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The combination of low dose of radiation and an anticancer drug is a potent strategy for cancer therapy. Nucleoside analogs are known to have a radiosensitizing effects via the inhibition of DNA damage repair after irradiation. Certain types of nucleoside analogs have the inhibitory effects on RNA synthesis, but not DNA synthesis, with multiple functions in cell cycle modulation and apoptosis. In this review, the most up-to-date findings regarding radiosensitizing nucleoside analogs will be discussed, focusing especially on the mechanisms of action.

To clarify the mechanisms of purvalanol A in the induction of apoptosis, we investigated whether purvalanol A influenced the RNA synthesis and expression of RNA polymerase II and signal transducer and activator of transcription 3 (STAT3). When MKN45 cells were treated with 30 micromol/l purvalanol A, mitochondrial dysfunction occurred before the induction of the apoptosis and the expression of antiapoptotic proteins survivin, Bcl-XL, and Bcl-2 was reduced. The treatment with parvalanol A was also shown to reduce not only mRNA for these proteins but also global RNA synthesis. The phosphorylation of the carboxy-terminal domain of RNA polymerase II, which was involved in transcriptional regulation, was strongly inhibited by purvalanol A, followed by the partial inhibition of the expression of RNA polymerase II. Furthermore, the phosphorylation at Tyr705 of STAT3, which is known to be a phosphorylation site for Janus kinase 2 (JAK2), was completely inhibited by purvalanol A early (3 h) after drug treatment, although the phosphorylation of STAT3 at Ser727, which is a phosphorylation site for Ras/Raf/MEK and extracellular signal-regulated protein kinase 1/2, was still detectable until late (12 h) after treatment. In addition, the tyrosine phosphorylation of JAK2 was efficiently inhibited by purvalanol A. These results suggest that the inhibition of JAK2/STAT3 and RNA polymerase II is crucial in the downregulation of antiapoptotic proteins leading to the apoptotic cell death induced by parvalanol A.

To investigate the mechanism of radioresistance of solid tumor cells, we created two expression vectors encoding Survivin mutants, T34A and D53A. When T34A and D53A were overexpressed in NIH3T3, A549 and HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of Survivin with Smac/DIABLO in the cells that overexpressed these mutants. Coimmunoprecipitation analysis revealed that mutant form of Survivin, D53A and T34A could bind to Smac/DIABLO, but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between Survivin and Smac/DIABLO through overexpression of T34A and D53A.

Our previous study showed that ionizing radiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines and that the death receptor of the TNF alpha-related apoptosis-inducing ligand TRAIL enhanced the apoptotic pathway (Hamasu et al., (2005) Journal of Radiation Research, 46:103-110). The present experiments were performed to examine whether treatment with TRAIL enhanced the cell killing in tumor cells exposed to ionizing radiation under hypoxia, since the presence of radioresistant cells in hypoxic regions of solid tumors is a serious problem in radiation therapy for tumors. When human lung carcinoma A549 cells were irradiated under normoxia and hypoxia, respectively, radiation-induced enhancement of expression of DR5 was observed under both conditions. Incubation in the presence of TRAIL enhanced the caspase-dependent and chymotrypsin-like-protease-dependent apoptotic cell death in A549 cells exposed to X rays. Furthermore, it was shown that treatment with TRAIL enhanced apoptotic cell death and loss of clonogenic ability in A549 cells exposed to X rays not only under normoxia but also under hypoxia, suggesting that combination treatment with TRAIL and X irradiation is effective for hypoxic tumor cells.

To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G2/M phase, followed by activation of the G2/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 microM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G2/M-phase regulatory proteins and reduced the fraction of the cells in the G2/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X(L) and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.

Oxidative damage due to ischemia/reperfusion has been implicated as one of the leading causes for delayed neuronal cell death in a number of neurodegenerative diseases, including stroke. The purpose of this research was to investigate whether oral administration of a fermented grain food mixture (AOB(R)) might offer protective effects against ischemia/reperfusion-induced neuronal damage in Mongolian gerbils, a model known for delayed neuronal death in the hippocampal CA1 region. Histological analysis revealed that AOB administration ad libitum for 3 weeks (preoperative administration) and 1 week (postoperative administration) dose-dependently suppressed the induction of transient ischemia/reperfusion-induced neuronal cell death. TUNEL assay also revealed that AOB suppressed it by inhibiting the induction of apoptosis. A significant increase of superoxide dismutase-like (SOD-like) activity was observed in the hippocampal CA1 region of the AOB-treated gerbil. Furthermore, immunoblot analysis showed that AOB administration down-regulated the expression of heat shock proteins HSP27 and HSP70 in the same region. These results indicated that oral administration of AOB protected against ischemia/reperfusion-induced brain injury by minimizing oxidative damage via its SOD-like activity and inhibiting apoptosis.

To clarify the role of lipid rafts in 2-chloro-2'-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca(2+)](i)) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca(2+) chelators BAPTA-AM and EGTA, and the L-type Ca(2+) channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca(2+) influx via the plasma membrane.

