RESUMEN
Progressive fibrosis of the interstitium is the dominant final pathway in renal destruction in native and transplanted kidneys. Over time, the continuum of molecular events following immunological and nonimmunological insults lead to interstitial fibrosis and tubular atrophy and culminate in kidney failure. We hypothesize that these insults trigger changes in DNA methylation (DNAm) patterns, which in turn could exacerbate injury and slow down the regeneration processes, leading to fibrosis development and graft dysfunction. Herein, we analyzed biopsy samples from kidney allografts collected 24 months posttransplantation and used an integrative multi-omics approach to understand the underlying molecular mechanisms. The role of DNAm and microRNAs on the graft gene expression was evaluated. Enrichment analyses of differentially methylated CpG sites were performed using GenomeRunner. CpGs were strongly enriched in regions that were variably methylated among tissues, implying high tissue specificity in their regulatory impact. Corresponding to this methylation pattern, gene expression data were related to immune response (activated state) and nephrogenesis (inhibited state). Preimplantation biopsies showed similar DNAm patterns to normal allograft biopsies at 2 years posttransplantation. Our findings demonstrate for the first time a relationship among epigenetic modifications and development of interstitial fibrosis, graft function, and inter-individual variation on long-term outcomes.
Asunto(s)
Atrofia/patología , Metilación de ADN , Fibrosis/patología , Rechazo de Injerto/genética , Fallo Renal Crónico/patología , Trasplante de Riñón/métodos , Túbulos Renales/patología , Adulto , Atrofia/metabolismo , Biomarcadores/metabolismo , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Fibrosis/metabolismo , Estudios de Seguimiento , Perfilación de la Expresión Génica , Tasa de Filtración Glomerular , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/epidemiología , Supervivencia de Injerto , Humanos , Fallo Renal Crónico/genética , Fallo Renal Crónico/cirugía , Pruebas de Función Renal , Túbulos Renales/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Factores de Riesgo , Trasplante HomólogoRESUMEN
The molecular basis of calcineurin inhibitor toxicity (CNIT) in kidney transplantation (KT) and its contribution to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA) were evaluated by: (1) identifying specific CNIT molecular pathways that associate with allograft injury (cross-sectional study) and (2) assessing the contribution of the identified CNIT signature in the progression to CAD with IF/TA (longitudinal study). Kidney biopsies from well-selected transplant recipients with histological diagnosis of CNIT (n = 14), acute rejection (n = 13) and CAD with IF/TA (n = 10) were evaluated. Normal allografts (n = 18) were used as controls. To test CNIT contribution to CAD progression, an independent set of biopsies (n = 122) from 61 KT patients collected at 3 and ~12 months post-KT (range = 9-18) were evaluated. Patients were classified based on 2-year post-KT graft function and histological findings as progressors (n = 30) or nonprogressors to CAD (n = 31). Molecular signatures characterizing CNIT samples were identified. Patients classified as progressors showed an overlap of 7% and 22% with the CNIT signature at 3 and at ~12 months post-KT, respectively, while the overlap was <1% and 1% in nonprogressor patients, showing CNIT at the molecular level as a nonimmunological factor involved in the progression to CAD.
Asunto(s)
Biomarcadores/sangre , Inhibidores de la Calcineurina/efectos adversos , Perfilación de la Expresión Génica , Rechazo de Injerto/clasificación , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Adulto , Aloinjertos , Área Bajo la Curva , Estudios Transversales , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Rechazo de Injerto/sangre , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/genética , Humanos , Fallo Renal Crónico/complicaciones , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Complicaciones Posoperatorias , Pronóstico , Estudios ProspectivosRESUMEN
Important progress has been made in improving short-term outcomes in solid organ transplantation. However, long-term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response. MicroRNAs (miRNAs) are a class of small single-stranded noncoding RNAs that function through translational repression of specific target mRNAs. MiRNA expression has been associated with different diseases and physiological conditions. Moreover, miRNAs have been detected in different biological fluids and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA detection, their disease specificity and the availability of accurate techniques for detecting and monitoring these molecules has encouraged a pursuit of miRNA biomarker research and the evaluation of specific applications in the transplant field. miRNA expression might develop as excellent biomarkers of allograft injury and function. In this minireview, we summarize the main accomplishments of recently published reports focused on the identification of miRNAs as biomarkers in organ quality, ischemia-reperfusion injury, acute rejection, tolerance and chronic allograft dysfunction emphasizing their mechanistic and clinical potential applications and describing their methodological limitations.
Asunto(s)
Biomarcadores/sangre , MicroARNs/sangre , Trasplante de Órganos , Rechazo de Injerto/sangre , Humanos , Daño por Reperfusión/sangre , Donantes de TejidosRESUMEN
Diagnosis and prediction of the severity of hepatitis C virus recurrence (HCVrec) after liver transplantation (LT) remain a challenge. MicroRNAs have been recently recognized as potential disease biomarkers. Archival liver biopsy samples from 43 HCV+ LT recipients were collected at clinical HCVrec time and at 3 years post-LT. Patients were classified as progressors (P = F0/F1) or nonprogressors (NP = F3/F4) according to the severity of fibrosis on the 3-year biopsy. Training (n = 27) and validation (n = 16) sets were defined. RNA was isolated from all biopsies at clinical HCVrec time, labeled and hybridized to miRNA-arrays. Progressors versus nonprogressors were compared using the two-sample t-test. A p-value ≤0.01 was considered significant. The ingenuity pathway analysis tool was used for microRNA and miRNA:mRNA ontology data integration. Nine microRNAs were differentially expressed between groups. A supervised cluster analysis separated samples in two well-defined groups (progressors vs. nonprogressors). Pathway analysis associated those microRNAs with hepatitis, steatosis, fibrosis, cirrhosis and T cell-related immune response. Data integration identified 17 genes from a previous genomic study as 9-microRNAs signature targets. Seven microRNAs were successfully validated in the validation set using QPCR. We have identified a 9-microRNA signature able to identify early post-LT patients at high risk of severe HCVrec during long-term follow-up.
