Fig. 3

FHIT methylation is equally represented in both germinal and tumor cells and is independent from HTLV-I proviral load. a HTLV-I proviral load does not dictate FHIT gene methylation. Proviral loads were determined for acute and chronic ATL patients’ samples (n = 131 M and n = 32 UM) and compared to the epigenetic status of the FHIT gene as determined by MSPCR. p-values were obtained by two-tailed, unequal variance T-tests. b Effective sorting of ATL and HD PBMCs from patients into CD25- and CD25+ fractions by FACS analysis. 5 HDs and 5 ATL patients were sorted based on CD25 surface expression. c HTLV-I proviral load confirms effective CD25 sorting of HD and ATL patients’ samples. Proviral load was determined by quantitative PCR for gDNA isolated from CD25- and CD25+ samples. HTLV-I positive proviral loads were found in CD25+ fractions, compared to CD25-. Non-HTLV-I infected, HDs, served as negative controls. d FHIT CpG island methylation occurs in both tumorigenic (CD25+) and non-tumorigenic (CD25-) leukemic T-cells from ATL patients’ samples. Five non-HTLV-I infected HDs and five ATL patients’ samples (3 acute, 1 chronic, and 1 smoldering) were sorted into CD25- and CD25+ fractions. Individual MSPCR bands amplifying the unmethylated (U) or methylated (M) product are demonstrated. Methylation of CpG islands in the oncogene, Rb, was used as a control to demonstrate the specificity of the FHIT methylated PCR product. e Representation of global BSG methylation pattern of two HDs (HD-1 through HD-2), three acute ATL patients (ATL-1 through ATL-3) and one smoldering ATL (ATL-4) patient’s sample. Patient cells were sorted into CD25- and CD25+ fractions, and gDNA was analyzed for FHIT and CDKN1A gene methylation. Unmethylated and methylated alleles are noted by white and black boxes, respectively. Circle graphs representing the percentage of methylated CpG islands in the FHIT and CDKN1A genes are shown