RETINOIC ACID AND CAMP DIFFERENTIALLY REGULATE HUMAN CHROMOGRANIN-A PROMOTER ACTIVITY DURING DIFFERENTIATION OF NEUROBLASTOMA-CELLS

We report the first evidence that differential transcriptional regulat ion of human chromogranin A (CHGA) gene expression occurs during in vi tro treatment of tumorigenic neuroblastoma (NB) cells with retinoic ac id (5 mu M) and/or dibutyryl-cAMP (1 mM). The CHGA gene encodes a tiss ue specific protein restricted to cells of the diffuse neuroendocrine system, but also widely expressed among NE tumours. We previously repo rted that CHGA as well as other neuroendocrine markers are modulated d uring NE differentiation in vitro. To investigate, at the molecular le vel, the mechanisms leading to NE tumour cell differentiation during t he treatment with biologically active compounds, we sequenced and func tionally characterised 2169 bp of a genomic DNA clone encompassing the 5' flanking region of the human CHGA gene. Computer-assisted analysis of the sequence revealed the presence of a cAMP responsive element at positions -56 to -49, and Spl binding sites at positions -181 to -176 and -216 to -210. Two novel 9 bp motifs, located at position -462 to -454 and -91 to -83 of the CHGA promoter were identified in the regula tory regions of two other neuroendocrine genes encoding for tyrosine h ydroxylase and neuropeptide Y. In addition, in the first 1000 bp of th e untranslated 5' region, we found the presence of several putative DN A binding sites of bHLH molecules, a protein family regulating tissue specific differentiation. Transient transfection experiments of chlora mphenicol acetyltransferase (CAT) deletion constructs, showed the pres ence of an active promoter within the first 455 bp upstream from the s tart site. This region conferred tissue specific expression to a CA Tr eporter gene. In addition, the transcriptional activity of this fragme nt was modulated during the induction of differentiation of NE cells t reated by retinoic acid and/or dibutyryl-cAMP. These observations prov ide preliminary data regarding CHGA transcriptional regulation in NE c ells, and indicate that retinoic acid and cAMP activate distinct, appa rently competitive, transcriptional pathways during NE cell differenti ation. The molecular characterisation of the mechanisms regulating CHG A expression in tumour and normal neuroendocrine tissue could lead to the identification of novel molecules potentially relevant for future gene therapy of NE tumours.