Frequent Methylation of p16^INK4A/p14^ARF Promoters in Tumorigenesis of Epstein-Barr Virus Transformed Lymphoblastoid Cell Lines

Abstract

Background: The data that the p16^INK4A gene is frequently inactivated in Burkitt lymphoma (BL) and that this event often accompanies the inactivation of p14^ARF in several tumours prompted us to examine the genetic and methylation status of both genes in BL and B-lymphoblastoid cell lines (LCLs). Materials and Methods: The existence of gene deletion, mutation and promoter methylation was investigated by single-strand conformational polymorphism, direct sequencing and methylation-specific PCR (MSP) analysis, respectively. Results: Sequencing of each exon of both tumour suppressor genes revealed p16^INK4A mutation only in 3 out of 11 BL, but 1 of them also affected the p14^ARF gene. MSP analysis of promoters showed p16^INK4A to be methylated and p14^ARF not to be methylated in each Epstein-Barr virus-positive BL cell line. Primary B-cells and de novo established LCLs had no genetic changes or methylated promoter of either gene. LCLs achieving the stage of immortalization usually showed methylation of both p16^INK4A and p14^ARF promoters. Conclusion: Our results suggest that, in contrast to p16^INK4A, inactivation of p14^ARF by either genetic change or promoter methylation has no importance in the development of BL cell lines, while its methylation has a central role in the immortalization of LCLs.