Article
Promoter methylation of the p16INK4a, p14ARF, p18INK4c and RB genes in human pituitary adenomas
Methylierung der Promotorregion der Zellzyklus inhibierenden Tumorsuppressorgene p16INK4a, p14ARF, p18INK4c und des Retinoblastomgens
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Published: | May 8, 2006 |
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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 p16INK4a gene was shown to be frequently altered in pituitary adenomas. p18INK4c knock-out and combined p18INK4c /p27 or p18INK4c /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 p16INK4a, p18INK4c 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 p16INK4a, p18INK4c and Rb loci. Mutations of the p18INK4c and p16 genes were screened by direct sequencing. Analysis for expression of the p16INK4a, p14ARF, p18INK4c 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 p16INK4a, p14ARF, p18INK4c and RB was ascertained by bisulphate modification and followed methylation-specific PCR.
Results: p16IN4a methylation was found in 6 (16%) adenomas and p14ARF methylation in 5 (13%) adenomas. Surprisingly 15 (40%) adenomas showed p18INK4c 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. p18INK4c promoter methylation appeared to be increased not significantly with life time. Nearly all p18INK4c methylated tumors showed loss of p18INK4c protein expression. LOH at the p18 gene locus was detected in 25% of pituitary adenomas, whereas the RB and p16INK4a loci were altered only in 10%. By sequence analysis, no mutations were detected. Absence of immunohistochemical staining correlated with LOH at the p18INK4c locus.
Conclusions: The p18INK4c locus is frequently altered in pituitary adenomas (24%). Methylation of the p18INK4c promoter was very frequently observed in 40% of all adenomas, whereas methylation of p16INK4a and RB promoters occurred in fewer than 15%.