Search

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G(0)/G(1), sub-S and sub-G(2)/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.

Doxorubicin is a widely used chemotherapeutic drug, effective on patients with acute lymphoblastic leukemia but associated with significant long term cardio-toxicity. Menadione (vitamine K3) and the flavonoid quercetin are known as strong apoptogens in human leukemia Jurkat T cells. We explored the potential synergic cytotoxic effects of doxorubicin in association with quercetin and Menadione in this cellular model for acute lymphoblastic leukemia. Cellular viability, apoptosis, necrosis oxidative stress and cellular cycle were determined by flow cytometry utilizing Jurkat lymphoblasts labeled with Annexin V-FITC/7-AAD, CM-H2DCFDA/7-AAD and propidium iodide respectively. Results indicate a dose-dependent oxidative-stress generation, cell cycle arrest and apoptosis induction by doxorubicin alone, correlated with a decrease of the required doses when the anticancer drug was associated with quercetin and menadione, hence supporting the theory of an additive cytotoxic effect on leukemia cells. Introducing QC-MD combinations in leukemia doxorubicin-based treatment could significantly increase the treatment’s efficacy. The main mechanism responsible for this effect appears to be the increase in DOX affinity for DNA, which enables lowering of the therapeutic dose.