Dactinomycin Impairs Cellular Respiration and Reduces Accompanying ATP Formation

The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin

Inhibition of Cellular Respiration by Doxorubicin

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)]. In solutions containing glucose and cells, [O(2)] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2-4 h of incubation with 20 microM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation

Effects of Cisplatin on Mitochondrial Function in Jurkat Cells

In this work, we measured the effects of pharmacological concentrations of cisplatin (cis-diaminedichloroplatinum II) on mitochondrial function, cell viability, and DNA fragmentation in Jurkat cells. The exposure of cells to 0-25 microM cisplatin for 3 h had no immediate effect on cellular mitochondrial oxygen consumption, measured using a palladium-porphyrin oxygen sensing phosphor. Similarly, the cell viability as measured by trypan blue staining was unchanged immediately following exposure to the drug, and no small DNA fragments, characteristic of drug-induced apoptosis, appeared. At 24 h after exposure to cisplatin, cellular respiration and viability decreased relative to controls and the amount of small DNA fragments, measured using quantitative agarose gel electrophoresis, was proportional to the concentration of cisplatin present during the drug exposure period. The small DNA fragments showed the banding pattern (with a spacing of approximately 300 bp) characteristic of drug-induced cell death by apoptosis. The changes in respiration and DNA fragmentation correlated linearly with the amount of platinum bound to DNA, determined by atomic absorption spectroscopy immediately following drug exposure. The oxygen consumption by beef heart mitochondria was not affected 0-24 h after exposure to 25 microM cisplatin or to solutions containing the monoaquated form of the drug, suggesting that the drug does not attack the mitochondrial respiratory chain directly. Cells exposed to the peptide benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone, which blocks apoptosis by the caspase pathway, showed a decrease in cisplatin-induced DNA fragmentation but not in the impairment of cellular respiration. Thus, although apoptosis is caspase-dependent, the impairment of cellular respiration is independent of the caspase system. Collectively, these results suggest that alteration in mitochondrial function is a secondary effect of cisplatin cytotoxicity in Jurkat cells

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture

Objectives: This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures.Methods: Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Results: In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min-1 mg-1 (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min-1 per 107 cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. Conclusion: The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation

Caspase Activation by Anticancer Drugs: The Caspase Storm

This study measures the time-dependence of cellular caspase activation by anticancer drugs and compares it with that of cellular respiration. Intracellular caspase activation and cellular respiration were measured during continuous exposure of Jurkat, HL-60, and HL-60/MX2 (deficient in topoisomerase-II) cells to dactinomycin, doxorubicin, and the platinum (Pt) compounds cisplatin, carboplatin, and oxaliplatin. Caspase activation was measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC). We show that this substrate rapidly enters cells where it is efficiently cleaved at the aspartate residue by specific caspases, yielding the fluorescent compound 7-amino-4-trifluoromethyl coumarin (AFC). Following cell disruption, released AFC was separated on HPLC and detected by fluorescence. The appearance of AFC in cells was blocked by the pancaspase inhibitor benzyloxycarbonyl-val-ala-asp-fluoromethylketone, thus establishing that intracellular caspases were responsible for the cleavage. Caspase activity was first noted after about 2 h of incubation with doxorubicin or dactinomycin, the production of AFC being linear with time afterward. Caspase activation by doxorubicin was delayed in HL-60/MX2 cells, reflecting the critical role of topoisomerase-II in doxorubicin cytotoxicity. For both drugs, caspase activity increased rapidly between 2 and 6 h, went through a maximum, and decreased after 8 h (“caspase storm”). Cisplatin treatment induced noticeable caspase activity only after 14 h of incubation, and the fluorescent intensity of AFC became linear with time at 16 h. Exposure of the cells to all of the drugs studied led to impaired cellular respiration and decreased cellular ATP, concomitant with caspase activation. Thus, the mitochondria are rapidly targeted by active caspases