title: Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.
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, dysregulation 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 responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours 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 restriction 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 patterns. 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 beta-cell or Leydig cell tumorigenesis.
authors.affiliation: Department of Endocrinology, St Bartholomew's Hospital, London EC1A 7BE, UK. m.korbonits@qmul.ac.uk
authors.forename: V V
authors.initials: VV
authors.lastname: Vax
authors.forename: R
authors.initials: R
authors.lastname: Bibi
authors.forename: S
authors.initials: S
authors.lastname: Diaz-Cano
authors.forename: M
authors.initials: M
authors.lastname: Gueorguiev
authors.forename: B
authors.initials: B
authors.lastname: Kola
authors.forename: N
authors.initials: N
authors.lastname: Borboli
authors.forename: B
authors.initials: B
authors.lastname: Bressac-de Paillerets
authors.forename: G J
authors.initials: GJ
authors.lastname: Walker
authors.forename: I I
authors.initials: II
authors.lastname: Dedov
authors.forename: A B
authors.initials: AB
authors.lastname: Grossman
authors.forename: M
authors.initials: M
authors.lastname: Korbonits
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015153:Blotting, Western / Q000379:methods; D016022:Case-Control Studies; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007340:Insulinoma / Q000737:chemistry / Q000378:metabolism; D007984:Leydig Cell Tumor / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins; D013736:Testicular Neoplasms / Q000378:metabolism
chemical_list: D011518:Proto-Oncogene Proteins; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases