Gamma-radiation-induced phosphorylation of p53 on serine 15 is dose-dependent in MOLT-4 leukaemia cells.

Molecular indicators of the absorbed dose of ionizing radiation are powerful tools in biodosimetry. The studies reported here were undertaken with the motivation to find such a marker among the mo lecules involved in ataxia-telangiectasia mutated kinase- dependent signalling induced by ionizing radiation (ATM-kinase, checkpoint kinase-2, protein p53, and oncoprotein Mdm2). In our previous work on T-lymphocyte leukaemia MOLT-4 cells we described the mentioned molecules of ATM-dependent pathway and none of them showed a pronounced dosedependent response. Here we employed Western blotting and ELISA assay to investigate the response of post-translationally modified p53 (particularly phosphorylated on serine 15) after gamma-irradiation. We have found the amount of phosphorylated p53 to be homogenously increased after irradiation by the doses of 0.5 to 7.5 Gy. The dose-dependent response was pronounced especially after the doses up to 3.0 Gy. The presented data indicate that p53 phosphorylated on serine 15 might be used as a potential biodosimetric marker.

Antileukemic activity of the combination of ionizing radiation with valproic acid in promyelocytic leukemia cells HL-60.

Valproic acid (VA) possesses anticonvulsant as well as anticancer properties of histondeacetylases inhibitor. Incubation of human promyelocytic leukemia cells HL-60 with VA leads to acetylation of nuclear histones H3 and H4. Using 2 mmol/l concentration we proved the expression of protein p21, which relates to the arrest of cell proliferation and decrease in number of cells in S phase of cell cycle. Treatment of HL-60 cells with VA causes their differentiation, proved as increase in CD11b expression. The most widely used method in cancer treatment is radiotherapy. 24 hours after irradiation by the therapeutical dose of 2 Gy, 56% of HL-60 cells are accumulated in G2 phase of cell cycle. VA had no influence on this accumulation, but 24 h-long pretreatment of cells with 1 mmol/l VA provoked higher decrease in cell number in S phase (18%) comparing with only irradiated cells (25%). The results of our work show that VA posseses radiosensitizing properties when applied 24 hours prior to irradiation and that during parallel long-term action of VA and IR the cells undergo differentiation and faster apoptosis induction. Radiosensitizing effect of VA is not caused by abrogation of G2/M cell cycle arrest, but VA induces p21 and leads to differentiation of HL-60 cells.

Effect of valproic acid and antiapoptotic cytokines on differentiation and apoptosis induction of human leukemia cells.

This work compares effect of histondeacetylase inhibitor, valproic acid (VA), on proliferation, differentiation and apoptosis induction in two human leukemic cell lines: HL-60 (human promyleocytic leukemia, p53 negative) and MOLT-4 (human T-lymphocyte leukemia, p53 wild type). Incubation with VA caused decrease in percentage of cells in S phase of cell cycle. The decrease was more intensive in HL-60 cells, where the cells in S phase were absent 6 days after the beginning of incubation with VA (4 mmol/l). 3-day-long incubation of HL-60 cells with 4 mmol/l VA caused differentiation of these cells, marked by increase in CD11b and co-stimulatory/adhesion molecule CD86, and induction of a significant apoptosis. Annexin V positive cells lost the CD11b antigen. 3-day-long incubation of MOLT-4 cells with VA (1-2 mmol/l) inhibited proliferation and decreased percentage of cells in S phase of the cell cycle. 90% of MOLT-4 cells are CD7 positive. This CD7 positivity is not changed during apoptosis induction (detected as Annexin V positivity). On the other hand, CD4 marker expression decreases after incubation with 1-2 mmol/l VA, but during apoptosis induction by 4 mmol/l VA, most of the apoptotic Annexin V positive cells were also CD4 positive. Using a clonogenic survival assay EC(50) for 3-day-long incubation with VA was determined. For HL-60 cells, the established EC(50) was 1.84 mmol/l, for MOLT-4 cells it was 1.76 mmol/l. Ability of VA to induce differentiation in HL-60 cells thus does not affect final cell killing. However, the elimination of the cells was considerably affected by presence of hematopoietic growth factors. 14-day-long incubation of HL-60 cells with VA in conditioned medium (source of IL-3, SCF, G-CSF) caused increase in EC(50) to 4 mmol/l, while in MOLT-4 cells (cultivation without conditioned medium), the EC(50) decreased to 0.63 mmol/l.