Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Melanócitos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Melanócitos/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Receptor Tipo 1 de Melanocortina/metabolismo , Receptores de Esteroides/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , alfa-MSH/metabolismo , alfa-MSH/farmacologiaRESUMO
Three ecdysteroid 7,9(11)-dien-7-ones (dacryhainansterone, 25-hydroxydacryhainansterone and kaladasterone) were prepared by dehydration of the corresponding 11a-hydroxy ecdysteroids (ajugasterone C, turkesterone and muristerone A, respectively). The biological activities of the dienones in the Drosophila melanogaster B(II) cell bioassay, which reflect the affinity for the ecdysteroid receptor complex, showed that the dienones retain high biological activity. Irradiation at 350 nm of the ecdysteroid dienones (100 nM) with bacterially-expressed dipteran and lepidopteran ecdysteroid receptor proteins (DmEcR/DmUSP or CfEcR/CfUSP), followed by loading with [(3)H]ponasterone A revealed that irradiation of dacryhainansterone or kaladasterone resulted in blocking of >70% of the specific binding sites. Thus, ecdysteroid dienones show considerable potential as photoaffinity analogues for ecdysteroid binding proteins.
Assuntos
Ecdisterona/análogos & derivados , Marcadores de Fotoafinidade/química , Receptores de Esteroides/química , Animais , Asteraceae/química , Bioensaio/métodos , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Drosophila melanogaster , Ecdisterona/agonistas , Ecdisterona/biossíntese , Ecdisterona/química , Ecdisterona/metabolismo , Ecdisterona/fisiologia , Ligantes , Receptores de Esteroides/isolamento & purificação , Receptores de Esteroides/efeitos da radiação , Análise Espectral/métodos , Análise Espectral/veterinária , Raios UltravioletaRESUMO
One hundred and five breast cancer patients with stage T3/4, N+/-, Mo were treated at random either with a pre- and postoperative chemotherapy (A) (5-drug-combination + tamoxifen) or with a pre- and postoperative radiotherapy (B). Paraffin embedded tissue samples were prepared from tumor material taken by biopsy prior to therapy as well as at surgery from patients of both groups to estimate the HER-2 oncogene copy numbers before and after treatment. In 53 and 50% of the pretherapeutic samples the HER-2 gene was amplified in groups A and B, respectively. In the post-therapeutic group 60% of the chemotherapy and 48% of the radiotherapy patients, respectively, had low or high HER-2 oncogene copy numbers. In addition, HER-2 amplification before and after therapy was estimated in 28 patients. An increase of oncogene copy numbers could be detected in 21% of the chemotherapy patients, and a decrease was noted in 11%. No radiotherapy patient showed a rise, but 11% a loss of copy numbers. Although amplification of HER-2 oncogene was not found to be associated with overall survival as it was in many studies before, it could still be a predictor of clinical outcome and the cause of mammary carcinomas developing into stage T3/4.
Assuntos
Neoplasias da Mama/terapia , Amplificação de Genes , Proteínas Oncogênicas Virais/genética , Oncogenes , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Neoplasias da Mama/cirurgia , Terapia Combinada , DNA de Neoplasias/análise , Feminino , Amplificação de Genes/efeitos dos fármacos , Amplificação de Genes/efeitos da radiação , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptor ErbB-2 , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/efeitos da radiação , Tamoxifeno/uso terapêuticoRESUMO
Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.
Assuntos
Marcadores de Afinidade/metabolismo , Calcitriol/metabolismo , Receptores de Esteroides/metabolismo , Animais , Ligação Competitiva , Núcleo Celular/química , Eletroforese em Gel de Poliacrilamida , Técnicas de Imunoadsorção , Intestinos/ultraestrutura , Peso Molecular , Fotólise , Receptores de Calcitriol , Receptores de Esteroides/efeitos da radiação , Suínos , Trítio , Raios UltravioletaAssuntos
Corticosterona/efeitos da radiação , Citosol/metabolismo , Fígado/efeitos da radiação , Nucleotídeos Cíclicos/efeitos da radiação , Receptores de Glucocorticoides/efeitos da radiação , Receptores de Esteroides/efeitos da radiação , Animais , Núcleo Celular/metabolismo , Corticosterona/metabolismo , Masculino , Nucleotídeos Cíclicos/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Glucocorticoides/metabolismoRESUMO
Photoactivation of the alpha,beta-unsaturated ketones of natural and synthetic steroid molecules by light of lambda greater than or equal to 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites.