A role for Sp and helix-loop-helix transcription factors in the regulation of the human Id4 gene promoter activity.

Abstract Id family helix-loop-helix (HLH) proteins are involved in the regulation of proliferation and differentiation of several cell types. To identify cis- and trans-acting factors that regulate Id4 gene expression, we have analyzed the promoter regulatory sequences of the human Id4 gene in transient transfections and gel mobility shift assays. We have identified two functional elements, both located downstream from the TATA motif, that control Id4 promoter activity. One element contains a consensus E-box, and we demonstrated that the protein complex binding to the E-box contains the bHLH-zip upstream stimulatory factor (USF) transcription factor. Enforced expression of USF1 leads to E-box-mediated stimulation of promoter activity. The E-box also mediated stimulatory effects of several bHLH transcription factors, and co-expression of Id4 blocked the stimulatory effect mediated by the bHLH factors. A second element is a GA motif, located downstream from the transcriptional start sites, mutation of which resulted in a 20-fold increase in transcriptional activity. Gel-shift analysis and transfections into DrosophilaSchneider SL2 cells showed that the repressor element is recognized by both Sp1 and Sp3 factors. These data suggest that Id4 transcription control is highly complex, involving both negative and positive regulatory elements, including a novel inhibitory function exerted by Sp1 and Sp3 transcription factors

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells.

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A- transfected cells. Further, we show that in Ret-transfected cells the suc1- associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade

Thymosin β-10 Gene Overexpression Is a General Event in Human Carcinogenesis

The β-thymosins comprise a family of structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. Recently, we have demonstrated the overexpression of thymosin β-10 (TB10) in rat thyroid transformed cell lines and in human thyroid carcinoma tissues and cell lines. To verify whether TB10 overexpression is a general event in the process of carcinogenesis, we have analyzed TB10 mRNA levels in human colon carcinomas, germ cell tumors of different histological types, breast carcinomas, ovarian carcinomas, uterine carcinomas, colon and esophageal carcinoma cell lines. Overexpression of the TB10 gene was detected in all of the neoplastic tissues and cell lines compared to the respective normal tissues. Moreover, the mouse model of skin carcinogenesis induced by the combined action of chemical carcinogens and phorbol esters was used to identify the stage of TB10 gene induction. The expression was almost undetectable in normal keratinocytes, its induction occurred even at the papilloma stage, however a further increased expression was observed in the carcinoma derived cell lines. Finally, immunohistochemical analysis of some breast, colon and ovary carcinoma samples by using specific anti-TB10 antibodies revealed the presence of the TB10 protein in all of the neoplastic tissues, but not in the respective normal tissues. Therefore the TB10 detection may be considered a potential tool for the diagnosis of several human neoplasias