Abstractp27 Kip1 is a candidate human tumour-suppressor protein, because it is able to inhibit cyclindependent kinases and block cell proliferation [1][2][3][4][5] . Abnormally low levels of the p27 protein are frequently found in human carcinomas, and these low levels correlate directly with both histological aggressiveness and patient mortality [6][7][8][9][10] . However, it has not been possible to establish a causal link between p27 and tumour suppression, because only rare instances of homozygous inactivating mutations of the p27 gene have been found in human tumours [11][12][13][14] . Thus, p27 Kip1 does not fulfill Knudson's 'two-mutation' criterion for a tumour suppressor gene 15 . Here we show that both p27 nullizygous and p27 heterozygous mice are predisposed to tumours in multiple tissues when challenged with g-irradiation or a chemical carcinogen. Therefore p27 is a multipletissue tumour suppressor in mice. Molecular analyses of tumours in p27 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Hence, p27 is haplo-insufficient for tumour suppression. The assumption that null mutations in tumour-suppressor genes are recessive excludes those genes that exhibit haplo-insufficiency.Tumour-suppressor proteins affect several cellular pathways, such as those controlling proliferation, apoptosis, differentiation and genomic integrity. Consequently, the identification of a tumour-suppressor gene is usually established genetically rather than by any particular functional criterion. The common operational definition of a tumoursuppressor gene requires the demonstration of mutations of both copies of a candidate gene in tumours 15 . Genetic and physical mapping of bi-allelic tumour specific mutations allows localization of tumour-suppressor genes, and provides evidence for a causal link between mutations in those genes and tumorigenesis. This approach has successfully demonstrated the tumour-suppressor function of the retinoblastoma protein (Rb), p53, INK4a, and others [16][17][18] . However, we show here that this definition is too restrictive because it excludes tumour suppressors such as p27 Kip1 that exhibit haplo-insufficiency.We studied the tumour-suppressor function of p27 Kip1 by examining the susceptibility of p27-deficient mice to tumorigenesis induced by two different DNA-damaging agents, gCorrespondence and requests for materials should be addressed to M.L.F. (mfero@fhcrc.org). -ethyl-N-nitrosourea (ENU). p27 −/− mice showed decreased tumour-free survival following g-irradiation compared with p27 +/+ controls (Fig. 1a). p27 +/− mice showed an intermediate sensitivity (see below). Thymic lymphomas were the most frequent tumours in wild-type mice (incidence 0.07), but their frequency was not significantly increased in p27 −/− animals (incidence 0.17). The increased mortality of p27-null mice was mainly due to the development of pituitary tumours and intestinal adenomas (Fig. 2). A separate group of mice was injected with a single dose of ENU, and again p27-nu...

The p27 Kip1 protein is a cyclin-dependent kinase inhibitor that blocks cell division in response to antimitogenic cues. p27 expression is reduced in many human cancers, and p27 functions as a tumor suppressor that exhibits haploinsufficiency in mice. Despite the well characterized role of p27 as a cyclin-dependent kinase inhibitor, its mechanism of tumor suppression is unknown. We used Moloney murine leukemia virus to induce lymphomas in p27؉͞؉ and p27؊͞؊ mice and observed that lymphomagenesis was accelerated in the p27؊͞؊ animals. To identify candidate oncogenes that collaborate with p27 loss, we used a high-throughput strategy to sequence 277 viral insertion sites derived from two distinct sets of p27؊͞؊ lymphomas and determined their chromosomal location by comparison with the Celera and public (Ensembl) mouse genome databases. This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27؊͞؊ tumors. We found that Myc activations occurred more frequently in p27؊͞؊ lymphomas than in p27؉͞؉ tumors. We also characterized insertions within two novel loci: (i) the Jun dimerization protein 2 gene (Jundp2), and (ii) an X-linked locus termed Xpcl1. Each of the loci that we found to be frequently involved in p27؊͞؊ lymphomas represents a candidate oncogene collaborating with p27 loss. This study illustrates the power of high-throughput insertion site analysis in cancer gene discovery.

Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.