DNA damage from oxidants: influence of lesion complexity and chromatin organization

DNA damage by reactive oxygen species results in a spectrum of DNA lesions including single-strand breaks (ssb) and double-strand breaks (dsb). However, most damage is not lethal, and the location and nature of the DNA damage, in addition to total number of breaks, are likely to be critical in determining ultimate survival. Generally associated only with ionizing radiation, multiply damaged sites (i.e., complex lesions and clusters of complex lesions in DNA) are more likely to be lethal because they are less easily repaired. We examined five drugs known to cause DNA adducts, strand breaks, and reactive oxygen species for their ability to produce complex lesions: 4-nitroquinoline-1-oxide (4NQO), H2O2, doxorubicin, Tirapazamine, and etoposide. As indicators of lesion complexity we compared 1) the ratio of ssb to dsb, 2) the rate of rejoining of single-strand breaks, 3) the relative lethality of the breaks (number of breaks per mean lethal dose), and 4) the ability to produce complex lesions. Tirapazamine, etoposide, and doxorubicin gave dsb/ssb ratios similar to that for X-rays, whereas 4NQO and H2O2 showed dsb/ssb ratios of 200 and 3250, respectively. The number of dsb per LD50 varied from 2.5 to 500 for different drugs. There was no apparent relation between ssb rejoining half-time (3.5-85 min) and relative lethality or lesion complexity. A modified (nonionic detergent) filter elution method confirmed that tirapazamine, like ionizing radiation, produced multiple dsb within single chromatin domains. These data indicate that complex lesions can be produced by a number of different chemicals and suggest that the damage that results in killing by these drugs may be related to production of multiply damaged sites in DNA.     

Oxidative stress induces DNA fragmentation and caspase activation via the c-Jun NH2-terminal kinase pathway in H9c2 cardiac muscle cells

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.     

Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).     

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

1. The effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells. 2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1-30 microM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 microM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 microM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO). 3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH. 4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. 5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event.     

Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.     

The intraoperative administration of ketorolac tromethamine in evaluating length of stay in a same day surgery unit

Relative biological effectiveness (RBE), as a function of linear energy transfer (LET), is evaluated for different types of damage contributing to mammalian cell reproductive death. Survival curves are analysed assuming a linear-quadratic dose dependence of lethal lesions. The linear term represents lethal damage due to single particle tracks, the quadratic term represents lethality due to interaction of lesions from independent tracks. RBE-LET relationships of single-track lethal damage, sublethal damage, potentially lethal damage and DNA double-strand breaks (dsb) are compared. Single-track lethal damage is shown to be composed of two components: damage that remains unrepaired in an interval between irradiation and assay, characterized by a very strong dependence on LET, with RBEs up to 20, and potentially lethal damage, which is weakly dependent on LET with RBEs < 3. Potentially lethal damage and sublethal damage depend similarly on LET as DNA dsb. The identification of these different components of damage leads to an interpretation of differences in radiosensitivity and in RBEs among various types of cells.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX

The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.     

Association of thymoma and severe intestinal strongyloidiasis

The G2 chromosomal radiosensitivity of murine SCID (severe combined immunodeficient) and normal fibroblasts has been investigated. We have also investigated the G2 response of these cell lines to the restriction endonuclease PvuII. We show that chromatid breaks are induced linearly with radiation dose in both cell lines and SCID cells are approximately 1.6 times as radiosensitive as normal murine fibroblasts when tested using a G2 assay with a 2 h sampling time. The disappearance of chromatid breaks with time after irradiation was first order with a half-time of approximately 1.5 h in both cell lines. Thus, although SCID cells are deficient in the rejoining of double-strand breaks (dsb), they show similar kinetics of disappearance of chromatid breaks with time as normal CB17 cells, indicating that the 'rejoining' of chromatid breaks does not reflect dsb repair. When CB17 and SCID cells were treated with PvuII, which generates dsb in cellular DNA in the presence of streptolysin O (as a porating agent), approximately 3 times more chromatid breaks were observed in SCID than CB17 cells. We conclude that SCID cells convert a higher number of dsb into chromatid breaks than do CB17 cells. The conversion process is interpreted in terms of the recently proposed 'signal' model, whereby a signal, resulting from a single dsb, triggers the cell to make a recombinational exchange which, if incomplete, gives rise to a visible chromatid break. In terms of the signal model, elevated conversion of dsb into chromatid breaks results from altered signalling and the disappearance of chromatid breaks with time following irradiation represents the completion of recombinational exchanges rather than repair of dsb.     

