Frequent deregulation of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma.

Alterations of the p16 gene in neuroblastoma are very rare. Pronounced expression of p16 at both the transcript and protein levels, however, was observed in 7 of 19 (39%) neuroblastoma cell lines and 2 of 6 (33%) primary neuroblastoma samples. As p16 expression is tightly controlled in a feedback loop with Rb, we investigated the possibility that changes in p16 expression were reflective of alterations of the downstream components in the G1 regulatory pathway. Two cell lines and one primary sample highly expressing p16 were shown to harbor CDK4 amplification. The cyclin D2 gene was infrequently expressed in neuroblastoma cell lines and did not correlate with p16 expression. Slight variations in the expression of CDK6, cyclins D1, D3 and E; and E2F1 and E2F2 among the cell lines were observed, without apparent correlation with p16 status. No mutations to the p16-binding site of CDK4 and CDK6 nor any mutations to the coding region of p16 itself were identified in neuroblastoma cell lines. Despite frequent N-myc amplification in these cell lines, no relationship with this gene was observed either. All cell lines contained Rb protein with varying degrees of phosphorylation, which bears no correlation with p16 expression. Overall, alterations of the G1 pathway in neuroblastoma included relatively frequent p16 expression and infrequent CDK4 amplification and cyclin D2 expression. Despite a reported feedback relationship between p16 expression and Rb/G1 deregulation, p16 expression in neuroblastoma cell lines is independent of Rb gene and phosphorylation status and, in contrast to other cell lines where expression of p16 leads to G1/S arrest, neuroblastoma cell lines proliferate in the presence of elevated levels of p16.