Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule

Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G₁ phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G₂/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.     

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-X_L, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.     

Radiosensitization by nitric oxide at low radiation doses

Nitric oxide was shown to radiosensitize anoxic V79 and CHO hamster cells and MCF7 and UT-SCC-14 human cells, measuring clonogenic survival and/or DNA damage in vitro at low radiation doses (0.1-5 Gy). Radiosensitization was easily detected after 2 Gy in anoxic V79 cells exposed to 40 ppm ( approximately 70 nM) nitric oxide, indicating that nitric oxide is a significantly more efficient radiosensitizer than oxygen. The yield of double-strand breaks (as gamma-H2AX foci) in V79 and MCF7 cells was doubled by irradiation in 1% v/v nitric oxide/N(2), and there was a longer repair time in cells irradiated in nitric oxide than in air or anoxia; single-strand breaks ("comet" assay) also appeared to be enhanced. Potent radiosensitization by nitric oxide is consistent with near diffusion-controlled reaction of nitric oxide with purine and pyrimidine radicals observed by pulse radiolysis, with nitric oxide reacting two to three times faster than oxygen with the 5-hydroxy-uracil-6-yl radical. Stable NO/base adducts were formed with uracil radicals. Effects on the radiosensitivity of cells exposed to as low as 40 ppm v/v nitric oxide after doses of 1-2 Gy suggest that variations in radiosensitivity in individual patients after radiotherapy might include a component reflecting differing levels of nitric oxide in tumors.     

Transformation depending on intermolecular homologous recombination is stimulated by UV damage in transfected DNA

DNA-mediated gene transfer (DMGT) was performed in DNA repair-proficient and UV-hypersensitive, repair-deficient Chinese hamster ovary (CHO) cell lines using the UV-irradiated thymidine kinase gene from herpes simplex virus (HSV-TK). Transformation frequencies in repair-deficient CHO cell lines declined relative to repair-proficient cells with increasing UV damage in transfected DNA; approximately 3-fold higher UV fluence was required to inactivate 50% of irradiated HSV-TK plasmid molecules in repair-proficient cells. In cotransfection experiments performed with pairs of HSV-TK plasmids containing linker insertion mutations in TK coding sequences, moderate UV damage in plasmid DNA enhanced the yield of TK+ transformants resulting from homologous recombination between HSV-TK sequences up to 4-fold. These results suggest that UV damage in DNA can stimulate transformation of mammalian cells dependent on intermolecular DNA homology.     

Hypersensitivity to cell killing and mutation induction by chemical carcinogens in an excision repair-deficient mutant of CHO cells

A strain of Chinese hamster ovary cells that is deficient in nucleotide excision repair, strain UV5, was compared with the normal parental CHO cells in terms of cytotoxicity and mutagenesis after exposure to several chemical carcinogens that are known to produce bulky, covalent adducts in DNA. Induced mutations were measured at the hprt locus using thioguanine resistance and at the aprt locus using azaadenine resistance. The compounds tested that required metabolic activation (using rat or hamster microsomal fractions) were 7,12-dimethylbenz(a)anthracene, 3-methylcholanthrene, benzo(a)pyrene, aflatoxin B1, 2-acetylaminofluorene, and 2-naphthylamine. The direct-acting compounds (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, N-acetoxy-2-acetylaminofluorene, and N-OH-2-naphthylamine were also studied. For all compounds except 2-naphthylamine and its active metabolite, the repair-deficient cells were significantly more sensitive to killing than the normal CHO cells. Mutation induction at both loci was also more efficient in UV5 cells in each instance where enhanced cytotoxicity was observed. By using tritium-labeled N-acetoxy-2-acetylaminofluorene, normal and mutant cells were shown to bind mutagen to their nuclear DNA with similar efficiency, and a greater amount of adduct removal occurred in the normal cells. From this study it is concluded that the use of excision repair-deficient CHO cells provides enhanced sensitivity for detecting mutagenesis and that a positive differential cytotoxicity response gives an indication of repairable, potentially lethal genetic damage.     

Individual and interactive effects of ocean acidification,global warming,and UV radiation on phytoplankton

Rising carbon dioxide (CO₂) concentrations in the atmosphere result in increasing global temperatures and ocean warming (OW). Concomitantly, dissolution of anthropogenic CO₂ declines seawater pH, resulting in ocean acidification (OA) and altering marine chemical environments. The marine biological carbon pump driven by marine photosynthesis plays an important role for oceanic carbon sinks. Therefore, how ocean climate changes affect the amount of carbon fixation by primary producers is closely related to future ocean carbon uptake. OA may upregulate metabolic pathways in phytoplankton, such as upregulating ß-oxidation and the tricarboxylic acid cycle, resulting in increased accumulation of toxic phenolic compounds. Ocean warming decreases global phytoplankton productivity; however, regionally, it may stimulate primary productivity and change phytoplankton community composition, due to different physical and chemical environmental requirements of species. It is still controversial how OA and OW interactively affect marine carbon fixation by photosynthetic organisms. OA impairs the process of calcification in calcifying phytoplankton and aggravate ultraviolet (UV)-induced harms to the cells. Increasing temperatures enhance the activity of cellular repair mechanisms, which mitigates UV-induced damage. The effects of OA, warming, enhanced exposure to UV-B as well as the interactions of these environmental stress factors on phytoplankton productivity and community composition, are discussed in this review.     

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.     

