MSP and bisulfite-DGGE analyses of DAP-kinase and p15^INK4B CpG island methylation in leukemic cell lines and AML samples.

(A) MSP analysis of DAP-kinase. The 3 cell lines (HL-60, MOLT-4, and Raji), as well as AML samples 5-4, 5-8, 4-11, and 5-56, were methylated in the DAP-kinase CpG island. (B) Bisulfite-DGGE analysis of DAP-kinase. Methylated and unmethylated alleles of DAP-kinase were collectively amplified from bisulfite-treated DNA with primers [40 GC]-AGAGTTAAAGTAGGGGATTTTGTTTTT (forward) and [5 GC]-AAATAAAAAACTCAAATCCCTCCCAAA (reverse), and subsequently resolved in a 10% denaturant/6% polyacrylamide-70% denaturant/12% polyacrylamide double-gradient gel, run at 58°C and 160V for 4.5 hours, and stained with ethidium bromide. HL-60 and MOLT-4 both contain methylated as well as unmethylated DAP-kinase alleles, whereas Raji only contains methylated alleles. None of the 10 AML samples were found to be methylated by bisulfite-DGGE. The faint bands in samples 4-2 and 5-29 were not reproducible and were thus interpreted as PCR artifacts. (C) Bisulfite-DGGE analysis ofp15^INK4B. HL-60 is unmethylated, MOLT-4 is fully methylated and Raji has both methylated and unmethylated p15^INK4B alleles. All AML samples are extensively methylated in thep15^INK4B CpG island with only a small fraction of unmethylated alleles present.