Effect of the histone deacetylase inhibitor trichostatin a in human peripheral blood lymphocytes as a function of donor age.

The histone deacetylase inhibitor trichostatin A (TSA) is a promising agent for the treatment of certain types of cancers alone or in synergistic combination with other anticancer agents. One of the advantages of the use of histone deacetylase inhibitors, such as TSA, is that its effects have been found to be more potent toward cancer cells compared to normal cells. The effect of anticancer agents on the immune system, and on lymphocytes in particular, is of major importance to the success of anticancer regimens. In this respect, information documenting the effect of such agents on normal lymphocytes compared to malignant cells may be of significant value for the successful designing of clinical protocols. Moreover, the parameter of age may be a factor in the differential effects of such protocols. Histone deacetylase inhibitors lead to the accumulation of acetylated histones and, depending on the cell type, may induce either apoptosis, cell cycle arrest, or differentiation. Previous work from our lab has shown that TSA induces the accumulation of histone H4 acetylation and apoptosis in human peripheral blood lymphocytes. In light of the above, we have extended our investigation of the effects of TSA on human lymphocytes to include the parameter of age, which has not been previously studied. Our results show that TSA induces apoptosis of lymphocytes from donors of all age groups, but no age-related changes in the levels of apoptosis are observed.

Cell mediated cytotoxicity assessment by relative changes in viable target absolute counts.

We measured absolute counts (cells/microl) of Calcein-AM stained target cells that remained viable [i.e. not permeable to the viability probe 7-AAD (7-Amino Actinomycin D)] following a four-hour incubation with effectors [NK, CTL (cytotoxic T lymphocytes)] without using beads or standards. The absolute counts were evaluated by a Cytoron Absolute (Ortho) flow cytometry apparatus. Median triplicate counts were compared at 0 and 4h, in single targets and increased effector/target ratios. The Coefficient of Variation (CV) of the cell concentration (cells/microl) at the beginning of the experiment was below 6%. The % changes of viable target counts were correlated to the effector/target ratios by linear regression. The methodology was applied in pairs: 17 for allogenic stem cell transplantation from unrelated donors, 3 of healthy unrelated individuals, 10 for measuring NK activity and 7 autologous. In 15/20 allogenic MLC (mixed lymphocytes culture) and for all NK assays the cytotoxicity was positive (p < 0.05, r2 > 0.9) while in 5/20 allogeneic MLC and in all autologous MLC the outcome was negative (p > 0.05, r2 < 0.4). The proposed method offers the advantage of combining absolute counts with flow cytometry analysis of viable targets, assessment of cell behavior during the cytotoxic phenomenon, the use of a small amount of cells and excellent sensitivity. Our method can estimate frequencies higher than 1:100 for CTL assays.