Asunto(s)
Hepatitis C/cirugía , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/cirugía , Trasplante de Hígado/efectos adversos , MicroARNs/genética , Complicaciones Posoperatorias , Adulto , Anciano , Biomarcadores/análisis , Biomarcadores/metabolismo , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Rechazo de Injerto , Supervivencia de Injerto , Hepacivirus/patogenicidad , Hepatitis C/complicaciones , Hepatitis C/virología , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/etiología , Cirrosis Hepática/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Estudios Prospectivos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteína Quinasa CDC2 , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Fragmentación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Células Mieloides/metabolismo , Células Mieloides/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Cultivo Primario de Células , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Vorinostat , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.
RESUMEN
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.
Asunto(s)
Apoptosis , Proteínas de Neoplasias/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Trofoblastos/metabolismo , Animales , Northern Blotting , Femenino , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Técnicas para Inmunoenzimas , Proteínas Luminiscentes/metabolismo , Proteínas de Neoplasias/genética , Embarazo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Trofoblastos/patología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We have previously demonstrated that expansion of activated tumor-sensitized T cells in interleukin (IL)-7/15 results in greater expansion and antitumor activity than expansion in IL-2. We sought to determine whether T cells exposed to IL-2 versus IL-7/15 exhibited distinct gene expression patterns. Lymphocytes were harvested from Pmel-1 mice immunized with B16-GMCSF melanoma cells, activated in vitro, and cultured in IL-2 or IL-7/15 for 1, 3 or 6 days. T cells were harvested and analyzed using microarray, real-time quantitative polymerase chain reaction (RT-QPCR) or sorted into T-cell subsets and analyzed. We found significant differences in gene expression for T cells cultured in IL-2 versus IL-7/15, starting on day 3. This was not a function of subset differentiation; when T cells were divided into subsets, the central memory (T(CM)), effector memory (T(EM)) and effector (T(E)) T cells cultured in the IL-2 more closely resembled each other than the identical phenotypic subset exposed to IL-7/15. Thus, the differences in gene expression induced by culture in IL-2 versus IL-7/15 do not merely reflect differences in the frequency of T(CM) versus T(EM) versus T(E) cells, but rather reflect that the gene expression levels of those T-cell subsets when exposed to different cytokines are fundamentally different.
Asunto(s)
Citocinas/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Inmunoterapia Adoptiva , Interleucina-15/farmacología , Interleucina-2/farmacología , Interleucina-7/farmacología , Melanoma/genética , Melanoma/terapia , Melanoma Experimental , Ratones , Ratones Transgénicos , Subgrupos de Linfocitos T/efectos de los fármacosAsunto(s)
Linfoma Plasmablástico/genética , Receptores de Antígenos de Linfocitos B/fisiología , Transcripción Genética , Adolescente , Adulto , Niño , Preescolar , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Femenino , Genes myc , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Masculino , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Proteínas Nucleares/genética , Transducción de SeñalRESUMEN
Previous cross-platform reproducibility studies have compared consistency of intensities as well as consistency of fold changes across different platforms using Pearson's correlation coefficient. In this study, we propose the use of measurement error models for estimating gene-specific correlations. Additionally, gene-specific reliability estimates are shown to be useful in prioritizing clones for sequence verification rather than selecting clones using a simple random sample. The proposed 'disattenuated' correlation may prove useful in a wide variety of studies when both X and Y are measured with error, such as in confirmation studies of microarray gene expression values, wherein more reliable laboratory assays such as real-time polymerase chain reaction are used.
Asunto(s)
Calibración , Bases de Datos Genéticas/estadística & datos numéricos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Análisis de Regresión , Neoplasias de la Mama/genética , Simulación por Computador , Femenino , Humanos , Neoplasias Ováricas/genética , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Expresión Génica , Proteínas de Neoplasias/genética , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Neoplasias Trofoblásticas/metabolismo , Neoplasias Uterinas/metabolismo , Secuencia de Aminoácidos , Coriocarcinoma/metabolismo , Femenino , Humanos , Mola Hidatiforme/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Embarazo , Receptores del Factor de Necrosis Tumoral/química , Alineación de Secuencia , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , TATA Box , Transactivadores/genética , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/química , Biblioteca de Genes , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Placenta/metabolismo , Transactivadores/químicaRESUMEN
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.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas Gestacionales/genética , Glicoproteínas beta 1 Específicas del Embarazo , Transcripción Genética , Animales , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Huella de ADN , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Glicoproteínas/genética , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Familia de Multigenes , Placenta/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Timidina Quinasa/genética , TransfecciónRESUMEN
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.