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:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear estrogen binding was characterized in HM-1, a malignant hamster melanoma cell line transplanted into male and female athymic mice following acute, subchronic, and chronic injection of estradiol. Nuclear binding was saturable, of high affinity (10(10) M-1) and readily soluble in low salt buffer. Saturation analyses revealed that [3H]estradiol in excess of 5.0 nM apparently bound to a second class of lower affinity (10(9) M-1), higher capacity cytosol sites. Enzyme-linked immunoassay with a specific monoclonal antibody (H222 Sp gamma) directed against the human estrogen receptor protein was in excellent agreement (r = 0.93) with values obtained using hydroxyapatite to separate bound from free ligand. Nuclear estrogen receptor content in HM-1 cells was increased maximally 1 h after acute s.c. injection of a low dose (0.1 microgram) of estradiol. The increase in nuclear receptor content was accompanied by an apparent rapid reduction in cytosol binding. Subchronic (3 days) and chronic exposure (35 days) to estradiol also produced a significant, dose-related increase in tumor nuclear estrogen receptor content. Cytosol binding for progestin was low (less than or equal to 2 fmol) to absent in HM-1 xenografts not exposed to estradiol. Subchronic and chronic exposure to estradiol induced a dose-related, specific, high affinity (10(9) M-1) cytosol binding protein for progestin(s) in HM-1 xenografts carried in male and female athymic mice. In contrast, progestin binding to nuclear receptor was not increased in estrogen-primed animals, nor did acute injection of progesterone (100 micrograms s.c.) increase the amount of saturable, high affinity (10(9) M-1) nuclear progestin receptor in control or estradiol-primed athymic mice. In contrast to the induction of progestin binding, tyrosinase activity was not altered by a similar exposure to estradiol when assayed at a saturating concentration of tyrosine. These observations suggest that the estrogen receptor in HM-1 cells may be functional but that pigmentary changes observed in mammals following chronic exposure to estradiol may not be mediated by a direct effect on the rate limiting enzyme of melanin synthesis.
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PMID:Effects of estradiol on estrogen receptor, progesterone receptor, and tyrosinase in hamster melanoma transplanted into athymic mice. 313 19

A model system is presented to explain how tyrosinase, an enzyme unique to pigmented cells such as normal and malignant melanocytes can oxidize [3H]-estradiol to radiolabeled products which closely resemble the tight binding of [3H]-estradiol to estrogen receptor. In the model system studied, tyrosinase oxidized [2,4,6,7-3H]-estradiol to [3H]-water and [3H]-estradiol metabolites, the latter of which formed ring-substituted conjugates with nucleophiles like monothioglycerol and BSA. Radiolabeled estradiol without tritium in the C-2 position (i.e. [6,7-3H]-estradiol) failed to liberate [3H]-water when exposed to tyrosinase but, nevertheless, did form ring-substituted [3H]-estradiol adducts with nucleophiles. The [3H]-water and the ring-substituted radiolabeled products possessed several characteristics of a genuine estrogen receptor protein in that they were resistant to dextran-coated charcoal (DCC) adsorption and their enzymatic formation was inhibited with non-steroidal estrogens like diethylstilbestrol. Other natural (estradiol) and synthetic (hydroxytamoxifen) estrogens which contain the phenol grouping also inhibited the enzymatic oxidation of [3H]-estradiol. Although it was difficult to differentiate estrogen receptor from tyrosinase using the conventional DCC assay system, several differences in these two proteins permitted a distinction to be made between them. First, tyrosinase oxidation of [3H]-estradiol was markedly inhibited by sulfhydryl reducing agents (monothioglycerol) that stabilize [3H]-estradiol binding to estrogen receptor. Second, estrogen receptor adsorbed by hydroxylapatite whereas tyrosinase did not, thus permitting the separation of these two proteins prior to incubation with [3H]-estradiol. We conclude that the [3H]-estradiol binding components in melanoma previously reported to be estrogen receptor probably represent instead the radiolabeled products of the tyrosinase-catalyzed oxidation of [3H]-estradiol.
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PMID:Estrogen receptor in malignant melanoma: fact or artefact? 631 60

