p16/INK4a and p15/INK4b Gene Methylation and Absence of p16/INK4a mRNA and Protein Expression in Burkitt's Lymphoma

Abstract

The fact that the p16/INK4a and p15/INK4b genes are frequently inactivated in human malignancies and that p16/INK4a null mice spontaneously develop B-cell lymphomas prompted us to examine the status of both genes in Burkitt's Lymphoma (BL). We found a low frequency of p16/INK4a and p15/INK4b deletions and mutations in BL cell lines and biopsies. However, p16/INK4a exon 1 was methylated in 17 out of 19 BL lines (89.5%) and in 8 out of 19 BL biopsies (42%) analyzed. p15/INK4b Exon 1 was also methylated, although at a lower frequency. p16/INK4a mRNA was readily detected in BL lines carrying unmethylated p16/INK4a, but not in those carrying methylated p16/INK4a. No p16/INK4a protein was detected in any of the BL lines and biopsies examined. In contrast, only one out of seven lymphoblastoid cell lines (LCLs) examined was methylated in p16/INK4a exon 1, and three out of the six LCLs with unmethylated p16/INK4a expressed detectable levels of p16/INK4a protein. Thus, the frequent p16/INK4a methylation in BL lines correlates with downregulation of p16/INK4a expression, suggesting that exon 1 methylation is responsible for silencing the p16/INK4a gene in BL.

Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

“…Although changes in phosphorylation of Rb could be mediated through any of the G1/S cyclins, CDKs and CKIs, p16 INK4A is known to be coordinately repressed in LCLs by EBNA-3A and EBNA-3C [41] , [42] , [44] , and indeed, we do see increases in p16 INK4A in LCLs. In Wp-R BL, however, p16 INK4A was undetectable irrespective of EBNA-3A expression, suggesting that p16 INK4a is likely to be irreversible epigenetic silenced as is often observed in Latency I BL [84] . Therefore, the changes in Rb phosphorylation and total Rb protein levels that occur in Wp-R BL following knockdown of EBNA-3A must be independent of p16 INK4a .…”

p16INK4a loss and sensitivity in KSHV associated primary effusion lymphoma

“…The incidence of p16INK4a loss found in lymphoma derived cell lines is frequently higher than in primary clinical samples. For example, p16INK4a was found to be methylated in 89.5% of Burkitts lymphoma derived cell lines but in only 42% of primary tumours (Klangby et al, 1998). By analogy, although defects in p16INK4a are found in a very high proportion of PEL-derived lines they may occur less frequently in primary material.…”

“…Homozygous deletion of p16INK4a occurs in acute lymphoblastic leukaemia (Hangaishi et al, 1996;Hebert et al, 1994), adult T cell leukaemia (Hatta et al, 1995;Yamada et al, 1997) and chronic myeloid leukaemia (Sill et al, 1995). CpG methylation, resulting in silencing of p16INK4a transcription, frequently occurs in highgrade non-Hodgkin's lymphoma (Pinyol et al, 1998;Villuendas et al, 1998), mucosa associated lymphoid tissue lymphoma (MALT) and Burkitt's lymphoma (Klangby et al, 1998).…”

Alterations of the P16 gene in uterine cervical carcinoma from Indian patients

“…Approximately 100 ng of genomic DNA was digested overnight with Hpa II and Msp I (Gibco BRL) separately using 30 units of enzyme per reaction ( 17 ) . Another mock digestion was performed per sample, where water was added instead of the enzymes.…”

“…The PCR product was then run on 2% agarose gel, stained with ethidium bromide, visualized under UV transilluminator and photographed. The gel was then transferred to nylon membrane (Gene Screen, NEN) and hybridized with random 32P‐labeled P16 cDNA probe for final confirmation of the PCR product ( 17 ) . All the samples showing P16 methylation were reproduced twice.…”