Southern blotting of the p15INK4B gene. (A) Results of cell lines are shown. The p15INK4B gene was intensively methylated in ML1 and partially methylated in Raji, but not in HL60. (B) Results from patients with various types of MDS are shown. Lane 1, OL (patient no. 4); lane 2, OL (patient no. 29); lane 3, RAEB (patient no. 29); lane 4, RA (patient no. 16); lane 5, RAEB (patient no. 2); lane 6, RA (patient no. 2); lane 7, RAEB (patient no. 21); lane 8, RAEB (patient no. 8); lane 9, OL (patient no. 23). Lanes 10 and 11 show the results of control B lymphocytes digested with both HindIII and Eco52I and with HindIII alone, respectively. Although lanes 10 and 11 contained the same amount of DNA extracted from the same samples, the 2.2-kb band was weaker than the 2.8-kb band. Lanes 2 and 3 and lanes 5 and 6 show the results at different stages in the same patients, respectively. Lanes 2, 4, 5, 6, and 9 retained the 2.8-kb band, implying methylation of the p15INK4B gene. (C) DNA samples from healthy volunteers (lanes 1 through 3) and patients with RA or RARS (lane 4, RARS patient no. 6; lane 5, RA patient no. 10; lane 6, RARS patient no. 20) were almost completely digested by the methylation-sensitive restriction enzyme Eco52I, implying the unmethylated status of their p15INK4B genes. We did not detect altered size of a cross-hybridizing fragment of the p15INK4B gene reported previously.12,16
