Objectives: To investigate the in vitro effect of hexamethylene bisacetamine (HMBA) on the proliferation, cell cycle regulation, and apoptosis of human leukemia cells and to study the relevant mechanisms.
Methods: K562 and U937 human leukemia cells were cultured and then suspensions thereof without HMBA or with HMBA of different concentrations (1, 2, and 4 mmol/L) were made. The optical absorbance value at 595 nm of these suspensions were tested every 24 hours. The suspensions were mixed with 4% trypan blue dye solution. Live and dead cells were observed with microscope and the live cell rate was calculated. Double staining of cellular cyclin A protein/DNA was used to detect the expression levels of cyclin A protein in cytoplasm. Fluorescein isothiocyanate (FITC)-labeled annexin V/Propidiumiodide) was used to detect the apototic cells fluorescence-activated cell sorter. Gel electrophoresis was conducted to detect DNA contents.
Result: After exposure to HMBA for 6 days, the optical absorbance values at 595nm of K562 leukemia cells of the control group and groups with 1, 2, and 4mmol/L HMBA were 1.03, 0.81, 0.58, and 0.36 respectively. After treatment of HMBA, the percentages of cyclin A positive K562 leukemia cells in control group and groups with 1, 2, and 4 mmol/L HMBA were 34.4% +/- 5.2%, 16.1% +/- 2.5%, 9.9% +/- 1.7%, and 7.6% +/- 2.0% respectively (P < 0.01). No drug-related apoptosis was found in both cell lines.
Conclusion: HMBA inhibits cell proliferation and reduces S-phase-fraction in both cell lines dose-dependently through down-regulating the expression of cyclin A. Apoptosis is not a consequence of the proceeding mitosis arrest and is induced p53-dependently by HMBA. In both leukemia cell lines (U937 and K562) lacking wt-p53, a nonapoptotic death pathway may exist during HMBA induction.