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.     

A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.     

Hydrogen peroxide effects in Escherichia coli cells

We analyzed DNA lesions produced by H2O2 under low iron conditions, the cross adaptive response and the synergistic lethal effect produced by iron chelator-o-phenanthroline, using different Escherichia coli mutants deficient in DNA repair mechanisms. At normal iron levels the lesions produced by H2O2 are repaired mainly by the exonuclease III protein. Under low iron conditions we observed that the Fpg and UvrA proteins as well as SOS and OxyR systems participate in the repair of these lesions. The lethal effect of H2O2 is strengthened by o-phenanthroline if both compounds are added simultaneously to the culture medium. This phenomenon was observed in the wild type cells and in the xthA mutant (hypersensitive to H2O2). E. coli cells treated with low concentrations of H2O2 (micromolar) acquire resistance to different DNA damaging agents. Our results indicate also that pretreatment with high (millimolar) H2O2 concentrations protects cells against killing, by UV and this phenomenon is independent of the SOS system, but dependent on RecA and UvrA proteins. H2O2 induces protection against lethal and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). H2O2 also protects the cells against killing by cumene hydroperoxide, possibly with the participation of Ahp protein.     

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.     

Measurement of DNA damage after exposure to 2450 MHz electromagnetic radiation

Recent reports suggest that exposure to 2450 MHz electromagnetic radiation causes DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in cells of rat brain irradiated in vivo (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995; Int. J. Radiat. Biol. 69, 513-521, 1996). Therefore, we endeavored to determine if exposure of cultured mammalian cells in vitro to 2450 MHz radiation causes DNA damage. The alkaline comet assay (single-cell gel electrophoresis), which is reportedly the most sensitive method to assay DNA damage in individual cells, was used to measure DNA damage after in vitro 2450 MHz irradiation. Exponentially growing U87MG and C3H 10T1/2 cells were exposed to 2450 MHz continuous-wave (CW) radiation in specially designed radial transmission lines (RTLs) that provided relatively uniform microwave exposure. Specific absorption rates (SARs) were calculated to be 0.7 and 1.9 W/kg. Temperatures in the RTLs were measured in real time and were maintained at 37 +/- 0.3 degrees C. Every experiment included sham exposure(s) in an RTL. Cells were irradiated for 2 h, 2 h followed by a 4-h incubation at 37 degrees C in an incubator, 4 h and 24 h. After these treatments samples were subjected to the alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-267, 1992). Images of comets were digitized and analyzed using a PC-based image analysis system, and the "normalized comet moment" and "comet length" were determined. No significant differences were observed between the test group and the controls after exposure to 2450 MHz CW irradiation. Thus 2450 MHz irradiation does not appear to cause DNA damage in cultured mammalian cells under these exposure conditions as measured by this assay.     

Loss of heterozygosity on chromosome 16 in sporadic Wilms'' tumour

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.     

Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats

Programmed cell death in the myocardium has been linked to ischemia reperfusion injury as well as to excessive mechanical forces associated with increases in ventricular loading. Moreover, hypoxia activates the suicide program of cardiac myocytes in vitro. Because the supplied portion of the ventricular wall is ischemic and subjected to high levels of systolic and diastolic stresses (acutely after coronary artery occlusion), apoptosis and necrosis may contribute independently to myocyte cell death after infarction. Therefore, myocardial infarction was produced in rats, and, after the determination of ventricular hemodynamics, the contribution of apoptotic and/or necrotic myocyte cell death to infarct size was measured quantitatively from 20 minutes to 7 days after coronary artery occlusion. Programmed cell death was assessed by the terminal deoxynucleotidyl transferase assay and by the electrophoretic detection of DNA laddering. Myocyte necrosis was evaluated by myosin monoclonal Ab labeling. Moreover, the expression of Bcl-2, Bax, and Fas proteins in myocytes was examined by immunocytochemistry. Myocyte cell death by apoptosis and necrosis comprised nearly 3 million myocytes at 2 hours. Apoptotic cell death involved 2.8 million cells and necrotic cell death only 90,000 myocytes. Apoptosis continued to represent the major independent form of myocyte cell death, affecting 6.6 million myocytes at 4.5 hours. Myocyte necrosis peaked at 1 day, including 1.1 million myocytes. DNA electrophoretic analysis confirmed these observations by showing nucleosomal ladders at 2-3 hours, 4.5 hours, 1 day, and 2 days after coronary artery occlusion. Myocytes showing both DNA strand breaks and myosin labeling were a prominent aspect of myocardial damage only after 6 hours. Finally, the expression of Bcl-2 and Fas in myocytes increased 18-fold and 131-fold, respectively. In conclusion, programmed myocyte cell death is the major form of myocardial damage produced by occlusion of a major epicardial coronary artery, whereas necrotic myocyte cell death follows apoptosis and contributes to the progressive loss of cells with time after infarction. The enhanced expression of Fas may be implicated in the activation of apoptosis in spite of the increase in Bcl-2, which tends to preserve cell survival.     

Clustered organization of S100 genes in human and mouse

Hydrogen peroxide (H2O2) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H2O2 effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H2O2-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H2O2 at 40 or 400 microM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 microM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H2O2. The 400 microM H2O2 severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H2O2 were significantly different. Exposure of LE cells to 20, 40, or 100 microM H2O2 for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H2O2 than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Bcl-2 prevents topoisomerase II inhibitor GL331-induced apoptosis is mediated by down-regulation of poly(ADP-ribose)polymerase activity

Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme responsible for DNA strand breaks, has been recently suggested to be crucial for apoptosis induced by a number chemotherapeutic drugs. In this study, we demonstrated that the PARP activity could be evidently elevated with a peak at 6 h when HL-60 cells were treated with a new anticancer drug GL331. Coincident with the peak of PARP activity, an apparent DNA fragmentation and apoptotic morphology were observed in cells treated with GL331. The subsequent apoptotic DNA fragmentation induced by GL331 could be completely blocked by transfecting cells with anti-sense PARP retroviral vector or by treating cells with PARP inhibitor, 3-aminobenzamide (3-AB). This blocking effect thus suggests that activation of PARP was critically involved in GL331-induced apoptosis. The fact that Bcl-2 has been found to antagonize cell death induced by a wide variety of agents, accounts for why we examined whether if Bcl-2 could antagonize GL331 effects. Interestingly, ectopic overexpression of Bcl-2 in either HL-60 or U937 cells caused in resistance towards GL331-elicited DNA fragmentation and cytotoxic effect. Additionally, Bcl-2 also attenuated the poly(ADP-ribosyl)ation of PARP itself as well as Histone H1 at the early period of drug treatment. However, Bcl-2 did not influence the extent of DNA strand breaks induced by GL331 in either control or Bcl-2-overexpressing cells. In addition, analysis of basal PARP activity in control and several Bcl-2 overexpressing clones revealed that Bcl-2 down-regulated PARP activity under the condition without DNA damages. Above findings suggest that poly(ADP-ribosyl)ation of nuclear targets is important for apoptosis induced by DNA-reactive anticancer drugs.     

Defense Mechanism Test and electrodermal activity

Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.