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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously, we demonstrated that the progestin components (19-nortestosterone derivatives) in oral contraceptives are able to stimulate human breast cancer cell proliferation via an estrogen receptor (ER)-mediated mechanism. We now examine RU486, an antiprogestin, to determine whether it has estrogenic properties because it is also a 19-nortestosterone derivative. We found that RU486 stimulated the growth of MCF-7 human breast cancer cells at a concentration of 10(-6) M, which is similar to the pharmacological concentration (micromolar range) found in women taking RU486. The antiestrogens 4-hydroxytamoxifen and ICI 164,384 blocked RU486-induced cell proliferation. The estrogenic activity of RU486 is not due to impurities or aromatization to estrogenic metabolites. To determine whether the proliferative action of RU486 was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene under the control of an estrogen response element derived from the Xenopus laevis vitellogenin 2A gene. We found that RU486 was able to induce chloramphenicol acetyltransferase activity at the concentrations that stimulated cell proliferation, and this induction was blocked by the addition of 4-hydroxytamoxifen and ICI 164,384. The estrogenic potential of RU486 to regulate ER target gene expression was also investigated. We found that, like 17 beta-estradiol (E2), RU486 was able to alter the expression and synthesis of progesterone receptor. The level of progesterone receptor (145 and 186 fmol/mg cytosol protein, respectively) was increased significantly compared to the control value (3 fmol/mg cytosol protein) with the addition of 10(-6) M RU486 or 10(-10) M E2, as determined by an enzyme immunoassay. The levels of transforming growth factor-beta 2 (TGF beta 2) and TGF beta 3 mRNA, but not TGF beta 1 mRNA, were decreased dramatically with the addition of 10(-6) M RU486. This is consistent with the effects of E2 on TGF beta expression. Therefore, RU486 has estrogen-like activities in its regulation of ER target gene expression. These results demonstrate that RU486 is a weak estrogen in human breast cancer cells and suggest that the RU486-induced cell proliferation is mediated via ER. The novel finding that RU486 exhibits some estrogen-like activity may be important for the interpretation of its action at high dosages as an abortifacient and also if RU486 is going to be evaluated clinically, again at high doses, for the treatment of breast cancer.
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PMID:Estrogenic actions of RU486 in hormone-responsive MCF-7 human breast cancer cells. 850 63

17 beta-Estradiol (E2) induces cathepsin D mRNA levels and intracellular levels of immunoreactive protein in MCF-7 human breast cancer cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression in this cell line; however, in cells cotreated with TCDD and E2, TCDD inhibited E2-induced cathepsin D mRNA levels, the rate of gene transcription, and levels of immunoreactive protein. The inhibitory responses were observed within 30 to 120 min after the cells were treated with TCDD. TCDD also inhibited E2-induced secreted alkaline phosphatase activity in aryl hydrocarbon (Ah)-responsive MCF-7 and wild-type mouse Hepa 1c1c7 cells cotransfected with the human estrogen receptor (hER) and the pBC12/S1/pac plasmid, which contains the 5' promoter region (-296/+57) of the cathepsin D gene and an alkaline phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 to -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-175/-181) xenobiotic responsive element (XRE); the role of this sequence in Ah responsiveness was investigated in gel electrophoretic mobility shift assays and with plasmid constructs containing a wild-type ER/Sp1 oligonucleotide or a mutant ER/Sp1-"XRE" oligonucleotide containing two C-->A mutations in the XRE sequence (antisense strand). In plasmid constructs which contained a chloramphenicol acetyltransferase reporter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chloramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-"XRE" plasmids. Electrophoretic mobility shift assays showed that the nuclear or transformed cytosolic Ah receptor complex blocked formation of the ER-Sp1 complex with the wild-type but not the ER/Sp1 mutant oligonucleotide. Moreover, incubation of the wild-type bromodeoxyuridine-substituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demonstrate that Ah receptor-mediated inhibition of E2-induced cathepsin D gene expression is due to disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.
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PMID:Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. 852 36

The c-Fes proto-oncogene encodes a myeloid-specific protein-tyrosine kinase that is expressed preferentially in differentiated myeloid cells, but not in early myeloblast progenitor cells. To examine the basis for the phenotypic expression of c-Fes, the transcription initiation sites of the human c-Fes gene were mapped in myeloid leukemia cells and regulatory elements in the genomic c-Fes sequence were characterized. Two major transcription initiation sites were found in the myeloid leukemia cell line THP-1 which delineated exon 1 to be 72-83 bp. When the activity of the CAT reporter gene under the control of the c-Fes promoter region, untranslated exon 1 and intron 1 was measured in TF-1, K562 and MCF-7 cells, only TF-1 cells exhibited chloramphenicol acetyltransferase activity. In contrast, all cell lines supported reporter gene activity when intron 1 was deleted. Deletion analyses revealed a negative regulatory region in intron 1, which was localized by Southwestern analysis and DNA footprinting to a 14 bp region. This negative regulatory region suppressed reporter CAT activity in K562 and TF-1 cells when inserted downstream to the SV40 early promoter. These results suggest that the tissue-specific expression of c-Fes may result, in part, from the negative regulation of transcription in myeloid and nonmyeloid cells.
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PMID:Transcriptional regulation of c-Fes in myeloid leukemia cells. 863 35

