Effects of BAPTA-AM and forskolin on apoptosis and cytochrome c release in photosensitized Chinese hamster V79 cells.

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3- and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]i chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]i and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.

Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells.

We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.

Concentration-dependent modes of cell death in Chinese hamster V79 cells after treatments with H2O2.

Deterioration in the clonogenic ability of Chinese hamster V79 cells was observed after treatments with low concentrations of H2O2 (0.06-0.2 mM) for 1 h and subsequent incubation for 6-8 days, whereas no loss of ability of the cells to exclude trypan blue after treatments with H2O2 for 1 h and subsequent incubation for 6 h was observed in this concentration range. The loss of the dye-exclusion ability became observable when the cells were treated with concentrations greater than 0.9 mM H2O2 for 1 h and subsequently incubated for 6 h. Agarose gel electrophoresis of DNA extracted from cells treated with 1-5 mM H2O2 showed specific regular fragmentation of DNA, suggesting that apoptotic cell death was induced. The induction of apoptotic cell death was further confirmed by observing the protective effects of several modifiers, a protein synthesis inhibitor (cycloheximide), a Ca(2+)-chelator (BAPTA-AM) and an antioxidative compound PBN), against cell survival and DNA fragmentation. From these results, it was concluded that apoptotic cell death was induced by H2O2 in Chinese hamster cells at high concentrations, where their clonogenic ability completely disappeared.

Characterization of free radicals in gamma-irradiated polycrystalline uridine 5'-monophosphate: a study combining ESR, spin-trapping and HPLC.

Free radicals generated in gamma-irradiated polycrystalline uridine 5'-monophosphate (5'-UMP) were studied by ESR, spin-trapping and high-performance liquid chromatography (HPLC). After gamma-irradiation at 0 degree C (70kGy), poly-crystalline 5'-UMP was dissolved in an anaerobic aqueous solution of 2-methyl-2-nitrosopropane as a spin trap at room temperature. Since an ESR spectrum consisting of several components was observed immediately after irradiation, these components were separated with reverse-phase HPLC in the ion-suppression mode and again analysed by ESR spectrometry. Although HPLC ultimately gave four spin-adducts, one component that was originally present disappeared during HPLC. Spin adducts due to two types of C6 radicals were identified. One of these was thought to be formed by electron addition and subsequent protonation at the C6 position, and the other was presumed to be produced by electron addition and subsequent protonation at the O4 position. The spin adducts derived from the C5 and C5' radicals were also identified. The spin adduct that disappeared during HPLC was thought to correspond to the C4'-centred radical. Computer simulation of ESR spectra was carried out to estimate the hyperfine splitting constants.

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.

Effects of 3'-deoxyadenosine (cordycepin) on the repair of X-ray-induced DNA single- and double-strand breaks in Chinese hamster V79 cells.

The ability of cordycepin to inhibit the repair of DNA strand breaks was examined with X-irradiated Chinese hamster V79 cells in log-phase culture. A filter elution technique revealed that 70 microM cordycepin did not inhibit the repair of single-strand breaks but inhibited the repair of double-strand breaks. These findings confirmed the fact that the increase in the lethality of cordycepin in X-irradiated cultured mammalian cells was attributable to unrepaired DNA double-strand breaks.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C] were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radical attack and the subsequent radical reactions in cytosine-related compounds were clarified.

Evidence for the formation of strand-break precursors in hydroxy-attacked thymidine 5'-monophosphate by the spin trapping method.

A method combining spin trapping, ESR, and HPLC was employed to obtain evidence for the formation of sugar radicals in OH-attacked TMP with special emphasis on the detection of strand-break precursors of DNA. OH radicals were produced by irradiating an N2O-saturated aqueous solution with X-rays. When an N2O-saturated aqueous solution containing TMP and a spin trapping reagent, MNP, was irradiated with X-rays, it was estimated on the basis of theoretical calculations using rate constants that 94% of the TMP radicals were induced by OH radicals. Since several spin adducts between TMP radicals and MNP, as well as the byproducts of the spin trapping reagent itself, were produced, reverse-phase HPLC was used to separate them. The presence of six spin adducts was confirmed by ESR examination. Further examination of these spin adducts by UV absorbance spectrophotometry showed the presence of a chromophore at 260 nm in three adducts. Since a gradual increase in the release of unaltered base from these adducts was observed when they were allowed to stand for 0-22 h at room temperature, they could be regarded as the spin adducts of sugar radicals and MNP. ESR spectra from the spin adducts were consistent with hydrogen abstraction radicals at the C1', C4', and C5' positions of the sugar moiety. These radicals appeared to be precursors of AP sites and strand breaks. In addition to these spin adducts, ESR spectra that were consistent with the spin adducts of base radicals (the C5 and C6 radicals) and MNP were observed.

Cytotoxic effect of acyclovir on cultured mammalian cells to which herpesvirus thymidine kinase gene was introduced.

