Relationship Of Differentiation And Histone Acetylation Of DADS-induced Human Leukemia Cell In Vitro And In Vivo

Objective:To investigate the differentiation and histone acetylation of human leukemia HL-60 cells induced by diallyl disulfide(DADS) in vitro and in vivo; to clarify the molecular mechanism of HL-60 cells differentiation inducted by DADS, and search the potential application value for DADS in the treatment of human myeloid leukemia.Methods:1. The differentiation and histone acetylation inducted by DADS on HL-60 cell in vitro: The differentiation of HL-60 cell was shown by the ability of NBT reduction activity and morphologic observation; The level of acetylated histone H3 n H4 and p21^WAF1 were tested by western blot.2. Establishment and evaluation of leukemia model: SCID mice were transplanted by ip injection with 5x10⁶ HL-60 cell respectively. Leukemic cell from peripheral blood , liver, spleen and marrow were detected by chromosome karyotype analysis and morphologic observation.3. The differentiation and histone acetylation inducted by DADS on human acute myeloid HL-60 cell transplantated in abdominal cavity of SCID mice in vivo: Collecting HL-60 cell with DADS-treated (A group) or without DADS-treated (B group) respectively, SCID mice were transplanted by ip injection with 5×10⁶ HL-60 cell and observed the condition of tumor formation. SCID mice of B group were divided into three groups randomly ( NS control, SB positive control and DADS-treated group). After putted to death, tumor volumes were measured with calipers and tumor inhibition ratio were calculated. HL-60 cell morphology changes were observed by HE stain and induction differentiation rates were calculated (count200 cells under elaeo-mirror), the cycle distribution of HL-60 cells in SCID mice was detected by flow cytometer. Toxicity was observed by measuring body weight; expression of acetylated histone ï¿¡^ H4 and p21WAF1 of tumour were tested by western blot.Results:1 .DADS had significant anti-proliferative effect on HL-60 cell in a concentration -dependent manner. The NBT reduction ability of HL-60 cell treated by DADS for 24h had no significant difference compared with control group (P>0.05) . But after 48h the apparently NBT reduction ablility treated by DADS could be seen and exhibited a time-dependent manner, expressing peak of induction differentiation at 1.25(agmL"' (PO.01) . SB can also significantly increase the NBT reduction ablility of HL-60 cells compared with control group, but no distinct difference between SB and 1.25 ugmL"'DADS.2. The expression of actylated histone H3^ H4 of HL-60 cell treated with DADS increased in a concentration-dependent manner and in a time-dependent manner. Compared with control, the expression of acetylated H3 - H4 and p21WAF1 increased 2.5 times^ 3.4 times or 3.8-4.1 times respectively after treated 1.25ugmL"'DADS for 48h; the expression of acetylated H3^ H4 increased 3.6 times or 4.3 times respectively after treated 2.5ugmL''DADS for 24h.3. HL-60 cell could perform leukemia transplantation tumor in the SCID mice via ip route. Tumor growed fastly and live time of SCID mice was 32³8d. Peripheral blood had no marked change at earlier period of disease, after three weeks, WBC start to increase and obviously increase at course of disease advanced stage, and we found human acute promyelocytic cell similar to HL-60 cell cultured in vitro. Chromosome caryotype analysis indicated that neoplastic cell > tumor cell in peripheral blood and HL-60 cell cultured in vitro had identical caryotype, all showed meta-centromere, the feature of mankind chromosome.4. HL-60 cells inoculated in right lower abdominal cavity of SCID mice were markedly inhibited by DADS, and tumor inhibition ratio added up to 66.2% at...