Inactivation of the cyclin-dependent kinase inhibitor p15 INK4b by deletion and de novo methylation with independence of p16 INK4a alterations in murine primary T-cell lymphomas Marcos Malumbres 1, *, Ignacio Pe´rez de Castro 2, *, Javier Santos 2 , Ba´rbara Mele´ndez 2 , Ramon Mangues 1 , Manuel Serrano 3 , Angel Pellicer 1 and Jose´ Ferna´ndez-Piqueras 2 1 Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA; 2 Laboratorio de Gene ´tica Molecular Humana, Departamento de Biologı´a, Facultad de Ciencias, Universidad Auto ´noma de Madrid, 28049 Madrid, Espan ˜a; and 3 Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724, USA A wide panel of murine induced T-cell lymphomas have been analysed for p16 INK4a or p15 INK4b alterations. Only one g-radiation-induced lymphoma showed p16 INK4a homozygous deletion and no other intragenic mutations were found in these INK4 genes. However, de novo methylation of the 5’ CpG islands of the murine p15 INK4b and p16 INK4a genes was found to be highly frequent. While p16 INK4a hypermethylation was found in 36% of the neutron-radiation-induced lymphomas and 15% of the g- radiation-induced lymphomas, de novo methylation of p15 INK4b occurs in 88% and 42% of these tumors respectively, correlating with deficient expression of the corresponding mRNA and allelic losses in the p15 INK4b and p16 INK4a chromosome location. These data represent, to our knowledge, the first report on the significant involvement of hypermethylation of these INK4 genes in murine primary tumors. Moreover, they show the importance of allelic losses and CpG island methylation of p15 INK4b gene inactivation and support a tumor suppressor role for p15 INK4b in T-cell lymphomas independent of p16 INK4a . Keywords: p15 INK4b ; p16 INK4a ; CDK inhibitors; T-cell lymphomas; DNA methylation Introduction The central role of cyclin/cyclin-dependent-kinase (CDK) complexes and their respective inhibitors in the modulation of cell cycle progression is widely recognized (Hunter and Pines, 1994; Gran˜a and Reddy, 1995). D-type cyclins (D1, D2, D3) are growth-factor- regulated genes whose products associate with CDK4 or 6 which phosphorylate the retinoblastoma protein (pRb). During early G 1 , the hypophosphorylated forms of pRb bind and inactive the E2F (E2F-1, -2, -3, and DP-1) transcription factors. Phosphorylation of pRb by the cyclin-CDK complexes leads to release of the transcription factors E2F in an active form. Active E2F binds to the promoter of genes that encode enzymes essential for S-phase and activates their transcription. Thus, complexes of the D-type cyclins with CDK4 and CDK6 are principal contributors to cell cycle initiation. Specific inhibitors of the cyclin/CDK complexes, the INK4 proteins, therefore act as negative regulators of the mammalian cell cycle (Hunter and Pines, 1994; Sherr and Roberts, 1995). Four members of the INK4 family have been identified, including p16 INK4a (MTS1, CDK4I, CDKN2; Serrano et al., 1993), p15 INK4b (MTS2; Hannon and Beach, 1994), p18 INK4c and p19 INK4d (Guan et al., 1994; Chan et al., 1995). The cyclin D/CDK complex inhibition prevents the phosphorylation of pRb, which thus remains active and blocks the release of the active transcription factors and the activation of G 1 /S-phase genes. The products of the INK4 genes have been shown to arrest cell cycle progression in the G 1 phase (Serrano et al., 1993; Guan et al., 1994; Hirai et al., 1995). Lack of the INK4 proteins may therefore produce inactivation of the retinoblastoma protein by the active cyclin/CDK complexes, inducing progression from G 1 to S. Support for this mechanistic model comes from the observation that cancer cells deficient in p16 INK4a have wild-type retinoblastoma protein and vice versa (Okamoto et al., 1994; Tam et al., 1994). Recently, Khleif et al. (1996) have reported that increase in the level of free E2F-1, produced by a functional loss of pRb and disruption of the pRb-E2F complex, leads to an inhibition of cyclin D1- dependent kinase activity and induces the expression of p16 INK4a , resulting in a regulatory feedback control of CDK activity. The molecular complexity of the p16 INK4a gene has recently been increased by the finding of alternative reading frames, giving rise to two partially dierent transcripts, p16 INK4a and p19 ARF , which are both functionally involved in the inhibition of cell cycle progression (Quelle et al., 1995a; see review by Larsen, 1996). Several studies have proposed a tumor suppressor role for p16 INK4a (Kamb et al., 1994; Serrano et al., 1995, 1996). Human p16 INK4a and p15 INK4b genes are tandemly linked within 30 kilobases (kb) on human chromosome 9p21, a region of common cytogenetic abnormalities in tumor cells (Okamoto et al., 1994; Kamb et al., 1994; Nobori et al., 1994). Homozygous deletions in the 9p21 region is the most common type of inactivation of p16 INK4a and in 80% of the cases involve both p16 INK4a and p15 INK4b genes (Stone et al., 1995). p16 INK4a point mutations and small deletions are also frequent in some tumors (Pollock et al., 1996), but Correspondence: A Pellicer *These authors contributed equally to this work Received 9 September 1996; revised 15 November 1996; accepted 18 November 1996 Oncogene (1997) 14, 1361 – 1370  1997 Stockton Press All rights reserved 0950 – 9232/97 $12.00