RESUMO
Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.
Assuntos
Expressão Gênica , Produtos do Gene tat/metabolismo , HIV-1/genética , Interleucina-6/biossíntese , Animais , Linfócitos B , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Primers do DNA , Feminino , Produtos do Gene tat/biossíntese , Genes tat , HIV-1/metabolismo , Células HeLa , Humanos , Interleucina-6/genética , Cinética , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging cancer. In this report, 43 melanoma patients (including 4 in situ melanoma patients) were tested for tyrosinase mRNA in blood by RT-PCR. All patients had melanoma thinner than 1.5 mm (stage I). Circulating melanoma cells were detected in 8 (18.6%) out of 43 MM patients tested: 5 (16.1%) of 31 patients with melanoma thinner than 0.76 mm and 3 (42.8%) out of 7 patients with melanoma thicker than 0.76 mm. Moreover, in the tyrosinase-negative group we found only 4/31 patients (13%) with histologic signs of regression, but in the tyrosinase-positive group, 3 out of 8 patients (37.5%) showed, at histologic examination, signs of regression. At the time of this analysis all the patients enrolled (tyrosinase-negative and tyrosinase-positive ones) were free of disease, probably due to the short median time of follow-up after the inclusion in the study. The presence of regression is an important cause of melanoma understaging and the tyrosinase test could represent an effective tool in order to achieve a realistic staging in this subgroup of melanoma patients. Probably, maximum sensitivity of the diagnostic RT-PCR approach to monitor MM patients with either localized or advanced disease could be achieved by using additional markers expressed with high frequencies in melanoma. We propose that one such marker could be the sign of regression.
Assuntos
Melanoma/sangue , Monofenol Mono-Oxigenase/genética , Células Neoplásicas Circulantes/patologia , RNA Mensageiro/sangue , Adolescente , Adulto , Idoso , Intervalo Livre de Doença , Neoplasias Oculares/sangue , Neoplasias Oculares/genética , Neoplasias Oculares/patologia , Feminino , Seguimentos , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Sobreviventes , Fatores de TempoRESUMO
The behaviour of catalase activity was studied in patients with respiratory insufficiency and the results assessed in the light of a theory that attributes a completely new teleological function to this substance.
Assuntos
Catalase/sangue , Eritrócitos/enzimologia , Insuficiência Respiratória/enzimologia , Adulto , Idoso , Contagem de Eritrócitos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Respiratória/sangueRESUMO
Three groups of subjects (I with infections of the lower urinary ways; II with chronic pyelonephritis; III healthy controls) were treated with pipemidic acid for 10 consecutive days and the behaviour of their urinary NAG and AAP was studied. It was discovered that the healthy group and the patients with infections of the lower urinary ways showed no significant variations in the urinary activity of the two enzymes following the administration of pipemidic acid. However there was a distinct reduction in both enzymes in patients with chronic pyelonephritis who presented demonstrable bacterial sensitivity to the drug. Apart from the fact that it produces no nephrotoxic effects at least in the doses used and for the treatment period adopted, the drug is therefore considered to reduce renal inflammation that is beneficial to individuals with chronic interstitial nephropathy.
Assuntos
Pielonefrite/enzimologia , Infecções Urinárias/enzimologia , Acetilglucosaminidase/urina , Adulto , Idoso , Aminopeptidases/urina , Antígenos CD13 , Doença Crônica , Diagnóstico Diferencial , Feminino , Glomerulonefrite/enzimologia , Glomerulonefrite/urina , Humanos , Masculino , Pessoa de Meia-Idade , Nefrite Intersticial/enzimologia , Nefrite Intersticial/urina , Pielonefrite/urina , Infecções Urinárias/urinaRESUMO
The activation of the human immunodeficiency virus, type 1 (HIV-1) by the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, and mitomycin C was observed in human B lymphocytes transiently transfected with plasmids in which the HIV-1 long terminal repeat (LTR) directed the expression of the bacterial chloramphenicol acetyltransferase gene. Deletion of the two NF-kappa B-binding sites of LTR abolished the HIV-1 activation induced by the three mutagens, while deletion of the three Sp1-binding sites slightly reduced it. Electrophoretic mobility shift assays revealed an increased binding to the kappa B sites of HIV-1 LTR in the nuclear extracts of human B lymphocytes upon mutagen treatment, while binding to Sp1 sites was unaffected. The TAR region was also involved in the mutagen-mediated activation of HIV-1 LTR inasmuch as a small deletion in the TAR sequence (nucleotides +34 to +37) greatly decreased the induction of HIV-1 expression. Moreover, an enhanced binding activity to the TAR DNA sequence (nucleotides +24 to +47) was observed in nuclear extracts of mutagen-treated lymphocytes. Thus, both the enhancer and the 5'-untranslated region of HIV-1 functionally cooperate in the mutagen-mediated induction of HIV-1 expression.
