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3. CDK R24C constitutively activated Cdk4 expressing transgenic mice

A Cdk knockout strain and a strain carrying the Cdk4R24C mutation found in human familial melanoma was generated as pre-existing know-how and used in the study. A mutant Cdk4 (R24C) will be analysed for susceptibility of tumour formation. These animals will be treated with different carcinogens and will be crossed with mice carrying mutations in the Ras or p53 pathways. Spontaneous or induced tumours will be analysed at the molecular level and the role of Cdk4 activity in tumour formation will be clarified using specific Cdk4 mutants and inhibitors. No spontaneous tumour formation was detected in mice deficient in Cdk4 (Cdk4 neo/neo). In order to determine tumour susceptibility in Cdk4 R24C mice, we aged a group of 180 knockin mice carrying the Cdk4 R24C allele either in homozygosity or heterozygosity. Mice were sacrificed at any sign of disease and tissue samples were recovered for histology and nucleic acid/protein analysis. Mice homozygous for the R24C mutation (Cdk4R24C/R24C) are born at the expected mendilan ratio, are fertile and develop normally. However a proportion of mutant mice die before the first 8 months of life and 80% of the mice are dead by month 15. Necropsy analysis shows a significant number and a wide spectrum of tumours in these animals, including malignancies of endocrine origin (55% incidency); epithelial non-endocrine (24% incidency); mesenchymatous (67%); and hematopoietic malignancies (3% incidency).

Detailed pathology was performed on histological sections. Hemangiosarcomas develop in 56% of the animals and are the major cause of death in Cdk4R24C/R24C mice. They are frequently observed at multiple sites (spleen, liver, subcutaneous) or accompanied with metastasis (54% of the cases). This phenotype agrees with a significant number of reports showing Cdk4 amplification or over-expression in human sarcomas. Leydig cell tumors, on the other hand, are the most frequent type of malignancy in the Cdk4R24C/R24C males (62% incidency). Tumours of the pancreatic endocrine cells are also frequent (31%) and correspond mainly to b cell tumours, as detected by immunostaining with insulin-, PP-, or somatostatin antibodies. Tumours of the pituitary were observed in 22% of the Cdk4R24C/R24C mice. These tumours have also been observed in mice heterozygous for the retinoblastoma knockout allele, and in mice deficient in p18INK4c or p27Kip1. Interestingly, pituitary tumours in these models develop in the pars intermedia, whereas in the Cdk4 R24C mice are mainly of adenohypophysis (pars distalis) origin (80% of pituitary tumours). Pituitary tumours were also immunostained with antibodies against the pituitary hormones (ACTH, Prolactine, LH, FSH and GH). Most of pituitary tumours in Cdk4 R24C mice correspond to adenomas with a variety of patterns of positivity for hormones, although some carcinomas usually positive for prolactine were also found (about 15% of pituitary tumours).

Cdk4 R24C mice develop a wide spectrum of other tumours affecting a variety of tissues such as liver, thyroid, lung, liver, gut, salivary gland, Harderian gland, kidney, and hematopoietic malignancies. A group of 50 mice were sacrificed at 14-16 months without any external sign of disease. Detailed necropsy and pathologic analysis of these animals indicated the existence of several tumours with a distribution similar to that described above. Sarcomas and pituitary tumours are significantly reduced in this group in agreement with the fact that these malignancies are the major cause of death in Cdk4 R24C mice. Since the Cdk4 R24C mutation occurs in heterozygosity in human melanoma, we also aged 25 Cdk4R24C/+ mice. These animals also succumbed to the same type of malignancies with a similar distribution to that described above, although with a slightly increased latency.

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