In the present study, using inhibitors of ceramide synthase (fumonisin B1), ketosphinganine synthetase (L-cycloserine), acid sphingomyelinase (D609 and desipramine) and neutral sphingomyelinase (GW4869), the role of ceramide in X-ray-induced apoptosis was investigated in MOLT-4 cells. The diacylglycerol kinase (DGK) assay showed that the intracellular concentration of ceramide increased time-dependently after X irradiation of cells, and this radiation-induced accumulation of ceramide did not occur prior to the appearance of apoptotic cells. Treatment with D609 significantly inhibited radiation-induced apoptosis, but did not inhibit the increase of intracellular ceramide. Treatment with desipramine or GW4869 prevented neither radiation-induced apoptosis nor the induced increase of ceramide. On the other hand, fumonisin B1 and L-cycloserine had no effect on the radiation-induced induction of apoptosis, in spite of significant inhibition of the radiation-induced ceramide. From these results, it was suggested that the increase of the intracellular concentration of ceramide was not essential for radiation-induced apoptosis in MOLT-4 cells.

The clonogenic ability (reproductive cell death) of Chinese hamster V79 cells was measured after treatment with X radiation and a newly developed anti-cancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106). Amplification in the loss of clonogenicity was observed compared to that obtained for cells exposed to X rays alone. Addition of benzyloxycarbonyl-val-ala-asp-fluoromethylketone (Z-VAD-FMK), a broad-spectrum caspase inhibitor, attenuated the increased lethality, but the dose-response curve obtained was found to merely revert to that obtained for cells exposed to X rays alone. Flow cytometric analysis showed that the number of cells arrested at the G2/M phase by X irradiation was decreased by co-treatment with TAS106, and instead the number of cells in the sub-G1 phase increased. Western blot analysis proved that TAS106 treatment down-regulated the expression of the G2/M arrest-related proteins cyclin B1, phospho-CDC2 and WEE1. From these results, it was concluded that (1) no apoptosis was included in the dose-response curve obtained from cells exposed to X rays alone, (2) X radiation induced a potentially apoptotic (proapoptotic) state in cells independent of the loss of their clonogenic ability, and (3) TAS106 enhanced the loss of their clonogenic ability by converting the proapoptotic cells to apoptotic cells through the abrogation of arrest at the G2/M phase.

The death receptors Fas and DR5 are known to be expressed not only in immune cells but also in various tumor cells. The aim of the present study was to determine whether X irradiation enhanced induction of apoptosis in Tp53 wild type and Tp53-mutated tumor cell lines treated with agonists against these death receptors. We showed that 5 Gy of X irradiation significantly up-regulated the expression of death receptors Fas and DR5 on the plasma membrane in gastric cancer cell lines MKN45 and MKN28, lung cancer cell line A549, and prostate cancer cell line DU145, and that subsequent treatments with agonistic molecules for these death receptors, Fas antibody CH11 and TRAIL, increased the formation of active fragment p20 of caspase 3 followed by the induction of apoptosis. This death-receptor-mediated apoptosis was independent of Tp53 status since MKN28 and DU145 were Tp53-mutated. The post-irradiation treatment of the cells with N-acetyl-L-cysteine (NAC) abolished the up-regulation of the expression of Fas and DR5 on the plasma membrane. NAC also attenuated the increase in the formation of p20 and the induction of apoptosis by agonistic molecules. These results suggested that the increase in the induction of apoptosis by combined treatment with X irradiation and CH11 or TRAIL occurred through a change of the intracellular redox state independent of Tp53 status in human carcinoma cell lines.

We investigated the effect of SCF, a c-kit ligand, on the radiosensitivity of HL60 cells. X-ray-induced apoptosis in HL60 cells was significantly lower in the presence of SCF than in the absence of SCF. This attenuation of X-ray-induced apoptosis by SCF was abolished by PD98059 (an ERK inhibitor), but not by wortmannin (a PI3-K inhibitor) or GF109203X (a PKC inhibitor). The expression of phospho-ERK1/2 (active form) and the ERK1/2-regulated expression of survivin were found to increase in cells treated with X irradiation and SCF. However, X irradiation alone induced down-regulation of the expression of phospho-ERK1/2. Our findings suggest that activation of c-kit by SCF confers radioresistance through up-regulation of ERK-dependent survivin expression in HL60 cells.

A brief period of ischemia, i.e. preconditioning of the middle cerebral artery territory, induces ischemic tolerance reducing the cerebral infarction volume caused by subsequent lethal ischemia. Nevertheless, little is known about the molecular mechanisms underlying this phenomenon. In the present study, we examined the involvement of the activation of Akt, a serine/threonine kinase, in the cerebral ischemic tolerance. Western blot analysis showed that Akt was activated in both non-preconditioned and preconditioned groups after ischemia for 1 hr, but the activation was long-lasting in the preconditioned rats. Immunohistochemical analysis demonstrated that the preconditioning-induced preventive effect on a rapid decrease in the activation level of Akt was due to the persistent activation of Akt in the penumbra region. In addition, TUNEL staining demonstrated that the preconditioning treatment inhibited the augmentation of neuronal death probably through apoptosis in the penumbra region to prevent the spread of infarction. Since the activation of Akt has been reported to protect cells from stress, the present results suggest that the preconditioning-induced persistent activation of Akt in the penumbra region plays an important role in ischemic tolerance of the brain.

Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates.

To clarify the role of the Golgi apparatus in photodynamic therapy-induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhodamine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (1O2) and superoxide anion (O2-). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O2- scavenger, but not by NaN3, a quencher of 1O2. Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl2, which are known as inhibitors of deoxyribonuclease (DNase) gamma, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, a chelator of Ca2+, but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O2- was responsible for TBR-induced apoptosis, and Ca(2+)-dependent and caspase-3-independent nuclease such as DNase gamma played an important role in apoptotic signaling triggered by Golgi dysfunction.

A post-irradiation treatment of the human leukemia cell line MOLT-4 with the antioxidant Trolox attenuated caspase-3 dependent apoptosis. The increase in the p53 expression and SAPK/JNK activation after X irradiation was also inhibited by a Trolox treatment, but the expression of BCL-2 and BAX, which would occur downstream from p53, was not changed. Studies on the effects of the intracellular calcium chelator BAPTA-AM on the induction of apoptosis and the activation of SAPK/JNK and caspase-3 proved that the chelation of calcium merely delayed the onset of radiation-induced apoptosis and the activation of SAPK/JNK and caspase-3. When the effects of the protein synthesis inhibitor cycloheximde on the apoptotic signaling pathways, including the activation of caspase family proteins and SAPK/JNK, were investigated, the expression of death receptor Fas through SAPK/JNK activation was found to be required for radiation-induced apoptosis. Finally, the relationship between the amounts of DNA dsb and induction of apoptosis was examined by irradiating BrdU-incorporated cells. An increase in DNA dsb caused by BrdU was found, but the induction of apoptosis was not enhanced. From these data, we could get no positive evidence for DNA as a target of X-rays and p53 as an indispensable factor to induced apoptosis in X-irradiated MOLT-4 cells.

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H2O2 and investigated the roles of protein kinase C delta (PKC delta) and Ca2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H2O2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC delta inhibitor) but not by BAPTA-AM (an intracellular Ca2+ chelator). PKC delta itself was activated through the phosphorylation at tyrosine residues. H2O2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC delta contributes to the accumulation of p53 and that Ca2+ plays a role in downstream signals of p53 leading to apoptosis in H2O2-treated BAEC.

Purpose: To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells., Materials and Methods: MOLT-4 cells pretreated with 0.5 microg/ml cycloheximide for 1h were exposed to 7.5Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively., Results: Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells., Conclusion: These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.

Severe acute respiratory syndrome (SARS) is caused by SARS-coronavirus (SARS-CoV). Infection of Vero E6 cells with SARS-CoV inhibits cell proliferation. Our previous study indicated that Akt, which is poorly phosphorylated in confluent cultures of Vero E6 cells, is phosphorylated and then dephosphorylated upon infection by SARS-CoV. In the present study, we showed that a serine residue of Akt was phosphorylated in Vero E6 cells in subconfluent culture and that Akt was dephosphorylated rapidly after SARS-CoV infection without up-regulation of its phosphorylation. Phosphorylation of glycogen synthase kinase-3β, which is one of the downstream targets of Akt, was prevented in SARS-CoV-infected cells. However, treatment with glycogen synthase kinase-3β small interfering RNA indicated that the glycogen synthase kinase-3β signaling pathway was not related to inhibition of cell proliferation. Treatment of Vero E6 cells with the phosphatidylinositol 3′-kinase/Akt inhibitor, LY294002, which induces dephosphorylation of Akt, inhibited cell proliferation. As shown in our previous studies, apoptosis occurred in virus-infected cells within 18 h postinfection. Cellular mRNA transcription, which was reported to be up-regulated in SARS-CoV-infected Caco-2 cells, was not up-regulated in virus-infected Vero E6 cells, partially as a result of apoptosis. These results suggested that inhibition of cell proliferation is regulated by both the phosphatidylinositol 3′-kinase/Akt signaling pathway and by apoptosis in SARS-CoV-infected Vero E6 cells. This is the first study to analyze SARS-CoV-induced cell growth inhibition. [ABSTRACT FROM AUTHOR]

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H 2 O 2 and investigated the roles of protein kinase C δ (PKCδ) and Ca 2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H 2 O 2 caused the accumulation of p53, which was inhibited by rottlerin (a PKCδ inhibitor) but not by BAPTA-AM (an intracellular Ca 2+ chelator). PKCδ itself was activated through the phosphorylation at tyrosine residues. H 2 O 2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKCδ contributes to the accumulation of p53 and that Ca 2+ plays a role in downstream signals of p53 leading to apoptosis in H 2 O 2 -treated BAEC. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-l-cystein (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-XL and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO–OOH was detected. This NADH/succinate-dependent production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances production from mitochondria to trigger cytochrome c release in A549 cells. [Copyright &y& Elsevier]