RESUMO
Gestational trophoblastic diseases, like the complete hydatidiform mole (CHM), are a group of human interrelated neoplasms whose etiology and progression is poorly understood at the molecular level. We have previously reported the cloning and expression of a new tumor necrosis factor receptor (TNF-R) related transcript, named CHMS-1 that encodes a potential death domain. Here we show that ectopic expression of the putative CHMS-1 death domain specifically induced apoptosis in a dose-dependent manner, in trophoblastic (JEG-3) and non-trophoblastic (COS-7) cells. We also investigated the expression of apoptosis-related molecules such as Bcl-2 and p53 and demonstrated that Bcl-2 is repressed in CHM while p53 is overexpressed in CHM compared with persistent gestational trophoblastic tumors. Altogether, these data indicate that the CHMS-1 death domain is able to trigger apoptosis, thus suggesting that this new entity might be an important inducer of molar regression mechanisms in women.
Assuntos
Apoptose , Proteínas de Neoplasias/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Trofoblastos/metabolismo , Animais , Northern Blotting , Feminino , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Técnicas Imunoenzimáticas , Proteínas Luminescentes/metabolismo , Proteínas de Neoplasias/genética , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Trofoblastos/patologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
To assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated with these placental proliferative disorders we performed differential display (DD) techniques. This strategy resulted in the isolation of four mitochondrial transcripts downregulated in benign, as well as in malignant, trophoblastic diseases encoding the cytochrome oxidase subunit I (COX I), the ATPase subunit 6, the 12S ribosomal RNA (12S rRNA) and the transfer RNA for phenylalanine (tRNA(Phe)). This expression pattern was confirmed by Northern blot in normal early placenta (NEP), complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and the human choriocarcinoma derived cell line JEG-3. Quantification of mitochondrial DNA by dot blot indicated that these changes in expression were not associated with a significant alteration in the number of mitochondrial genome. In addition, a reduction in the mitochondrial transcription factor A (mtTFA) mRNA level was observed in benign as well as in malignant trophoblastic diseases in correlation with the decrease in the mitochondrial transcript levels. Furthermore, Western blot analysis for COX-I showed a close parallelism with the expression level of the cognate RNA. Taken together, these data demonstrate that a significant change in mitochondrial transcription is associated with the phenotypic alteration present in GTDs.
Assuntos
Coriocarcinoma/genética , DNA Mitocondrial/genética , Expressão Gênica , Mola Hidatiforme/genética , Prostaglandina-Endoperóxido Sintases , Neoplasias Trofoblásticas/genética , Neoplasias Uterinas/genética , Adenosina Trifosfatases/genética , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Mitocondrial/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Humanos , Isoenzimas , Proteínas de Membrana , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/análise , RNA de Transferência de Fenilalanina/genética , Análise de Sequência de DNA , Homologia de Sequência , Células Tumorais CultivadasRESUMO
The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.
Assuntos
Glicoproteínas/metabolismo , Proteínas da Gravidez/genética , Glicoproteínas beta 1 Específicas da Gravidez , Transcrição Gênica , Animais , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Glicoproteínas/genética , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Placenta/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Timidina Quinase/genética , TransfecçãoRESUMO
Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.
Assuntos
Expressão Gênica , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Neoplasias Trofoblásticas/metabolismo , Neoplasias Uterinas/metabolismo , Sequência de Aminoácidos , Coriocarcinoma/metabolismo , Feminino , Humanos , Mola Hidatiforme/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Gravidez , Receptores do Fator de Necrose Tumoral/química , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , TATA Box , Transativadores/genética , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/química , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/química , Biblioteca Gênica , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Fases de Leitura Aberta , Placenta/metabolismo , Transativadores/químicaRESUMO
Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.