Assuntos
Alquilantes/farmacologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Linfócitos/microbiologia , Mutagênicos/farmacologia , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Metanossulfonato de Etila/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Metanossulfonato de Metila/farmacologia , Mitomicina/farmacologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp1/metabolismo , Ativação Viral/efeitos dos fármacos , Ativação Viral/genéticaRESUMO
The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-kappaB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-kappaB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-alpha. The TAR NF-kappaB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-kappaB enhancer upstream of the TATA box in the NF-kappaB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-kappaB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-kappaB inducers; (ii) deletion or base pair substitutions of the TAR NF-kappaB site significantly reduce the HIV-1 LTR activation by NF-kappaB inducers; (iii) deletions of both the NF-kappaB sites upstream of the TATA box and the TAR NF-kappaB site abolish the activation of HIV-1 LTR in response to NF-kappaB inducers. Moreover, the p50 p65 NF-kappaB complex binds to the TAR NF-kappaB sequence and trans-activates the TAR NF-kappaB-directed expression. The identification of an additional NF-kappaB site in the HIV-1 LTR points to the relevance of NF-kappaB factors in the HIV-1 life cycle.
Assuntos
Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV/genética , HIV-1/genética , NF-kappa B/metabolismo , Sequência de Bases , Linhagem Celular , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Deleção de Sequência , Transcrição Gênica , Ativação TranscricionalRESUMO
In this study, we addressed the question of whether carcinogens affected the expression of interleukin-1 alpha (IL1 alpha), interleukin-1 beta (IL1 beta), and interleukin-6 (IL6) genes and the production of the relative proteins. Primary cultures of human monocytes were exposed to the alkylating agents mitomycin C (Mit C), methylmethane sulfonate (MMS), and ethylmethane sulfonate (EMS) and tested for the production of IL1 alpha, IL1 beta, and IL6 proteins, as well as for the expression of IL1 alpha, IL1 beta, and IL6 transcripts. The production of IL1 beta and IL6 was significantly augmented by all the three chemicals after 24-48 h of treatment. IL1 alpha was also increased by Mit C and MMS. By Northern blotting analysis, the increased expression of IL1 alpha, IL1 beta, and IL6 genes was shown to occur at 30 min of Mit C and MMS treatment and to decline after 8 h. Similarly, EMS up-regulated the expression of IL1 beta and IL6 genes. The mutagen-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was due to the enhanced half-life of IL1 alpha, IL1 beta, and IL6 mRNAs rather than to the increased rate of gene transcription. These results suggest that carcinogens, in addition to causing DNA mutations and rearrangements, may also affect cell growth and differentiation by enhancing the expression of cytokine genes.
Assuntos
Carcinógenos/farmacologia , Dano ao DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/genética , Interleucina-6/genética , RNA Mensageiro/metabolismo , Células Cultivadas , Metanossulfonato de Etila/farmacologia , Humanos , Interleucina-1/biossíntese , Interleucina-6/metabolismo , Metanossulfonato de Metila/farmacologia , Mitomicina/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA Mensageiro/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The AP-2 transcription factor plays a pivotal role in regulating the expression of several genes involved in tumor growth and progression of melanoma. We determined, by Western blot, variation in the level of expression of AP-2 and three of its downstream targets, c-kit, E-cadherin, and p21 in several human melanoma cell lines and, by immunohistochemistry, in a group of 99 histological samples including benign and malignant melanocytic lesions. A significant negative correlation between AP-2 expression level and tumor thickness was found. Moreover, AP-2 expression was positively associated with E-cadherin and c-kit expression. In contrast, there was a significant negative association between AP-2 and p21 expression levels. These findings suggest that p21 is independent of AP-2 transactivator function during the latest phases of melanoma progression. Finally, AP-2, c-kit, E-cadherin, and p21 expression levels did not show to be able to distinguish between dysplastic nevi and nevi without dysplasia. We conclude that changes in the expression of these proteins are involved in the later phases of melanoma progression, and may be responsible for the transition from local invasive melanoma to metastasis.
Assuntos
Caderinas/biossíntese , Ciclinas/biossíntese , Proteínas de Ligação a DNA/biossíntese , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-kit/biossíntese , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/biossíntese , Western Blotting , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Feminino , Humanos , Imuno-Histoquímica , Masculino , Nevo/metabolismo , Fator de Transcrição AP-2 , Células Tumorais CultivadasRESUMO
We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors. This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta.