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1.
Oncogene ; 26(51): 7185-93, 2007 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-17525741

RESUMEN

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G(2)/M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G(2)/M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50(cdc37), an Hsp90 co-chaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Quinasas CDC2-CDC28/metabolismo , División Celular , Regulación hacia Abajo/efectos de los fármacos , Fase G2 , Glioblastoma/metabolismo , Glioblastoma/patología , Proteínas HSP90 de Choque Térmico/fisiología , Rifabutina/farmacología , Fosfatasas cdc25/metabolismo , Calpaína/antagonistas & inhibidores , Línea Celular Tumoral , Humanos
2.
Clin Transl Oncol ; 8(5): 306-12, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16760004

RESUMEN

Exocrine pancreatic cancer is one of the neoplasias with a worse prognosis, with conventional treatments having little impact on disease outcome. Research and genomic high-throughput technology is continuously expanding our knowledge of pancreas cancer biology. Characterization of genetic and epigenetic alterations in pancreatic tumors has allowed a better understanding of the progression model of the disease at the molecular level. The development of new therapeutic approaches with target- oriented agents is been tested in the preclinical and clinical settings. This review updates the current available data on pancreatic cancer molecular biology.


Asunto(s)
Genes Supresores de Tumor , Oncogenes , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Aberraciones Cromosómicas , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes p16 , Genes p53 , Genes ras , Humanos , Proteínas de Neoplasias/genética , Síndromes Neoplásicos Hereditarios/genética , Pronóstico
3.
Clin Cancer Res ; 3(12 Pt 1): 2405-14, 1997 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9815641

RESUMEN

It is increasingly recognized that drug-resistant cells undergo transitions not directly linked to "classical" drug resistance. We examined the expression of growth factors, growth factor receptors, and the estrogen receptor in 17 drug-resistant and 2 revertant human breast cancer sublines to provide an understanding of the phenotypic changes that occur and how these changes could affect the biology of the cell. These sublines were derived from five parental human breast cancer cell lines (MCF-7, ZR75B, T47D, MDA-MB-231, and MDA-MB-453). The expression of estrogen receptor was absent or decreased in 6 of the 15 resistant MCF-7, ZR75B, and T47D sublines. Increases of as much as 49-fold compared to parental levels were observed in transforming growth factor alpha, epidermal growth factor receptor, c-erbB2, and/or c-erbB3 mRNA expression in 14 of the 17 resistant sublines. Altered amphiregulin and insulin-like growth factor-I receptor expression was observed in nine and four drug-resistant sublines, respectively. No major alterations were observed in epidermal growth factor and c-erbB4 expression. Few alterations were observed in two sublines derived from estrogen receptor-negative cells. Higher levels of phosphotyrosine residues were detected in a subset of the resistant sublines, indicating an increased tyrosine kinase activity in these cells. Interestingly, decreased growth rates were observed in all of the sublines, despite up-regulated growth factor-related gene expression. Taken together, these data suggest that loss of estrogen receptor, increased expression of growth factor pathway genes, and decreased growth rate regularly occur in drug-resistant breast cancer cells. Although we do not know whether the altered expression of growth factor pathway genes is linked as a cause or a consequence of the reduced growth rate, it is well established that decreased growth rate confers drug resistance. These phenotypic changes in drug-resistant human breast cancer cells could serve to initiate, support, or extend the drug resistance.


Asunto(s)
Antineoplásicos/toxicidad , Neoplasias de la Mama/genética , Resistencia a Múltiples Medicamentos/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Genes erbB-2 , Proteínas Proto-Oncogénicas/genética , División Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , Femenino , Humanos , Proto-Oncogenes , ARN Mensajero/genética , Receptor ErbB-2/genética , Receptor ErbB-3 , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Transcripción Genética , Células Tumorales Cultivadas
4.
Mol Endocrinol ; 3(11): 1782-7, 1989 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-2608058

RESUMEN

We have previously demonstrated that regulation of estrogen receptor (ER) expression in MCF-7 breast cancer cells is a complex process involving transcriptional and posttranscriptional regulation by estradiol. Treatment of MCF-7 cells with estradiol results in the down-regulation of receptor expression; posttranscriptional suppression of receptor mRNA appears to be the predominant mechanism. To determine whether posttranscriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we have used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the posttranscriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis. A study of the effects of estradiol on the steady-state levels of nuclear and cytoplasmic receptor mRNA suggest that posttranscriptional suppression is a nuclear event.


Asunto(s)
Neoplasias de la Mama/genética , Estrógenos , Regulación Neoplásica de la Expresión Génica , Neoplasias Hormono-Dependientes/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Receptores de Estrógenos/fisiología , Neoplasias de la Mama/patología , Núcleo Celular/análisis , Cicloheximida/farmacología , Citoplasma/análisis , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hormono-Dependientes/patología , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Receptores de Estrógenos/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo
5.
Mol Endocrinol ; 2(12): 1157-62, 1988 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-3216858

RESUMEN

The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.


Asunto(s)
Estradiol/farmacología , Receptores de Estrógenos/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Línea Celular , Femenino , Humanos , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/ultraestructura
6.
Endocrinology ; 138(4): 1498-505, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9075708

RESUMEN

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.


Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Estradiol/farmacología , Femenino , Semivida , Humanos , Progesterona/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Transcripción Genética , Células Tumorales Cultivadas
7.
Endocrinology ; 137(10): 4322-30, 1996 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8828492

RESUMEN

Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.


Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/patología , Carcinoma/patología , Femenino , Humanos , Concentración Osmolar , ARN Mensajero/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Transcripción Genética , Células Tumorales Cultivadas
8.
Endocrinology ; 136(12): 5659-65, 1995 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7588321

RESUMEN

Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.


Asunto(s)
Neoplasias de la Mama/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes de Retinoblastoma , Neoplasias Hormono-Dependientes/genética , Femenino , Humanos , Insulina/farmacología , ARN Mensajero/análisis , Células Tumorales Cultivadas
9.
Endocrinology ; 136(9): 3983-92, 1995 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7649107

RESUMEN

The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.


Asunto(s)
Neoplasias de la Mama/química , Estrógenos/farmacología , Glicoproteínas/análisis , Glicoproteínas/genética , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Acetato de Tetradecanoilforbol/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Anfirregulina , Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Brioestatinas , Medios de Cultivo Condicionados , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Familia de Proteínas EGF , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoquinolinas/farmacología , Lactonas/farmacología , Macrólidos , Piperazinas/farmacología , Alcamidas Poliinsaturadas , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Estrógenos/agonistas , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Células Tumorales Cultivadas
10.
J Endocrinol ; 165(2): 371-8, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10810301

RESUMEN

The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.


Asunto(s)
Neoplasias de la Mama/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Estrógenos/genética , Transducción de Señal/efectos de los fármacos , Androstadienos/farmacología , Sitios de Unión , Estradiol/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Femenino , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Factores de Tiempo , Transcripción Genética , Células Tumorales Cultivadas , Tirfostinos/antagonistas & inhibidores , Tirfostinos/farmacología , Wortmanina
11.
J Endocrinol ; 180(3): 487-96, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15012603

RESUMEN

Results presented in this study demonstrate that treatment of MCF-7 cells with taxol resulted in induction of estrogen receptor-alpha (ER alpha) gene transcription with a subsequent increase in ER alpha mRNA; this effect was promoter specific since taxol did not affect total transcription in MCF-7 cells and lacked an effect on transcription of the human acidic ribosomal phosphoprotein protein PO, progesterone receptor, and pS2 genes. In contrast to the increase in transcription of the ER alpha gene, taxol inhibited translation of the ER alpha mRNA. This effect is also transcript specific since taxol did not alter total protein synthesis and did not affect the concentration of progesterone receptor protein in the cell. The overall result of taxol treatment was to decrease the concentration of ER alpha protein in the MCF-7 cells. Evidence is presented that the effects of taxol on ER alpha gene transcription may be mediated through the induction of p53.


Asunto(s)
Neoplasias de la Mama/metabolismo , Moduladores de los Receptores de Estrógeno/uso terapéutico , Paclitaxel/uso terapéutico , Receptores de Estrógenos/genética , Transcripción Genética/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Receptor alfa de Estrógeno , Femenino , Expresión Génica/efectos de los fármacos , Semivida , Humanos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/análisis , Receptores de Estrógenos/análisis , Estimulación Química , Factores de Tiempo
12.
J Endocrinol ; 180(3): 497-504, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15012604

RESUMEN

The results presented here demonstrate that p53 upregulates estrogen receptor-alpha (ER alpha) expression in the human breast cancer cell line MCF-7. Two approaches were used to alter the activity of p53 in the cells. In the first approach, stable transfectants expressing an antisense p53 were established. In the stable clones, expression of antisense p53 resulted in a decrease in the expression of ER alpha protein. In the second approach, MCF-7 cells were transiently transfected with wild-type p53. Overexpression of p53 increased the amount of ER alpha. To determine whether the effects of p53 on the expression of ER alpha were due to changes in transcription, deletion mutants of the ER alpha promoter were used. This experimental approach demonstrated that p53 up-regulates ER alpha gene expression by increasing transcription of the gene through elements located upstream of promoter A. Transfection assays using p53 mutants further demonstrated that the p53-induced increase in ER alpha gene transcription was not dependent on the ability of p53 to bind to DNA but on its ability to interact with other proteins.


Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación de la Expresión Génica , Genes p53 , Regiones Promotoras Genéticas , Receptores de Estrógenos/genética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , ADN sin Sentido/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno , Femenino , Vectores Genéticos/administración & dosificación , Humanos , ARN Mensajero/análisis , Transfección/métodos
13.
Biochem Pharmacol ; 33(13): 2033-9, 1984 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-6378207

RESUMEN

The possible participation of enzymatic methylation reactions in the process of insulin release was investigated in rat pancreatic islets. The combination of 3-deazaadenosine and DL-homocysteine impaired the incorporation of 3H-methyl from L-[methyl-3H]methionine into endogenous islet proteins and phospholipids, but failed to affect turnover in the phosphatidylinositol cycle. The inhibitors of methylation decreased insulin release evoked by D-glucose or the combinations of D-glucose and gliclazide, L-leucine and L-glutamine, or Ba2+ and theophylline. The inhibitors of methylation did not impair either the oxidation of D-glucose or affect its capacity to decrease K+ conductance, stimulate Ca2+ inflow and provoke 45Ca accumulation in pancreatic islets. It is proposed that, in the process of insulin secretion, a methyl acceptor protein and/or phospholipid play(s) a limited modulatory role in the coupling of cytosolic Ca2+ accumulation to exocytosis.


Asunto(s)
Homocisteína/farmacología , Insulina/metabolismo , Ribonucleósidos/farmacología , Tubercidina/farmacología , Animales , Calcio/metabolismo , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Metabolismo de los Lípidos , Metilación , Oxidación-Reducción , Fosfatidilinositoles/metabolismo , Proteínas/metabolismo , Ratas
14.
J Steroid Biochem Mol Biol ; 66(3): 113-20, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9719445

RESUMEN

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.


Asunto(s)
Estradiol/farmacología , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Neoplasias de la Mama , Centrifugación por Gradiente de Densidad , Cicloheximida/farmacología , Estabilidad de Medicamentos , Inhibidores Enzimáticos/farmacología , Semivida , Humanos , Cinética , Magnesio/farmacología , Pactamicina/farmacología , Hormonas Placentarias/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura , Células Tumorales Cultivadas
15.
Adv Exp Med Biol ; 330: 143-53, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8368130

RESUMEN

One of the most prevalent of cancers, breast cancer, is characterized by hormonal control of its growth. Expression of the estrogen receptor (ER) in MCF-7 breast cancer cells appears to be a complex process involving multiple steps subject to hormonal regulation by estrogen. Treatment of MCF-7 cells with estradiol results in the suppression of estrogen receptor protein. By 6 hours, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA and remained suppressed for 24-48 hours. Similar results were obtained with an estrogen receptor binding assay. Estrogen treatment also resulted in a decrease of receptor mRNA to approximately 10% of control values by 6 hours. Estrogen receptor remained at the suppressed level for up to 48 hours. Transcription run-on experiments demonstrated a transient decrease of about 90% in receptor gene transcription after 1 hour. By 3-6 hours transcription increased approximately 2-fold and remained elevated for at least 48 hours. These data suggest that estrogen suppresses ER mRNA by inhibition of ER gene transcription at early times and by a post-transcriptional effect on receptor mRNA at later times. To determine whether post-transcriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study the regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the post-transcriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis.


Asunto(s)
Neoplasias de la Mama/genética , Estrógenos/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Hormono-Dependientes/genética , Receptores de Estrógenos/biosíntesis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cicloheximida/farmacología , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Receptores de Estrógenos/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos
18.
Biochem Int ; 8(3): 445-52, 1984 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-6383400

RESUMEN

Glucose provokes a transient stimulation of phospholipid methylation in rat pancreatic islets, possibly by increasing phospholipid methyltransferase activity. The association of DL-homocysteine and 3-deazaadenosine inhibits phospholipid methylation. The methylation of phospholipids may play a role in the stimulus-secretion coupling for glucose-induced insulin release.


Asunto(s)
Islotes Pancreáticos/metabolismo , Fosfolípidos/metabolismo , Animales , Glucosa/farmacología , Islotes Pancreáticos/efectos de los fármacos , Cinética , Metilación , Metiltransferasas/metabolismo , Fosfatidil-N-Metiletanolamina N-Metiltransferasa , Fosfatidiletanolamina N-Metiltransferasa , Ratas
19.
J Cell Biochem ; 76(4): 605-14, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10653980

RESUMEN

The role of insulin-like growth factor-I (IGF-I) in regulating estrogen receptor-alpha (ER-alpha) gene expression and activity was investigated in the human breast cancer cell line MCF-7. Treatment of cells with 40 ng/ml IGF-I resulted in a 60% decrease in ER-alpha protein concentration by 3 h, and the amount of ER-alpha remained suppressed for 24 h. A multiple-dose ligand-binding assay demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease of 50% in estradiol-binding sites with no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the absence of IGF-I (K(d) = 3 x 10(-10) +/- 0.5 x 10(-10) M) was similar to the dissociation constant in the presence of IGF-I (K(d) = 6 x 10(-10) +/- 0.3 x 10(-10) M). The decrease in ER-alpha protein concentration was paralleled by an 80% decrease in the steady-state amount of ER-alpha mRNA by 3 h. The IGF-I induced decrease in ER-alpha mRNA was due to the inhibition of ER-alpha gene transcription. When an 128-base pair ER-alpha-promoter-CAT construct was transfected into MCF-7 cells, treatment with IGF-I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER-alpha, treatment with IGF-I induced two endogenous estrogen-regulated genes, progesterone receptor and pS2, by 4- and twofold, respectively. The pure antiestrogen ICI-164, 384 blocked this induction, suggesting that ER-alpha mediates the effects of IGF-I. Transient co-transfections of wild-type ER-alpha and an estrogen response element-CAT reporter into COS-1 cells demonstrated that IGF-I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3-kinase inhibitors blocked the IGF-I effects on ER-alpha expression and activity, suggesting that these kinases may be involved in the cross-talk between the IGF-I and ER-alpha pathways.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptores de Estrógenos/genética , Animales , Sitios de Unión/efectos de los fármacos , Células COS , Inhibidores Enzimáticos/farmacología , Estradiol/análogos & derivados , Estradiol/metabolismo , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno , Genes Reporteros , Humanos , Alcamidas Poliinsaturadas , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas , Proteínas/genética , ARN Mensajero/efectos de los fármacos , Receptores de Progesterona/genética , Transfección , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor
20.
Horm Res ; 32 Suppl 1: 242-9, 1989.
Artículo en Inglés | MEDLINE | ID: mdl-2693328

RESUMEN

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.


Asunto(s)
Neoplasias de la Mama/fisiopatología , Neoplasias Mamarias Experimentales/fisiopatología , Animales , Membrana Basal/fisiología , División Celular/fisiología , Línea Celular , Femenino , Humanos
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