We have investigated whether or not the cellular content of reactive platinum, aside from total cellular and DNA-bound platinum, is a measure of the growth inhibitory potential of a given platinum complex. Human MCF-7 breast cancer cells, after treatment with cisplatin [cis-diamminedichloroplatinum(II)] and several 1,2-diphenylethylenediamineplatinum(II) complexes at a fixed dose of 3 microM, were analyzed for their contents of platinum in total cells, isolated nuclei, chromosomal DNA, and the cellular pool of reactive platinum, and compared with ED50-values. Platinum was measured by atomic absorption. Reactive platinum was identified after its reaction with calf thymus DNA that had been added to the cells before their lysis. The amounts of platinum binding to chromosomal DNA were related to previously established ED50-values, and such a correlation could not be found for platinum in total cells, nuclei, and, especially, reactive platinum. The observed differences in the platinum contents of DNA were referred to variations in the rate of adduct formation rather than repair because two representative platinum complexes were indistinguishable by their effects on the chloramphenicol acetyltransferase (EC 2.3.1.28) transfection system. One of the other platinum complexes accumulated, showing an increased growth inhibition in support of this interpretation with regard to the other platinum complexes. During prolonged treatment of MCF-7 cells with the platinum(II) complexes, pools of reactive platinum were found to persist even after drug depletion in the culture medium. This suggested a hitherto unrecognized cellular storage and availability of reactive platinum.
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PMID:Cellular distribution and cellular reactivity of platinum (II) complexes. 867 10

The antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in several cell lines using transient transfection assays and constructs containing 5'-regulatory sequences from the estrogen (E2)-responsive vitellogenin (Vit) A2 gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. TCDD significantly inhibited CAT activity induced by E2 in MCF-7 human breast cancer cells transiently transfected with 5'-deletion plasmids containing the homologous promoter [(-821/+14)- and (-482/+14)-CAT] or the heterologous thymidine kinase (tk) promoter [(-821/-87)tk-, (-482/-87)tk-, (-397/-87)tk-, and (-331/-87)tk-CAT]. In parallel experiments using wild-type mouse Hepa 1c1c7 and human HeLa cells cotransfected with a human estrogen receptor expression plasmid, TCDD also inhibited E2-induced CAT activity. The role of the nuclear Ah receptor complex was confirmed by results of the following studies using MCF-7 or mouse Hepa 1c1c7 cells transiently transfected with E2-responsive Vit A2 gene 5'-promoter constructs: (i) for a series of Ah receptor ligands, there was a correlation between their antiestrogenic activity in MCF-7 cells and their rank order binding affinity for the Ah receptor; (ii) alpha-naphthoflavone, an Ah receptor antagonist, inhibited the antiestrogenic activity of TCDD in MCF-7 cells; and (iii) TCDD inhibited E2-induced CAT activity in Ah-responsive wild-type but not in Ah-nonresponsive class 2 mutant Hepa 1c1c7 cells. The antiestrogenic activity of TCDD was also observed in cells which transiently overexpressed the human estrogen receptor (ER), suggesting that the mechanism does not involve downregulation of the ER by TCDD.
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PMID:Inhibition of estrogen-induced activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-7 human breast cancer and other cell lines transfected with vitellogenin A2 gene promoter constructs. 901 89

The role of transforming growth factor-beta1 (TGFbeta1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pM TGFbeta1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nM in TGFbeta1-treated cells compared to 0.155 nM in control cells. Treatment with TGFbeta1 did not influence the half-life of the ER. In TGFbeta1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFbeta1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (approximately 75%) at 6 h. By 24 h, a small recovery in the amount of messenger RNA was observed. Transcription run-on experiments demonstrated a decrease of approximately 70% in the level of ER gene transcription at 3 h. Transient transfections using an ER promoter-chloramphenicol acetyltransferase construct demonstrated that after TGFbeta1 treatment, chloramphenicol acetyltransferase activity decreased by 50%, suggesting that TGFbeta1 inhibition of the ER gene transcription is mediated through the ER promoter. Although treatment with TGFbeta1 decreased the ER concentration, the growth factor had no effect on the activity of ER, as measured by its effects on estradiol induction of progesterone receptor and pS2, suggesting that TGFbeta1 does not inhibit proliferation of MCF-7 cells by blocking ER activity.
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PMID:The role of transforming growth factor-beta in the regulation of estrogen receptor expression in the MCF-7 breast cancer cell line. 907 8

A 114 bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of beta-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.
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PMID:Putative regulatory sequence in human papillomavirus type 16 E2 open reading frame. 915 49

