Figure 1
Surface CD44 expression and effects of ULMW-HA on MLL+ALL cell lines. (a) Surface CD44 expression. MLL+ALL cell lines were stained with normal IgG (open cytograms) or anti-CD44 mAb (filled cytograms). Cytograms of CD44-high KOPN1 and CD44-very low KOCL58 cell lines were representatively shown. (b) % Inhibition of thymidine uptake. Cell lines were divided into CD44-high (>95% positive; n=6) and CD44-very low (<5% positive; n=2) groups, and compared % inhibition of thymidine uptake at day 4 after ULMW-HA stimulation (2.5 mg/ml). The CD44-high group cell lines showed significantly higher inhibition (P<0.005 by Student’s t-test). (c) Changes in % inhibition of thymidine uptake. The MLL+ALL cell line expressing CD44 at high levels, KOPB26, was cultured (0.5 × 105 per well) in triplicate in the presence or absence of various concentrations (1.0, 2.5 and 5.0 mg/ml) of ULMW-HA for 4 days (left panel), and at a concentration of 2.5 mg/ml of ULMW-HA for 2, 3 and 4 days (right panel), and 5 h-thymidine uptake was examined. The % inhibition in the presence of HA was calculated as {1-[(cpm of HA+)/(cpm of HA-)]} × 100. The results are representative from three separate experiments and are shown as mean±S.E. The significance of differences between HA- and HA+ culture conditions at day 4 (left panel) and at days 2, 3 and 4 (right panel) was analyzed by Student’s t-test (*P<0.005, **P<0.001). (d) Establishment of the CD44 knockdown (KD) cell line and its thymidine uptake after ULMW-HA stimulation. The established cell line KOPB26CD44KD by genome editing (open cytogram) showed surface CD44 expression at very low levels (<10%) when compared with the parent cell line KOPB26 (filled cytogram) by flow cytometric analysis (left panel). The parent KOPB26 and KOPB26CD44KD cell lines were cultured in triplicate in the presence or absence of ULMW-HA (2.5 mg/ml) for 4 days and thymidine uptake was examined (right panel). The results are representative from three separate experiments and are shown as % thymidine uptake (mean±S.E.) when thymidine uptake of the parent cell line in the absence of HA is regarded as 100%. The differences were analyzed by Student’s t-test. NS, not significant. (e) Surface CD44 expression in B-precursor and T-cell leukemia cell lines. Leukemia cell lines were stained with normal IgG (open cytograms) or anti-CD44 mAb (filled cytograms). Cytograms of surface CD44 expression in two B-precursor (KOPN41, KOPT54) and two T-cell (KOPT5, KOPTK1) leukemia cell lines were representatively shown. (f) Effects of ULMW-HA on thymidine uptakes of B-precursor and T-cell leukemia cell lines. Ten B-precursor ALL cell lines (four Philadelphia chromosome-positive, four TCF3-PBX1 positive and two ETV6-RUNX1) and five T-ALL cell lines were cultured (0.5 × 105 per well) in the presence or absence of ULMW-HA (2.5 mg/ml) for 4 days, and the % inhibition in the presence of ULMW-HA was calculated. The X and Y axes represent fold MFI of surface CD44 expression and % inhibition of thymidine uptake, respectively
