RESUMO
Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.
Assuntos
Catecóis/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Nitrilas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirfostinas , Transporte Biológico , Carcinoma de Células Escamosas , Catecóis/síntese química , Catecóis/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática , Receptores ErbB/efeitos dos fármacos , Humanos , Cinética , Estrutura Molecular , Nitrilas/síntese química , Nitrilas/farmacologia , Proteínas Tirosina Quinases/metabolismoRESUMO
Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.
Assuntos
Imunidade/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Terapia PUVA , Animais , Antígenos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Epitopos , Imunoterapia Adotiva , Técnicas In Vitro , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteínas Recombinantes/farmacologia , Linfócitos T/efeitos dos fármacosAssuntos
Anticorpos Monoclonais , Fosfoproteínas/isolamento & purificação , Tirosina/análogos & derivados , Animais , Especificidade de Anticorpos , Linhagem Celular , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/farmacologia , Feminino , Haptenos , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Fosfoproteínas/metabolismo , Fosfotirosina , Radioimunoensaio/métodos , Tirosina/análise , Tirosina/imunologiaRESUMO
One of the more prominent clinical treatments for skin diseases such as psoriasis and vitiligo involves the use of a combination of psoralens and UV light, a procedure referred to as PUVA chemotherapy. This drug regimen markedly alters epidermal cell growth and differentiation. In many cell types, an early cellular event following treatment of cells with PUVA is inhibition of binding of epidermal growth factor (EGF) to its receptor. To examine the mechanism underlying this effect, we used A431 cells, a human epidermal cell line known to express large numbers of EGF receptors. We found that exposure of A431 cells to PUVA caused a dramatic inhibition of EGF-stimulated EGF receptor tyrosine kinase activity. Inhibition required intact cells and did not appear to be mediated by protein kinase C, because this inhibition was apparent in cells in which the enzyme was down-regulated by phorbol ester pretreatment and in cells treated with inhibitors of protein kinase C. Inhibition of tyrosine kinase activity by PUVA was distinct from other inhibitors of EGF receptor function in that it was associated with a rapid increase in the amount of phosphate incorporated into serine residues of the EGF receptor. This suggested that PUVA-induced serine phosphorylation may mediate EGF receptor kinase activity. These results demonstrate that alterations in EGF receptor function may contribute to the therapeutic efficacy of PUVA in photo-chemotherapy.
Assuntos
Receptores ErbB/efeitos dos fármacos , Terapia PUVA , Proteínas Tirosina Quinases/antagonistas & inibidores , Linhagem Celular , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Fosforilação , Proteína Quinase C/fisiologia , Psoríase/tratamento farmacológico , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region III (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72, 38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31-37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus EIII promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the cAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by cAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein.
Assuntos
Adenoviridae/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Precoces de Adenovirus , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Ligação a DNA/genética , Genes Virais , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Peso Molecular , Sondas de Oligonucleotídeos , Proteínas Oncogênicas Virais/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Estruturais Virais/genéticaRESUMO
Dr1, a repressor of class II genes, regulates transcription by a novel mechanism. Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex. By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo. In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays. Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo. By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression. One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues. The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression. We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo.
Assuntos
Genes MHC da Classe II , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Regulação da Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfoproteínas/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TATA Box , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes MHC da Classe II , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Testes de Precipitina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIA , Fator de Transcrição TFIIB , Fatores de Transcrição/genéticaRESUMO
Transcription factor IIA has been shown to interact with the TATA-binding protein and to act early during preinitiation complex formation. The human factor is composed of three subunits (alpha, beta, gamma). A human cDNA clone encoding the largest subunit of TFIIA (alpha) was isolated. The recombinant alpha polypeptide, together with the beta and gamma subunits, was capable of reconstituting TFIIA activity. Studies using antibodies raised against recombinant alpha polypeptide demonstrate that TFIIA can be an integral component of the preinitiation complex. We demonstrate that TFIIA not only interacts with TBP but also can associate with the TFIID complex. Functional assays establish that TFIIA has no apparent role in basal transcription but plays an important role in activation of transcription. Interestingly, amino acid sequence analyses of the beta-subunit demonstrate these residues to be entirely contained within the carboxyl terminus of the cDNA clone encoding the alpha-subunit.
Assuntos
DNA Complementar/isolamento & purificação , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIA , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery.
Assuntos
Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Ciclina H , Ciclinas/metabolismo , Detergentes , Células HeLa , Humanos , Técnicas Imunológicas , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/isolamento & purificação , RNA Polimerase II/metabolismo , Proteínas Recombinantes/metabolismo , Sarcosina/análogos & derivados , Fator de Transcrição TFIIH , Fatores de Transcrição/química , Transcrição Gênica , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A Dr1-associated polypeptide (DRAP1) was isolated from HeLa cells and found to function as a corepressor of transcription. Corepressor function requires an interaction between DRAP1 and Dr1. Heterodimer formation was dependent on a histone fold motif present at the amino terminus of both polypeptides. Association of DRAP1 with Dr1 results in higher stability of the Dr1-TBP-TATA motif complex and precluded the entry of TFIIA and/or TFIIB to preinitiation complexes. DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index. Analysis of DRAP1 in the developing brain of rat demonstrated undetectable levels of DRAP1 in actively dividing cells but high levels of DRAP1 expression in differentiated non dividing cells. Dr1 was immunodetected in all cells analyzed. A model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed.