Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours V V Vax 1,5 , R Bibi 1 , S Diaz-Cano 2 , M Gueorguiev 1 , B Kola 1 , N Borboli 1 , B Bressac-de Paillerets 3 , G J Walker 4 , I I Dedov 5 , A B Grossman 1 and M Korbonits 1 1 Department of Endocrinology, St Bartholomew’s Hospital, London EC1A 7BE, UK 2 Department of Histopathology, St Bartholomew’s Hospital, London EC1A 7BE, UK 3 Service de Génétique, Institut Gustave Roussy, 94805 Villejuif Cedex, France 4 Human Genetics Laboratory, Queensland Institute of Medical Research, Brisbane 4029, Australia 5 Department of Neuroendocrinology, Russian Center of Endocrinology, Moscow 107036, Russia (Requests for offprints should be addressed to M Korbonits, Department of Endocrinology, Endocrine Oncology, St Bartholomew’s Hospital, 59 Bartholomew Close, Unit 1·1, London EC1A 7BE, UK; Email: m.korbonits@qmul.ac.uk) (V V Vax and R Bibi contributed equally to this work) Abstract Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16 INK4A (encoded by CDKN2A), in turn regulate CDK4. In particular, dys- regulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16 INK4 inhibition and are respon- sible for melanoma susceptibility in some kindreds. How- ever, ‘knock-in’ mice homozygous for the CDK4 R24C mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adeno- mas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tu- mours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restric- tion digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote pat- terns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory ‘hot-spot’ on the CDK4 gene, causing various endocrine tumours in CDK4 R24C/R24C mice, are not a major factor in sporadic pituitary, insulin -cell or Leydig cell tumorigenesis. Journal of Endocrinology (2003) 178, 301–310 Introduction Abnormalities in cell cycle stimulators and inhibitors are increasingly recognized as crucial factors in tumorigenesis. Examples of cell cycle stimulators include the cyclins and cyclin-dependent kinases (CDKs), whilst examples of inhibitors of the cell cycle include the retinoblastoma protein (Rb) and the CDK inhibitors (INK4 and Cip/Kip group; Fig. 1) (Pavletich 1999). In G1 phase, various mitogenic signals activate the cell cycle via the cyclin D/CDK4 complex which, in turn, can be inhibited by the CDK inhibitor p16 INK4a (p16, encoded by CDKN2A) via binding to CDK4. Mutation in the CDK4 gene was first described in melanoma cells (Wölfel et al. 1995): the mutation involved an arginine to cysteine change (ArgCys) at codon 24 (R24C) as a result of a single nucleotide change (CGT to TGT). This mutation is an activating mutation, as it results in growth advantages 301 Journal of Endocrinology (2003) 178, 301–310 0022–0795/03/0178–301  2003 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org