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

Abstract

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 E 2 F1 and E 2 F2 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 G 1 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/G 1 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 G 1 /S arrest, neuroblastoma cell lines proliferate in the presence of elevated levels of p16. Int. J. Cancer 80:145-154, 1999. Wiley-Liss, Inc.Cell growth is a tightly regulated process that involves the interaction of many regulatory proteins working in concert with each other to direct a timely and orderly progression through the cell cycle (for review: Scherr, 1996). Each phase of the cell cycle is regulated by the interactions of specific cyclins with their respective cyclin-dependent kinases (CDKs). For the early G 1 -S phase transition, it involves the interactions of the cyclin D (D1, D2 and D3) proteins with CDK4 and -6, while the late G 1 -S phase is regulated through the interaction of cyclin E with CDK2. The activated cyclin/CDK complex results in phosphorylation of the retinoblastoma (Rb) protein. In G 0 and early G 1 , Rb exists predominantly in an under-phosphorylated form that acts as a negative regulator of the cell cycle by sequestering transcription factors such as E 2 F (Kouzarides, 1995). In the hyperphosphorylated state, Rb releases these factors, enabling them to activate transcription and drive the cell into S phase. The CDKs are subject to regulation by a large family of homologous proteins known collectively as cyclin-dependent kinase inhibitors (CDKIs), which inhibit CDK activity through protein-protein interactions. The p16 CDKI appears to be tightly regulated...

The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia and drug-resistant primary leukemia and cancer cell lines

“…In this study, AN-9 was effective in inhibiting the growth of HL60 myeloid and neuroblastoma clones that harbored multiple genetic alterations 32 and displayed a multidrug-resistant phenotype. [20][21][22] Moreover, AN-9 was equally effective in arresting proliferation of all the primary leukemia cells tested, including a doxorubicin-resistant T-ALL, a clinically refractory relapsed AML, and a relapsed infant ALL characterized by an 11q23 rearrangement and a very poor prognosis.…”

Pharmacologic inhibition of cyclin-dependent kinase 4/6 activity arrests proliferation in myoblasts and rhabdomyosarcoma-derived cells

“…Both Rb and Cdk4 are detectable in 13 Ewing's sarcoma cell lines examined (55). Cdk4 mRNA is detectable in 19 neuroblastoma cell lines and in all six primary tumors tested (56). Targeted therapeutic strategies depend on the expression of the relevant protein, but the expression of the protein itself may not necessarily correlate with effects of its inhibition.…”

Lack of evidence for mutations or deletions in the CDKN2A/p16 and CDKN2B/p15 genes of Brazilian neuroblastoma patients

“…Our results suggest that deletion is not an important mechanism of CDKN2A/ p16 gene inactivation in neuroblastomas. Few mutations in the CDKN2A/p16 gene have been described in neuroblastomas, either in neoplasms or in cell lines, and most represent polymorphisms or substitutions that do not affect the protein structure (10)(11)(12)15). The only significant alteration was a nonsense mutation in codon 52 described by Takita et al (3).…”