Analysis of p16, p15, and p14 gene methylation and mRNA expression in 3 diffuse large BCL. The methyl-sensitive restriction enzymes used for REP are indicated (HpaII, KspI, NaeI, and EagI); digestion with the non–methyl-sensitive enzyme MspI serves as a negative control; undigested DNA (Control) serves as a positive control. For MSP, 2 primer sets specific for the methylated (M1 and M2) and 2 for the unmethylated (U1 and U2) bisulfite-modified p16gene are used. Expression of p16, p15, and p14 mRNA is analyzed by RT-PCR. The p16 gene is methylated in cases 33 and 36 but unmethylated in case 30. In case 36, p16 methylation is detected by REP but not by MSP (A and B). The p15 gene is methylated in cases 33 and 36 but not in case 30. In case 33, all assessed restriction sites are methylated. In case 36, all sites exceptEagI are methylated (C). The p14 gene is unmethylated in all 3 cases (D). GAPDH and p14 mRNA is present in all cases; p15 mRNA is detectable in case 30 but not in cases 33 and 36, whereas p16 mRNA is present in cases 30 and 36 but not in case 33 (E).