Dual mechanism of daunorubicin-induced cell death in both sensitive and MDR-resistant HL-60 cells

Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance. This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation. However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis. To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity. Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells. However, at equitoxic doses, similar cell death pathways were observed. In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5–1 μM and delayed mitotic cell death at 0.1 μM. These concentrations are within the clinical dose range. Similarly, HL-60/Vinc cells underwent apoptosis at 50–100 μM daunorubicin and mitotic cell death at 10 μM. These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells. Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent. Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias. © 1999 Cancer Research Campaig

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle

Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing links. The Claude Bernard Lecture 2009

Insulin resistance is a hallmark of type 2 diabetes mellitus and is associated with a metabolic and cardiovascular cluster of disorders (dyslipidaemia, hypertension, obesity [especially visceral], glucose intolerance, endothelial dysfunction), each of which is an independent risk factor for cardiovascular disease (CVD). Multiple prospective studies have documented an association between insulin resistance and accelerated CVD in patients with type 2 diabetes, as well as in non-diabetic individuals. The molecular causes of insulin resistance, i.e. impaired insulin signalling through the phosphoinositol-3 kinase pathway with intact signalling through the mitogen-activated protein kinase pathway, are responsible for the impairment in insulin-stimulated glucose metabolism and contribute to the accelerated rate of CVD in type 2 diabetes patients. The current epidemic of diabetes is being driven by the obesity epidemic, which represents a state of tissue fat overload. Accumulation of toxic lipid metabolites (fatty acyl CoA, diacylglycerol, ceramide) in muscle, liver, adipocytes, beta cells and arterial tissues contributes to insulin resistance, beta cell dysfunction and accelerated atherosclerosis, respectively, in type 2 diabetes. Treatment with thiazolidinediones mobilises fat out of tissues, leading to enhanced insulin sensitivity, improved beta cell function and decreased atherogenesis. Insulin resistance and lipotoxicity represent the missing links (beyond the classical cardiovascular risk factors) that help explain the accelerated rate of CVD in type 2 diabetic patients