|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 2004 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2003 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Bone morphogenetic proteins(BMPs) are the members of TGF-β superfamily, and have been shown to be involved in neural crest cell differentiation. However, it is still unclear whether BMPs could promote neuronal differentiation or survival in human neuroblastoma cells. We heve found that BMP2 has a potent neurotrophic function in certain neuroblastoma cell lines. BMP receptors (I and II) were expressed in 5 neuroblastoma cell lines that we examined including SH-SY5Y, RTBM1, LA-N-5, SK-N-AS and IMR32. The treatment of neuroblastoma cell lines with 1 nM BMP2 induced a marked phosphorylation of Smad1/5 within 30 min. Among them, RTBM1 and LA-N-5 displayed a growth arrest and a subsequent neuronal differentiation in response to BMP2. In contrast, BMP2 induced a growth arrest in SK-N-AS cells, but did not promote a neuronal differentiation. In RTBM1 cells, BMP2-induced neuronal differentiation was associated with a significant decrease in the expression level of DAN, which is mapped to chromo
some 1p35 and its gene product acts as an antagonist of BMP. Immunoblot analysis revealed that BMP2 treatment caused a down-regulation of both p53 and p73. On the other hand, cyclin-dependent protein kinase inhibitor p27^<KIP1> was markedly increased in response to BMP2. Considering that BMP signal suppresses the expression of p73 which has an ability to regulate the expression levels of DAN, it is likely that these could exist a feedback mechanism by which BMP is regulated.
Although p53 is rarely mutated in neuroblastoma cells, we found a short form of p53 which is largely expressed in cytoplasm of SK-N-AS cells. The antibody that can recognize the extreme COOH-terminal region of p53 didn't detect p53 in SK-N-AS cells. These results suggest that p53 in SK-N-AS cells lacks a COOH-terminal region including nuclear localization signal(NLS). SH-SY5Y cells carrying a wild-type p53 responded to the cisplatin and underwent apoptosis, which was associated with the induction of p21^<WAF1> and bax. On the other hand, cispatin treatment induced G2/M arrest in SK-N-AS cells. Neither the stabilization of p53 nor induction of bax were detected during the cisplatin-mediated G2/M arrest. However, the expression of p21^<WAF1> were induced in response to cisplatin. Therefore, it is possible that cisplatin-resistant phenotype of SK-N-AS cells is attributed to the aberrant form of p53. Less