RESUMO
The constitutive androstane receptor (CAR, NR1I3) transcriptionally activates cytochrome P450 2B6, 2C9, and 3A4 when activated by xenobiotics, such as phenobarbital. Information on the human CAR promoter was obtained by searching the NCBI human genome database. A contig (NT026945) corresponding to a fragment of chromosome 1q21 was found to contain the complete CAR gene. These data were confirmed using chromosomal in situ hybridization. Both primer extension and 5'-rapid amplification of the cDNA end PCR analysis were carried out to determine the transcriptional start site of human CAR, which was found to be 32 nucleotides downstream of a potential TATA box (CATAAAA). In addition, we found that the 5'-untranslated region of CAR mRNA is 110 nucleotides shorter than previously reported. Using genomic PCR, we amplified and cloned approximately 4.9 kb (-4711/+144) of the CAR gene promoter. The activity of this promoter was measured by transient transfection. Deletion analysis suggested the presence of a glucocorticoid responsive element in its distal region (-4477/-4410). From cotransfection experiments, mutagenesis, and gel shift assays, we identified a glucocorticoid response element at -4447/-4432 that was recognized and transactivated by the human glucocorticoid receptor. Finally, using the chromatin immunoprecipitation assay, we demonstrated that the glucocorticoid receptor binds to the distal region of CAR promoter in cultured hepatocytes only in the presence of dexamethasone. Identification of this functional element provides a rational mechanistic basis for CAR induction by glucocorticoids. CAR appears to be a primary glucocorticoid receptor-response gene.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Fatores de Transcrição/genética , Células Cultivadas , Clonagem Molecular , Receptor Constitutivo de Androstano , Hepatócitos , Humanos , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Ativação Transcricional/fisiologiaRESUMO
Chromosome studies were carried out in long-term (142 and 184 d) human lymphocyte in vitro cultures in order to investigate the cytogenetic status of aging lymphocytes. The female donors were subdivided into three subgroups according to their age: 20-40 year-old (three individuals), 70-90 year-old (five persons), and centenarians (three persons). Besides some aneuploidy and structural abnormalities, telomere fusions were detected in all donor cells, and associations of acrocentric chromosomes were found in six persons in the three age-groups. Clonal trisomy 2 was present in three individuals (two from the 70-90 year-group and one centenarian with a clone +2, +8). While telomeric fusions and acrocentric associations seem to be more related to in vitro aging, trisomy 2 also appears dependent on the age of the cell donors.
Assuntos
Cromossomos Humanos , Linfócitos/ultraestrutura , Trissomia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Translocation t(1;22)(p13;q13) is associated with a peculiar subtype of acute megakaryocytic leukemia (M7) occurring in infants. We have recently characterized a fusion gene, OTT-MAL, resulting from this translocation. We now report three additional cases and show that this gene fusion is present in all five t(1;22) cases studied to date. Nucleotide sequence analysis of two translocation breakpoints suggests a nonhomologous end joining mechanism in the genesis of this translocation and reveals a noncanonical topoisomerase II-like consensus sequence within the OTT gene. FISH and PCR techniques described in this work are useful for identifying t(1;22) associated with M7.
Assuntos
Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 22/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas/genética , Proteínas de Ligação a RNA , Translocação Genética/genética , Sequência de Aminoácidos , Sequência de Bases , Coloração Cromossômica , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Leucemia Megacarioblástica Aguda/diagnóstico , Masculino , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genéticaRESUMO
The most frequent oncogenic activation events characterized in childhood T acute lymphoblastic leukemia (T-ALL) result in the transcriptional activation of genes coding for transcription factors. The main genes are TAL1/SCL, a member of the basic region helix-loop-helix gene family, and HOX11L2, a member of the homeobox-containing protein family. To gain insight into the pathogenesis of this type of hematologic malignancy, we analyzed 28 T-ALL samples. SIL-TAL1/SCL fusion was detected in 6 patients; expression of HOX11L2 was observed in 6 patients and of HOX11 in 3 patients. With one exception, these activations did not occur simultaneously in the same patients, and they allowed the subclassification of 50% of the patients. SIL-TAL1 fusion was detected in association with HOX11 expression in one patient and with a t(8;14) (q24;q11) in another. High expression of LYL1, LMO2, or TAL1 was observed mainly in samples negative for HOX11L2 expression. HOX11L1 and HOX11 expression were observed in one instance each, in the absence of detectable chromosomal abnormality of their respective loci, on chromosomes 2 and 10, respectively. HOX11L2 expression was associated with a chromosome 5q abnormality, the location of the HOX11L2 locus in each case tested. Finally, our data show that HOX11L2 expression was a suitable marker for minimal residual disease follow-up and was significantly associated with relapse (P =.02).