The murine gene p27Kip1 is haplo-insufficient for tumour suppression

Abstract

p27Kip is a candidate human tumour-suppressor protein, because it is able to inhibit cyclin-dependent kinases and block cell proliferation. 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. 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. Thus, p27Kip1 does not fulfil Knudson's 'two-mutation' criterion for a tumour-suppressor gene. Here we show that both p27 nullizygous and p27 heterozygous mice are predisposed to tumours in multiple tissues when challenged with gamma-irradiation or a chemical carcinogen. Therefore p27 is a multiple-tissue 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.

Figure 1

Tumour-induced mortality according to p27 genotype. a, γ-irradiation- induced tumour mortality (all tumour types) is increased in both p27^−/− and p27^+/−mice. b, ENU-induced tumour mortality is increased in p27^−/− mice. Non-tumour-related mortality was censored at the time of death (see Methods).

Figure 2

Cumulative occurrence of specific tumour types. The mean number of tumours (± s.e.m.) per mouse (tumour multiplicity) following treatment with either γ-irradiation or ENU is plotted for 27^+/+ (black bars), p27^+/− (grey bars), and p27^−/−(striped bars) mice. a, Intestinal adenomas; b, lung adenomas; and d, tumours of the female reproductive tract. (Asterisks indicate □ , 0X05 compared with wild- type by Wilcoxon rank sum test.) c, The occurrence of pituitary tumours is plotted as percentage incidence. (Asterisk indicates P< 0.05 compared with wild-type controls by chi-squared test.)

Figure 3

Analysis of tumours from p27^+/− mice. a, Representative Southern blots of ENU-induced intestinal and lung adenomas are shown with the genotype of the mouse listed above. The wild-type allele corresponds to the 2.5-kb band, and the knockout allele to the 2.1-kb band. No evidence of allelic loss is seen in p27^+/− tumours. b-d, Immunoblots of p27 protein expression in normal intestine, lung and pituitary, compared with p27 expression in tumours arising in these tissues following ENU treatment (intestine and lung) and γ-irradiation (pituitary). In c, d, the expression of p27 was quantified by densitometry followed by normalization to the amount of tubulin present in each sample (see Methods). Plotted are levels of protein in tumours relative to levels in normal, wild-type tissue. kD (kiloDalton), apparent relative molecular mass. e, Immunostaining of p27 protein in a p27^+/− pituitary tumour induced by g-irradiation (compared with p27−/− control). The p27 protein is localized to the nuclei of the tumour cells. f, Southern blot of spontaneous pituitary adenomas, with the corresponding genotype of the mouse listed above. g,Immunoblot of p27 protein expression in the same p27^+/− tumours as in f. The amount of p27 protein is similar to that present in normal p27^+/− pituitary tissue.