Melanomas from 20 patients were evaluated for estrogen and progesterone receptors. No restriction as to patient's age, sex, race, or menstrual status was made. Fifteen of the tumors were melanin producing. Of the 20 tumors examined, seven contained more than 3 fm/mg protein of specifically inhibitable estrogen binding. None of the tumors reported here demonstrated either a 4S or 8S binding protein as shown by sucrose density gradient analysis. All tumors in which estradiol binding was observed were melanin producing, whereas none of the amelanotic melanomas (five tumors) bound this steroid. Purified tyrosinase appeared to mimic the estrogen binding detected in melanoma cytosols, as demonstrated with the dextran-coated charcoal technique. Although the binding of estradiol to tyrosinase was inhibited by DOPA, the binding of estradiol to estrogen receptor preparations was not. These studies represent an extensive of previous studies of sex steroid binding in melanomas and suggest that gradient analysis and DOPA inhibition studies should be included in the evaluation of the estrogen binding phenomenon in human melanoma.
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PMID:Sex steroid receptor analysis in human melanoma. 677 5

We have previously described tyrosinase-like proteins of rat uterine nuclear extracts with type II estrogen binding characteristics. In this paper we have been able to affinity label these polypeptides with radio-iodinated estradiol. The major label at approximately 33-38 kDa comigrates with a approximately 36 kDa tyrosinase immunoreactive band assessed by autoradiograms and Western blots following electrophoresis. A minor label was also detected at approximately 45 kDa. The label is attenuated by excess quercetin hence these proteins are believed to represent putative type II estrogen binding sites that bind this bioflavonoid. These estrogen binding proteins are distinct from the estrogen receptor as judged by immunoblotting. The affinity crosslinking will be a useful approach in the purification of tyrosinase like proteins.
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PMID:Estrogen affinity crosslinking to tyrosinase-like immunoreactive proteins of rat uterine nuclear extracts. 803 12

Normal human melanocytes were cultured selectively with F12 culture medium supplemented with growth hormones, phorbol ester and 1% of fetal calf serum. The estrogen receptors were analyzed using hydroxylapatite-column assay with tritiated 17-beta-estradiol as the binding ligand. Phenol red- free medium was used when the changes in cell numbers, melanin content and tyrosinase were assessed after incubating with physiological concentration of 17-beta estradiol (10(-12) and 10(-9) M). It was found that the melanocytes contained both cytosol (5.42 +/- 1.11 fmol/mg protein) and nuclear (59.13 +/- 17.12 fmol/mg protein) estrogen receptor. In response to estradiol, the cell number increased but both the melanin content and the tyrosinase activity decreased in a dose related pattern. These data suggested the presence of estrogen receptor with biological function in normal human melanocytes.
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PMID:Effects of estrogen and estrogen receptor in normal human melanocytes. 814 84

Although tamoxifen is approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, several studies in animal models have shown that tamoxifen is heptocarcinogenic, and in humans, tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen carcinogenesis could involve metabolism of tamoxifen to 3,4-dihydroxytamoxifen followed by oxidation to a highly reactive o-quinone which has the potential to alkylate and/or oxidize cellular macromolecules in vivo. In the study presented here, we synthesized the 3,4-dihydroxytamoxifen, prepared its o-quinone chemically and enzymatically, and studied the reactivity of the o-quinone with GSH and deoxynucleosides. The E (trans) and Z (cis) isomers of 3,4-dihydroxytamoxifen were synthesized using a concise synthetic pathway (four steps). This approach is based on the McMurry reaction between the key 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone and propiophenone, followed by selective removal of the methylenedioxy ring of (E, Z)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-(3, 4-methylenedioxyphenyl)-2-phenyl-1-butene with BCl(3). Oxidation of 3,4-dihydroxytamoxifen by activated silver oxide or tyrosinase gave 3,4-dihydroxytamoxifen-o-quinone as a mixture of E and Z isomers. The resulting o-quinone has a half-life of approximately 80 min under physiological conditions. Reaction of the o-quinone with GSH gave two di-GSH conjugates and three mono GSH conjugates. Incubation of 3,4-dihydroxytamoxifen with GSH in the presence of microsomal P450 gave the same GSH conjugates which were also detected in incubations with human breast cancer cells (MCF-7). Reaction of 3, 4-dihydroxytamoxifen-o-quinone with deoxynucleosides gave only thymidine and deoxyguanosine adducts; neither deoxyadenosine nor deoxycytosine adducts were detected. Preliminary studies conducted with human breast cancer cell lines showed that 3, 4-dihydroxytamoxifen exhibited cytotoxic potency similar to that of 4-hydroxytamoxifen and tamoxifen in an estrogen receptor negative (ER(-)) cell line (MDA-MB-231); however, in the ER(+) cell line (MCF-7), the catechol metabolite was about half as toxic as the other two compounds. Finally, in the presence of microsomes and GSH, 4-hydroxytamoxifen gave predominantly quinone methide GSH conjugates as reported in the previous paper in this issue [Fan, P. W., et al. (2000) Chem. Res. Toxicol. 13, XX-XX]. However, in the presence of tyrosinase and GSH, 4-hydroxytamoxifen was primarily converted to o-quinone GSH conjugates. These results suggest that the catechol metabolite of tamoxifen has the potential to cause cytotoxicity in vivo through formation of 3,4-dihydroxytamoxifen-o-quinone.
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PMID:Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifen-o-quinone. 1064 67