The cytotoxic effects of acyclovir, which is a purine nucleoside analogue and is known as an antibiotic substance, were examined on three lines of rat skin fibroblast FR cells; normal FR cells, FRtk- cells which are deficient in the activity of thymidine kinase (tk) and FRtk-HSVtk+ cells which were prepared by introducing herpes simplex virus' tk gene to FRtk- cells. When FRtk-HSVtk+ cells growing exponentially were incubated in the presence of acyclovir for 4 h, the surviving fractions of the cells decreased in a concentration-dependent manner. Whereas, decrease of the surviving fractions was almost indiscernible in both FR cells and FRtk- cells at the whole ranges of drug-concentrations tested. These results indicate that acyclovir is phosphorylated by the herpes simplex virus' tk and becomes toxic to FRtk-HSVtk+ cells. This also means that FRtk-HSVtk+ cells are useful for the investigation of the biological activity of nucleoside analogues.

Elevated glutathione accelerates oxidative damage to erythrocytes produced by aromatic disulfide.

It has been shown that certain dogs have erythrocytes characterized by an inherited high concentration of reduced glutathione (GSH), five to seven times the normal level (high-GSH RBCs). We examined whether increased GSH in dog erythrocytes leads to increased protection against oxidative damage induced by acetylphenylhydrazine (APH) and/or 4-aminophenyl disulfide (4-AD). When erythrocytes were incubated with 30 mmol/L APH, the Heinz body count was appreciably higher in normal RBCs than in high-GSH RBCs, while there was no difference in the increase of the methemoglobin (metHb) concentration in both RBCs. In contrast, both the Heinz body count and metHb production were much higher in high-GSH RBCs than in normal RBCs when erythrocytes were incubated with 4-AD. Furthermore, the generation of the superoxide in erythrocytes treated with 4-AD, which was measured by spin trapping combined with electron spin resonance (ESR), was obviously higher in high-GSH RBCs than in normal RBCs. These results clearly indicate that erythrocyte GSH is an important defense against oxidative damage induced by certain compounds such as APH, but that, in contrast, elevated GSH appears to accelerate oxidative damage to erythrocytes produced by aromatic disulfides, such as 4-AD, which generated a superoxide in erythrocytes via its redox reaction with GSH.

OH-induced free radicals in purine nucleoside monophosphates: e.s.r. and spin-trapping.

Free radicals produced by the reactions of OH radicals with six purine nucleoside monophosphates (3'-AMP, 5'-AMP, 5'-dAMP, 3'-GMP, 5'-GMP and 5'-dGMP) were investigated by a method combining e.s.r. spin-trapping and high-performance liquid chromatography (HPLC). The N2O-saturated aqueous solutions of purine nucleoside monophosphates, containing 2-methyl-2-nitrosopropane as a spin-trap, were X-irradiated and the resulting spin-adducts were separated by reverse-phase HPLC in the ion suppression mode. The separated spin-adducts were characterized by e.s.r. spectrometry and UV spectrophotometry. Consequently, the radicals due to H-abstraction at the C4' position of the sugar moiety were identified arising from 5'-dAMP and 5'-dGMP. In all cases, e.s.r. spectra consisting of a secondary doublet were observed and assigned to the radical due to H-abstraction at the C5' position of the sugar moiety.

Induction of DNA double-strand breaks in Chinese hamster V79 cells by 2-chlorodeoxyadenosine.

2-Chlorodeoxyadenosine was found to induce DNA double-strand breaks as well as cell death in log-phase Chinese hamster V79 cells. The induction of DNA double-strand breaks, measured by a neutral elution technique, was observed after a 2-h incubation of the cells in the presence of 5 microM of 2-chlorodeoxyadenosine, but these breaks were almost rejoined by a subsequent 1-h incubation, even though this drug was present in the medium during incubation. This repair was prevented by the addition of nicotinamide, which is known to inhibit poly(ADP-ribose) synthesis that is strongly associated with the DNA ligation, but not prevented by the addition of 9-beta-D-arabinofuranosyladenine (araA), which is known to inhibit DNA polymerization. These results suggest that the repair of CdA-induced double-strand breaks is achieved by ligation alone without DNA polymerization. When 35 microM of cycloheximide and 1.3 mM of dibutyryl cAMP were added to the medium, it was found that the induction of double-strand breaks by 2-chlorodeoxyadenosine was suppressed, while the cytotoxicity of 2-chlorodeoxyadenosine measured by colony-forming ability was not interfered with. These results suggest that the induction of DNA double-strand breaks is not associated with the cytotoxicity of this drug.

Metabolic effects of 3'-deoxyadenosine (cordycepin) and 2-halo-3'-deoxyadenosine on repair of X-ray-induced potentially lethal damage in Chinese hamster V79 cells.

Using cultured Chinese hamster V79 cells, an attempt has been made to examine the phosphorylation of cordycepin and its 2-halo derivatives (2-chloro-3'-deoxyadenosine, 2-bromo-3'-deoxyadenosine, and 2-iodo-3'-deoxyadenosine) by adenosine kinase, within the cells, and to correlate it with their cytotoxicities and abilities to inhibit the repair of X-ray-induced potentially lethal damage (PLDR). Of all compounds, only cordycepin was found to be phosphorylated and showed both potent cytotoxicity and ability to inhibit PLDR.