Methylation and expression of p16INK4A in multiple myeloma tumors and cell lines. Purification of MM tumor cells, growth of cell lines (EJM, OPM-2, PE-1, Delta-47, ANBL6, KMS12-BM, JIM-3, H1112, KHM-11, KMS-11, H929, 8226, Karpas-620, LP1, SKMM-2, JJN-3), and preparation of RNA and cDNA have been described previously.2 The methylation status of p16 was determined as described elsewhere.3 Quantitative real-time PCR was done using USF2 as an internal control.4 The p16 primers (reference sequence L27211) were from exon 1 (nt 163-183) and exon 2 (nt 218-205), and the p16 probe was 5′VIC-CATGACCTGGATCGGCCTCGGA-TAMRA-3′. The data are calculated as ΔCt = Ct.p16-Ct.USF2, and then normalized as ΔΔCt = sample.ΔCt − EJM.ΔCt (∼ 0-1 in most experiments). The results are shown as −ΔΔCt and cover a more than 1000-fold range, with all values less than −10 shown as −10. MM indicates all MM tumors; MM-U and MM-M, MM tumors with unmethylated and methylated p16, respectively; and HMCL, MM cell lines. The boxed symbols indicate the 4 unmethylated HMCLs. The mean is shown as a horizontal line. The results for the MM and MM-M groups differ by a t test (P = .04).
