Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibito rs of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RE f unction, and set up the RB/p16 tumor suppressive pathway, which is abrogate d frequently in human neoplasms, either through inactivation of the RE or p 16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncopro teins. In hematological malignancies, deletion of p16/p15 locus has been sh own to be highly specific to lymphoid malignancies, and more particularly t o T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subset s of ALL, deletions of p16 and p15 are relatively rare events. To investiga te whether these genes are inactivated by methylation of the 5' CpG islands , we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemi a (AML) patients by Southern blot, polymerase chain reaction (PCR) and West ern blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell l ines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 met hylation were found to have lost p16 protein expression. The p15 gene was m ethylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines wi thout homozygous deletion of p15, and 15 (52%) of 29 AML patients. These re sults revealed the frequent methylation of p16 and p15 genes in B-ALL and A ML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RE protein, we found no expressio n of RE in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was fo und in all the cell lines with expression of RE. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common ge netic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia. (C) 1999 Elsevier Science Lt d. All rights reserved.