Growth factors coordinately regulate a variety of genes associated with pathological states including tumor invasion and metastasis. Overexpressed epidermal growth factor receptor (EGFR) on tumor cell surfaces is associated with enhanced cell attachment and migration into extracellular matrices, which promotes tumor aggressiveness. We have demonstrated that epidermal growth factor (EGF) up-regulates the cell surface adhesion molecule CD44 at both the mRNA and protein levels on mouse fibroblasts expressing full-length wild-type EGFR (NR6-WT) but not on EGFR-deficient cells (NR6-P). This increases cell attachment to hyaluronic acid. In this investigation, transcriptional regulation of CD44 by EGF was confirmed by defining an EGF-regulatory element. By employing human CD44 gene promoter-chloramphenicol acetyltransferase (CAT) constructs transfected into NR6-WT cells, EGF inducibility was observed within a 120-base pair (bp) DNA fragment located 450 bp upstream of the RNA initiation site. Differential EGF inducibility was found among different cell lines chosen, indicating a 3.2- and 1.8-fold enhancement in DU145 cells carrying exogenous wild-type EGFR and in MCF-7 cells, respectively, while minimal EGF induction was found in cervical cancer HeLa cells. Utilizing gel shift assays, a time-dependent increase of DNA-protein complex formation was found upon EGF stimulation in NR6-WT cells but not in NR6-P cells. Based upon these observations, a novel 22-bp EGF regulatory element (ERE) (5'--604CCCTCTCTCCAGCTCCTCTCCC-583-3') was isolated from the CD44 gene promoter. This ERE conferred DNA-protein binding ability in vitro, as well as the full functional recovery of EGF inducibility of CAT activity when linked to a homologous CD44 promoter or a SV40 promoter driving a CAT reporter gene. A two-base mutation of the ERE completely eliminated its binding activity as well as its EGF inducibility of CAT expression. Our studies indicate that EGF induces CD44 gene expression through an interaction between a specific ERE and putative novel transcriptional factor so as to regulate cell attachment to extracellular matrix.
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PMID:Epidermal growth factor induces CD44 gene expression through a novel regulatory element in mouse fibroblasts. 916 42

Several studies have reported a correlation between expression of the estrogen receptor (ER) and aryl hydrocarbon (Ah) responsiveness in human breast cancer cell lines. MDA-MB-231 cells are ER-negative and Ah-nonresponsive; however, initial studies showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA levels (5.8-fold) and chloramphenicol acetyltransferase activity (2.6-fold) in high passage (Hp, >50 passages) cells transiently transfected with an Ah-responsive plasmid. In contrast, no induction responses were observed in low passage (Lp, <20 passages) cells. The Ah responsiveness of Hp compared to Lp MDA-MB-231 cells was associated with a >2-fold increased expression of the Ah receptor in Hp cells. Further analysis revealed that the apparent molecular weight of the Ah receptor mRNA transcript and immunoreactive protein were comparable in Lp MDA-MB-231 and Ah-responsive human HepG2 cells. In contrast, RT-PCR analysis of the Ah receptor nuclear translocator (Arnt) protein showed that HepG2 cells expressed the expected 2.6-kb transcript, whereas a 1.3-kb transcript was the major product in MDA-MB-231 cells. Western blot analysis confirmed that HepG2 cells primarily expressed a 97-kDa wild-type form of Arnt, whereas a dominant 36-kDa variant was expressed in MDA-MB-231 cells. Complete sequence analysis of the variant form of Arnt revealed a major deletion of the C-terminal region of the protein (aa 330 to 789). Like HepG2 cells, the wild-type 2.6-kb transcript was detected in ER-positive (Ah-responsive) MCF-7 cells, whereas the low-molecular-weight variant Arnt was dominant in ER-negative MDA-MB-231, MDA-MB-435, and Adriamycin-resistant MCF-7 cells. These results suggest that expression of this protein may be useful as a prognostic factor in breast cancer.
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PMID:Aryl hydrocarbon (Ah) nonresponsiveness in estrogen receptor-negative MDA-MB-231 cells is associated with expression of a variant arnt protein. 932 85

The expression of aromatase in human breast tumors was studied using the reverse transcription-polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis revealed that exons I.3 and PII are the two major exons I present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells (ASCs). Recently, the regulatory properties of a 696-base pair region that contains promoter II, and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Detailed DNase 1 footprinting analysis, DNA mobility shift assays, and chloramphenicol acetyltransferase (CAT) functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study further revealed that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in ASCs. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in ASCs, respectively. It was also found that in several breast cancer cell lines, including MCF-7, the promoter activities of both promoter II and promoter I.3 were found to be suppressed by a negative regulatory element, a silencer, present in the 162 bp fragment which is located upstream from promoter II and downstream from promoter I.3. The precise position of the silencer element (termed S1) was localized by deletion mutation and DNase 1 footprinting analysis, and the silencing activity of S1 on promoter I.3 (in B1 fragment) was confirmed by CAT plasmid transfection experiments. UV crosslinking experiments are being performed to examine the regulatory proteins interacting with the silencer element. These studies serve as the basis for the further characterization of the regulatory mechanism of aromatase expression in human breast cancer and ASCs.
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PMID:Gene regulation studies of aromatase expression in breast cancer and adipose stromal cells. 936 1


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