gms | German Medical Science

Objective: The cyclin-dependent kinase inhibitors represent tumour suppressor genes and are frequently altered in a variety of tumor types including brain tumours. Among them the p16^INK4a gene was shown to be frequently altered in pituitary adenomas. p18^INK4c knock-out and combined p18^INK4c /p27 or p18^INK4c /p53 knock-out-mice develop pituitary adenomas at high frequencies. The purpose of our study was to evaluate the frequency of genomic alterations, promoter methylation status, and expression of p16^INK4a, p18^INK4c and Rb genes in pituitary adenomas.

Methods: Genomic DNA from blood and tumour was extracted from 38 pituitary adenomas. LOH-analysis was performed using PCR-based microsatellite marker for the p16^INK4a, p18^INK4c and Rb loci. Mutations of the p18^INK4c and p16 genes were screened by direct sequencing. Analysis for expression of the p16^INK4a, p14^ARF, p18^INK4c and RB proteins was investigated by immunohistochemistry. Detection of apoptosis was performed using the TUNEL assay. The methylation status of the CpG islands of the genes p16^INK4a, p14^ARF, p18^INK4c and RB was ascertained by bisulphate modification and followed methylation-specific PCR.

Results: p16^IN4a methylation was found in 6 (16%) adenomas and p14^ARF methylation in 5 (13%) adenomas. Surprisingly 15 (40%) adenomas showed p18^INK4c methylation, whilst none of the adenoma demonstrated a methylated RB gene promoter. The methylation status was not dependent on the hormone production of the tumors. p18^INK4c promoter methylation appeared to be increased not significantly with life time. Nearly all p18^INK4c methylated tumors showed loss of p18^INK4c protein expression. LOH at the p18 gene locus was detected in 25% of pituitary adenomas, whereas the RB and p16^INK4a loci were altered only in 10%. By sequence analysis, no mutations were detected. Absence of immunohistochemical staining correlated with LOH at the p18^INK4c locus.

Conclusions: The p18^INK4c locus is frequently altered in pituitary adenomas (24%). Methylation of the p18^INK4c promoter was very frequently observed in 40% of all adenomas, whereas methylation of p16^INK4a and RB promoters occurred in fewer than 15%.