1. ¹Angiogenesis Laboratory, Ospedale Pediatrico Bambino Gesù, Roma, Italy
2. ²Istituto di Biologia e Patologia Molecolari CNR, Sapienza Università di Roma, Roma, Italy

Correspondence: Dr R Rota, Angiogenesis Laboratory, Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio 4, Roma 00165, Italy. E-mail: rota@opbg.net; Dr S Nasi, Istituto di Biologia e Patologia Molecolari CNR, Sapienza Università di Roma, P. le A. Moro 5, Roma 00185, Italy. E-mail: sergio.nasi@uniroma1.it

Received 31 July 2008; Revised 20 January 2009; Accepted 21 February 2009; Published online 30 March 2009.

Abstract

Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27^Kip1 and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.