Expression and role of Brn3 family of transcription factors in human neuroblastomas
Brn-3a and Brn-3b are members of the POU-domain containing transcription factors. Both are found in specific neurons of the CNS and PNS. In mouse and human neuroblastoma cell line models, high levels of Bm-3a are found in differentiating neurons whereas high levels of Bm-3b are present in actively proliferating cells. The reciprocal expression of these two transcription factors in neuroblastoma cells is paralleled by their differential regulation on gene promoters. While Bm-3a activates several gene promoters whose products are associated with neuronal differentiation, Bm-3b has repressive effects on the activity of most of these promoters. Stable cell line systems were developed to test the effects of these factors in human neuroblastoma cell line, IMR-32, where the levels of Brn-3a and Bm-3b have been manipulated. While results from IMR-32 constitutively over-expressing Bm-3a remain to be validated, manipulation of Bm-3b showed profound effects on the growth characteristics of these cells. Thus, over-expression of Bm-3b caused increased growth rate, saturation density, proliferation, anchorage independent colonies, and enhanced migration compared to the mock transfected cells. Furthermore, cells with elevated levels of Brn-3b exhibited a stronger ability to form tumours in xenograft mouse models compared to controls. IMR-32 cells with reduced Bm-3b levels exhibited a significant decrease in growth and proliferation parameters. Moreover, Bm-3b over-expressing cells failed to respond to differentiation stimulus such as all-trans-retinoic-add and continued to proliferate in its presence, while empty vector control and cells with reduced Bm-3b levels ceased to proliferate when grown in the presence of this differentiation stimulus. The levels of cell cycle regulator protein, cyclin D1 were found to be elevated in Brn-3b over-expressing IMR-32 cells, with a concomitant decrease in cells with reduced levels of this protein. Using transactivation studies, we have shown for the first time that Bm-3b is able to activate the cyclin D1 gene promoter (5'-A TTT CTA TGA-3'), and this activation is dependent on a specific octamer sequence located between positions -240 and -231 from the transcriptional start site in this promoter. Taken together these studies are of fundamental importance in devising targeted therapeutic strategies for aggressive stages of human neuroblastomas.