Consistent inactivation of p19(Arf) but not p15(Ink4b) in murine myeloid cells transformed in vivo by deregulated c-Myc.

Cyclin-dependent kinase inhibitors p16(INK4a) and p15(INK4b), encoded by the CDKN2A and B loci, play an important role in negative regulation of the cell cycle. Furthermore, p19(ARF) also encoded by the CDKN2A locus, has been shown to regulate positively the p53 pathway leading to growth arrest and apoptosis. All three genes have been inactivated in human tumors. In myeloid cells, p15(INK4b) mRNA is upregulated during cytokine-induced differentiation and/or growth arrest, and hypermethylation of the p15(INK4b) gene promoter region is a common event in acute myeloid leukemia. In the present study, we examined murine monocyte/macrophage tumors with deregulated c-myc for evidence of Ink4 gene inactivation. p15(Ink4b) mRNA and protein were detected in the majority of leukemias, and p16(Ink4a) mRNA and protein were highly expressed in two of them. pRb was in a hypophosphorylated state in most of the neoplasms indicating that the Cdk inhibitors that were expressed in the cells were functional. The observed expression of p15(Ink4b) is inconsistent with their proliferation state, although it might be expected to be expressed owing to the maturity of the cells. These data suggest, therefore, that deregulated c-Myc bypasses the pRb restriction point and cell cycle arrest in these tumors. An examination of p19(Arf) exons revealed deletions of the gene in up to 94% of the tumors. Since this gene shares exon 2 with p16(Ink4a), it is often difficult to determine which gene is the relevant tumor suppressor. However, the loss of only the p19(Arf)-specific exon 1 beta was observed in a tumor that had normal p16(Ink4a) protein expression. In addition, the p19(Arf)-specific exon was deleted in another tumor that expressed a functional chimeric protein, p15Ex1-p16Ex2-3; it was demonstrated here that this fusion protein is capable of inducing G1 arrest. These data overall supports the hypothesis that the critical inactivation event in these hematopoietic neoplasms is elimination of p19(Arf), and not Ink4 function.