Increased sensitivity to sodium salicylate-induced apoptosis in drug-resistant leukemia L1210 cells.

Mouse leukemia L1210 cells selected for resistance to deoxyadenosine contain ribonucleotide reductase that is not feedback inhibited by dATP. These deoxyadenosine-resistant cells (Y8) also do not express p53 protein but do have WAF1 and Gadd45 mRNA and protein. The Y8 cells show increased sensitivity to DNA damaging agents and kinase inhibitors. In these studies we show that in the presence of sodium salicylate (NaSal), the parental wild-type (WT) cells block in G2/M phase of the cell cycle while the Y8 cells show a marked increased in the G0/G1 population of cells. The Y8 cells are more sensitive to apoptosis induced by NaSal than the WT cells. NaSal treatment causes the induction of caspase-3-like activity in Y8 cells but no induction of caspase-3 activity in the WT cells. The caspase inhibitor, Ac-DEVD-CHO, decreased the percentage of Y8 cells in the early apoptotic fraction, but this decrease was reflected by an increase in the percent of cells in the late apoptotic/necrotic fraction. SB20358, a p38-MAP kinase inhibitor did not protect the Y8 cells from NaSal-induced apoptosis indicating that the p38-MAP kinase pathway was not involved in the NaSal-induced apoptotic pathway in the p53-independent Y8 cells.

Characterization and quantitation by flow cytometry of membranous microparticles formed during activation of platelet suspensions with ionophore or thrombin.

The typical data presentation by flow cytometry of platelet suspensions stimulated with calcium ionophore A-23187, thrombin, C5b-9, or other agonists shows a unimodal decrease (or 'left-shift') in forward angle light scatter. Many reports in the literature interpret these findings as indicative of the appearance of small membranous microparticles generated from the platelets as part of the activation response. Investigators may attempt to quantify the microparticles by calculation of the percentage of counts falling below a gate set around the forward angle light scatter distribution of fresh, non-activated platelets. We believe that this approach can lead to erroneous results unless the total particle count in the sample is also determined. The true change in total particle count in a platelet sample is easily estimated on the flow cytometer by adding a known amount of fluorescent beads to the platelet suspension and noting a change in the ratio of bead versus non-bead event counts as a result of the stimulus added. Using this technique under settings considered routine for platelet analysis on a FACScan flow cytometer (Becton-Dickinson), we have found that particle counts increased very little (less than a doubling) in platelet suspensions stimulated with 1-10 microM A-23187 or 0.01-0.5 U/ml thrombin or with sera from patients with diagnosed heparin-induced thrombocytopenia (HIT). Only by employing high sensitivity settings for signal thresholding on orthogonal light scatter, combined with fluorescence gating on high prevalence surface antigens, were we able to detect significant increases (5- or 6-fold) in total particle count in the same experiments. The new events we observed were separated by a decade or more (log scale) from intact platelets on the light scatter plots and fluorescence histograms in a bimodal distribution. We postulate that the unimodal-shifted population of events seen under routine settings after stimulation with ionophore is really degranulated platelets, and only the much smaller new modal subpopulation represents microparticles released as new entities into the platelet suspension. We conclude that without high sensitivity settings for data acquisition, it is most likely incorrect to claim that the left-shifted events on flow cytometry light scatter plots appearing contiguous with the distribution of activated platelets are released microparticles.

Apoptosis induced by inhibitors of nucleotide synthesis in deoxyadenosine-resistant leukemia L1210 cells that lack p53 expression.

An L1210 cell line (Y8) selected for resistance to deoxyadenosine contains ribonucleotide reductase that is not subject to inhibition by dATP. In addition, the Y8 cells have other phenotypic expressions that include increased sensitivity to apoptosis induced by various agents such as radiation, doxorubicin, anisomycin and roscovitine. The Y8 cells were found to be more sensitive to apoptosis induced by methotrexate (MTX), tiazofurin (TZ), deoxyguanosine (dGuo) and N-(phosphonoacetyl)-L-aspartate (PALA). Deoxyguanosine, at concentrations that did not cause apoptosis in the Y8 cells, prevented the apoptotic response of the Y8 cells to MTX and TZ. Deoxycytidine had no effect. Since caspase-3 activation is involved in apoptotic pathways, the effects of the caspase-3 inhibitor, Ac-DEVD-CHO, were studied on the dGuo-, MTX- or TZ-induced apoptosis in the Y8 cells. Ac-DEVD-CHO caused a marked decrease in the fraction of cells in the early phase of apoptosis. However, there was a corresponding increase in the fraction of cells in the late apoptotic/necrotic stages of cell death. This is in marked contrast to the dGuo-induced decrease in apoptosis seen in the MTX- and TZ-treated Y8 cells in which there were no increases in the late apoptotic/necrotic fraction of cells. These data show that alterations of nucleotide pools in the Y8 cells cause marked increases in the apoptotic response which may indicate that the Y8 cells are much more susceptible to the effects of misincorporation of nucleotides into DNA than are the parental WT L1210 cells.