG2 cells and MCF-7 cells. These results suggest that the 3-methylcholanthrene-responsive Cyp1A2 element may be regulated in a tissue-specific manner.
Mol Pharmacol 1989 Jul
PMID:The human cytochrome Cyp1A2 gene contains regulatory elements responsive to 3-methylcholanthrene. 274 32

Since sex steroid hormones and growth factors are known to modulate the proliferation of breast tumors, we have studied the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, which contain low levels of ER, and in two sublines of MCF-7 cells which contain high ER levels. The mRNA levels were measured by Northern blot analysis using lambda OR8, a cDNA probe for ER, and protein levels were measured by hormone binding or Western blot analysis. Treatment of T47D cells with estradiol (E2) caused a 2.5-fold increase in ER mRNA (6.6 kilobases) levels after 48 h. The progestin R5020 evoked a marked decrease in ER mRNA and protein levels to 20% of control values, while the antiprogestin RU38,486 caused no change in ER. In MCF-7 cells, the effect of E2 on ER levels was dependent on the prior growth history of the cells. In cells grown in low estrogen [5% charcoal-dextran-treated calf serum with phenol red for 8 yr (MCF-7-K2)], which are still E2 responsive, treatment with E2, the antiestrogen LY117018, or both produced little change in ER mRNA or protein; in contrast, ER mRNA and protein were reduced by E2 to 40% and 50% of control levels, respectively, in MCF-7 cells (denoted MCF-7-K1) which had been maintained routinely in medium containing 5% calf serum. This decrease in ER mRNA was dose dependent; 10(-11) E2 reduced levels to 60%, and 10(-10) M E2 evoked the maximal drop to 40% of the control level in 2 days. LY117018 alone did not alter ER mRNA levels in these cells, but it completely prevented the down-regulation of ER by E2. Administration of progestin, but not antiprogestin, along with E2 partially prevented the decrease in ER evoked by E2. Addition of epidermal growth factor or insulin-like growth factor-I to MCF-7-K1 cells, which increased cell proliferation, had no detectable effect on ER levels. Treatment with transforming growth factor-beta, which decreased cell proliferation, reduced ER by about 20%.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1989 Feb
PMID:Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors. 278 42

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.
Mol Endocrinol 1988 Feb
PMID:Alterations in phosphoinositide metabolism associated with 17 beta-estradiol and growth factor treatment of MCF-7 breast cancer cells. 284 May 72

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.
Mol Pharmacol 1986 Aug
PMID:Uracil nucleotide synthesis in a human breast cancer cell line (MCF-7) and in two drug-resistant sublines that contain increased levels of enzymes of the de novo pyrimidine pathway. 287 77

A cDNA library has been constructed from the poly(A)+ mRNA of oestrogen-stimulated ZR-75-1 human breast cancer cells. Screening by differential hybridization has identified eight clones which are stimulated between 4- and 16-fold by oestrogen. Two clones (pLIV-1) that are stimulated 4-fold, hybridize to three different mRNA species. A further five recombinants encode for a mRNA 600 bp long which is induced greater than 16-fold and have been shown to cross-hybridize to the oestrogen-responsive clone, pS2, isolated from the MCF-7 breast cancer cell line. Oestradiol was shown to be without detectable effect upon the expression of mRNA for dihydrofolate reductase, which is reported to be oestrogen regulated in MCF-7 cells. Actin gene expression is also unresponsive to oestradiol in ZR-75-1 cells. These results suggest that pLIV-1 represents a previously unidentified mRNA that may be involved in the oestrogen-regulated growth of ZR-75-1 human breast cancer cells.
Mol Cell Endocrinol 1988 Oct
PMID:Effects of oestrogen on the expression of a 4.4 kb mRNA in the ZR-75-1 human breast cancer cell line. 290 3

Using a combination of hormone-binding assays, immunologic techniques, and mRNA hybridizations we have measured the estrogen receptor (ER) content and studied the hormonal regulation of ER mRNA in one estrogen responsive and one estrogen unresponsive breast cancer cell line, MCF-7 and T47Dco, respectively. Estradiol binding could be detected in cytosol from MCF-7 cells but not in T47Dco cells. However, when measured by an enzyme-linked immunosorbent assay, T47Dco cells were found to contain approximately 15 fmol ER/mg cytosolic protein or 10% of the ER content in MCF-7 cells. Immunologically reactive ER in T47Dco cells was indistinguishable in size (approximately equal to 68 KD) from the ER in MCF-7 cells, as shown by Western blotting using a monoclonal antihuman ER antibody. Quantification of ER mRNA in MCF-7 and T47Dco cells indicated that T47Dco cells contained approximately 50% of the ER mRNA levels found in MCF-7 cells. This basal level of ER mRNA in T47Dco cells was not decreased by estradiol treatment, as opposed to in MCF-7 cells where estradiol caused 40-60% decrease in the ER mRNA expression. Also, estradiol did not increase the progesterone receptor (PR) mRNA levels in T47Dco cells whereas in MCF-7 cells an approximately 5-fold increase of the PR mRNA levels occurred after estradiol treatment. However, incubation of the cells with the synthetic progestin R5020 decreased the ER mRNA levels to approximately the same degree in both cell lines. In conclusion, we have shown that estrogen down-regulates ER mRNA and up-regulates PR mRNA in MCF-7 cells. Neither of these estrogenic effects were seen in T47Dco cells. It appears that the steroid-resistance in T47Dco cells does not occur as a consequence of a complete absence of ER mRNA or protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1989 Jan
PMID:Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells. 291 47

The in vitro secretion of immunoreactive somatomedin C/insulin-like growth factor I (IR Sm-C/IGF-I) by two human breast cancer cell lines, MCF-7 and EVSA-T has been studied. IR Sm-C/IGF-I concentration showed a linear increase in serum-free culture media over 72 h of incubation for both cell lines, and a close correlation with cell number (P less than 0.001). To characterize this immunoreactivity, a pool of conditioned media collected after 72 h of incubation was dialyzed overnight against 1 M acetic acid, lyophilized, and gel filtered on a Sephadex G-50 column. Fractions were determined for Sm-C/IGF-I content and for the presence of a specific carrier for Sm-C/IGF-I. Two peaks of Sm-C/IGF-I-like immunoreactivity were evidenced, the first in the high molecular weight region and the second corresponding to the molecular weight of the free peptide. The first peak evidenced also a specific binding ability for radioiodinated Sm-C/IGF-I, suggesting that the activity found in this region could be interpreted as interference of the specific free binding sites in the immunoassay. Analysis of this peak by polyacrylamide gel electrophoresis demonstrated the presence of a specific binding for Sm-C/IGF-I in a molecular weight range between 35,000 and 45,000 Da, which was not modified in reducing conditions. The binding activity was competitively inhibited by addition of cold Sm-C/IGF-I but not by insulin excess.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1987 Dec
PMID:Partial characterization of somatomedin C-like immunoreactivity secreted by breast cancer cells in vitro. 296 40

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.
Mol Endocrinol 1988 May
PMID:Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro. 297 Nov 35

Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.
Mol Cell Biol 1986 Mar
PMID:5' Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene. 302 36


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