The mechanisms of estrogen and progesterone in human cutaneous pigmentation are largely unknown. The molecular identification of estrogen receptor (ER) and progesterone receptor (PR) in the human melanocytes is of great importance to understand the mechanisms. We performed immunocytochemistry analysis and demonstrated that ER and PR were expressed in the cytoplasms and nuclei of human melanocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis confirmed the expression of ER and PR at the transcriptional level. Despite of the presence of ER and PR, the physiological and pregnant levels of estrogen and progesterone showed inconsistent effects on the proliferation and tyrosinase activity of cultured human melanocytes. These results suggest that human melanocytes express ER and PR, which have a donor-specific action in human pigmentation. Further studies are needed to elucidate the induction mechanism and functions of these receptors, and the role of estrogen and progesterone in melanocytes.
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PMID:Donor specific response of estrogen and progesterone on cultured human melanocytes. 1185 May 90

Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors alpha and beta, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17beta-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17beta-equilenin. Because 17beta-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated equilenin derivatives in vitro, we studied the relative ability of the 17beta-hydroxy-4-halogenated equilenin derivatives to induce estrogenic effects in the ovariectomized rat model. The 4-fluoro derivative showed higher activity than 4-chloro and 4-bromo derivatives as demonstrated by inducing higher vaginal cellular differentiation, uterine growth, and mammary gland branching. However, 17beta-hydroxy-4-fluoroequilenin showed a lower estrogenic activity than 17beta-hydroxyequilenin and estradiol, which could be due to alternative pharmacokinetic properties for these compounds. These data suggest that the 4-fluoroequilenin derivatives have promise as alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity.
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PMID:Effect of halogenated substituents on the metabolism and estrogenic effects of the equine estrogen, equilenin. 1280 57

Although approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, the selective estrogen receptor modulator (SERM) tamoxifen has been associated with an increased risk of endometrial cancer in women. With an understanding of the potential carcinogenic mechanisms of these compounds, SERMs could in principle be designed or selected for use that avoids these problems. Acolbifene (EM-652) is a fourth-generation SERM and the active form of the ester prodrug EM-800. As a pure antagonist of breast tumor development and growth, acolbifene does not stimulate endometrial tissue. However, acolbifene was found in this investigation to form two kinds of quinone methides, either through chemical or through enzymatic oxidation. One was a classical acolbifene quinone methide, which was formed by oxidation at the C-17 methyl group, and the other was a diquinone methide involving the oxidation of two phenol groups. The half-life of the classical quinone methide was determined to be 32 +/- 0.4 s at physiological pH and temperature. The quinone methides reacted with glutathione (GSH) to form five mono-GSH conjugates and five di-GSH conjugates. The majority of GSH conjugates resulted from reaction of the classical acolbifene quinone methide with GSH. Incubations of acolbifene with GSH and either tyrosinase or human and rat liver microsomes also produced acolbifene quinone methide-GSH conjugates. In addition to reaction with GSH, the classical acolbifene quinone methide was also shown to react with deoxynucleosides. One of the major deoxynucleoside adducts was identified as the deoxyadenosine adduct resulting from reaction of the classical acolbifene quinone methide with the exocyclic amino group of adenine. Acolbifene could also induce DNA damage in the S30 breast cancer cell line. These data imply that the classical electrophilic acolbifene quinone methide might contribute to the potential toxicity of acolbifene.
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PMID:Bioactivation of the selective estrogen receptor modulator acolbifene to quinone methides. 1572 Jan 21

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.
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PMID:Characterization of melanocyte-specific inducible Cre recombinase transgenic mice. 1667 22


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