RESUMO
Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the tunicate Ecteinascidia turbinata currently under phase II clinical trials for its potent anticancer activity. ET-743 binds DNA in the minor groove and forms covalent adducts with some sequence specificity. It selectively inhibits in vitro binding of the CCAAT box factor NF-Y. In this study, we assayed ET-743 function in vivo on the HSP70 promoter. On heat induction, the drug blocks transcription rapidly at pharmacological concentrations and in a CCAAT-dependent manner, whereas the activity of the CCAAT-less simian virus 40 promoter is not affected. The effect is exerted at the mRNA level. The distamycin-like alkylating tallimustine is inactive in these assays. Binding of NF-Y and of the heat-shock factor is normal in ET-743-treated cells. Run-on analysis of several endogenous genes further proves that the drug has rapid, profound, and selective negative effects on transcription. Thus, this marine-derived compound is a promoter-specific, transcription-interfering agent.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Dioxóis/farmacologia , Isoquinolinas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Células 3T3 , Animais , Proteínas Estimuladoras de Ligação a CCAAT , DNA/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Camundongos , Regiões Promotoras Genéticas , Tetra-Hidroisoquinolinas , TrabectedinaRESUMO
The 'promiscuous' E2A gene, at 19p13.3, is fused with two different molecular partners, PBX1 and HLF, following two chromosome translocations recurrent in childhood pre-B ALL. We have identified a novel gene, FB1, by virtue of its fusion with E2A and by a combination of molecular techniques. FB1 was localized on 19q13.4, suggesting that the novel chimera originated by a cryptic rearrangement of chromosome 19. Two FB1 transcripts, of 1.2 kb and 1.1 kb, are differentially expressed at low level in a variety of human tissues, including hemopoietic cell lines from different lineages. Accordingly, FB1 cDNA displays high homology with a number of cDNA clones from different human tissues. High homology was found also with cDNA clones from mouse and rat, suggesting that the sequence might be conserved at least among mammals. The function of the putative FB1 protein, however, is currently unknown as database sequence comparisons have failed to reveal strong homology with known proteins. The E2A/FB1 fusion appears to be a recurrent feature of pre-B ALLs, suggesting that it might have a role in the development and/or progression of leukemogenesis.
Assuntos
Proteínas E2 de Adenovirus/genética , Proteínas de Ligação a DNA/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Criança , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 19 , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genéticaRESUMO
Pheochromocytomas are adrenal medullary tumors which arise from the transformation of neural crest-derived cells. In the course of studies of mice transgenic for an SV40 T-gene ectopically expressed in the adrenal medulla, we observed the occurrence of large, mainly bilateral tumors in a high proportion of transgenic animals. From these tumors we established immortalized cell lines which grow in vitro at 32 degrees C (the permissive temperature for the tsA58 T-protein encoded by the transgene), but not at 38 C. These cells demonstrate characteristics of both neuronal (160 kd neurofilament) and endocrine (chromogranins) cells. The expression of Mash-1 and ret supports their initial characterization as early bipotential neuro-endocrine progenitors.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Antígenos Transformantes de Poliomavirus/genética , Camundongos Transgênicos/genética , Neoplasias das Glândulas Suprarrenais/mortalidade , Medula Suprarrenal/patologia , Animais , Northern Blotting , Divisão Celular/efeitos dos fármacos , Cromogranina A , Cromograninas/metabolismo , Citocinas/farmacologia , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica , Camundongos , Mutação , Fatores de Crescimento Neural/farmacologia , Crista Neural/citologia , Crista Neural/patologia , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.