Effect ofγ-irradiated carbohydrates on isolated synaptic membranes

The effect of-irradiated solutions of carbohydrates, mainly glucose, upon Na⁺, K⁺-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe²⁺ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe²⁺-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.     

Human cytochrome P450 reductase can act as a source of endogenous oxidative DNA damage and genetic instability

Studies with repair-deficient mice and other experiments suggest that oxidative DNA modifications are generated in all types of cells even under physiological conditions and that this type of endogenous DNA damage contributes to spontaneous cancer incidence. However, the cellular sources of reactive oxygen species that are relevant for nuclear oxidative DNA damage are largely unknown. Here, we report that expression of human NADPH-cytochrome P450 reductase (hOR) in cultured V79 Chinese hamster cells gives rise to elevated basal levels of oxidative purine modifications after depletion of glutathione. Also, the basal levels of micronuclei are increased in the hOR-expressing cells, and again the effect is enhanced when the antioxidant defense system of the cells is diminished by depletion of glutathione. The oxidative DNA damage is increased when duroquinone, a substrate of hOR, is added, both in the presence and absence of glutathione. In contrast, hOR-expressing cells are similarly sensitive as the parental cells when oxidative DNA damage and micronuclei are induced by a mechanism independent of hOR, i.e., exposure to bromate. The results identify hOR as a potential source of endogenous oxidative DNA damage and subsequent genetic instability in mammalian cells.     

Characterization and antimutagenic activity of soybean saponins

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.     

Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 chinese hamster cells after treatment with colcemid,diamide, carbaryl and methyl mercury

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30–40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.     

Influence of poly(ADP-ribose) synthesis inhibitors on the repair of sublethal and potentially lethal damage in gamma-irradiated mammalian cells

The influence of poly(ADP-ribose) synthesis inhibitors on mammalian cell radiosensitivity was investigated. Four different inhibitors were studied: 3-methoxybenzamide, 3-aminobenzamide, 6-aminonicotinamide and nicotinamide. When exponentially growing or plateau phase cells are incubated before irradiation with non-toxic concentrations of these compounds, their radiosensitivity is enhanced except in the case of nicotinamide. The poly(ADP-ribose) inhibitors do not modify the repair of sublethal damage, but totally suppress the repair of potentially lethal damage, as shown by the survival of CHO cells and of a human osteosarcoma cell line.     

Non-enzymatic fast repair of DNA oxidative damage might also exist in cells

Many studies have shown that in a chemical system natural polyphenols can rapidly repair DNA oxidative damage. In this paper we report that in a cellular system the non-enzymatic fast repair activities of two natural polyphenols might also exist. The viability of a Chinese hamster ovary cell line (AA8) highly expressing the XRCC1 gene (a DNA repairing protein) treated with H2O2 is significantly higher than that of a normal Chinese hamster ovary cell line (CHO). Following inhibition of the enzymatic repair system by different inhibitors--methoxyamine (MX), 3-aminobenzamide (3AB) or nicotinamide (NIC)--DNA oxidative damage by H2O2 increased 2-5-fold in both cell lines. However, when natural polyphenols--rosmarinic acid (RA) or verbascoside (VER)--were added, DNA oxidative damage was significantly reduced. This decrease of DNA oxidative damage by RA or VER is not due to their scavenging activity for reactive oxygen species (ROS) because cells suffered from heavy ROS throughout the whole experimental process. Therefore, the decrease of DNA damage might be due to their non-enzymatic fast repair mechanisms. Further investigation showed that H2O2 induced a drop in the mitochondrial membrane potential (MMP), and that RA and VER were able to attenuate the drop. Previous studies have shown that H2O2 initiates a chain of events in cells, involving mtDNA damage, a drop in MMP and loss of repair activity. These results, taken together with our present results, suggest that the non-enzymatic fast repair mechanism exists not only in chemical systems but also might exist in cells.     

The free-radical theory of ageing--older, wiser and still alive: modelling positional effects of the primary targets of ROS reveals new support

The continuing viability of the free-radical theory of ageing has been questioned following apparently incompatible recent results. We show by modelling positional effects of the generation and primary targets of reactive oxygen species that many of the apparently negative results are likely to be misleading. We conclude that there is instead a need to look more closely at the mechanisms by which free radicals contribute to age-related dysfunction in living systems. There also needs to be deeper understanding of the dynamics of accumulation and removal of the various kinds of molecular damage, in particular mtDNA mutations. Finally, the expectation that free-radical damage on its own might cause ageing needs to be relinquished in favour of the recognition that the free-radical theory is just one of the multiple mechanisms driving the ageing process.     

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.Key words: AKT, DNA repair, KU-60019, temozolomide     

Seasonal changes in blood cells and biochemical parameters in the Mongolian hamster (<Emphasis Type="Italic">Allocricetulus curtatus</Emphasis>)

It was shown previously that the Mongolian hamster (Allocricetulus curtatus) is a mammalian species with irregular short hibernation. The purpose of the present study was to determine how this status affects seasonal changes in the biochemical and hematological parameters in A. curtatus males under a natural temperature and light regime. It was found that a reduction in circulating white blood cells, specifically lymphocytes, neutrophils, eosinophils, basophils, and monocytes, occurred in winter and bilirubin levels increased in spring. These characteristics make Mongolian hamsters closer to the true hibernating species. At the same time, the character of seasonal changes in the number of red blood cells, glucose, total protein, creatinine, and albumin is closer to species with torpor.     

Semiconservative and unscheduled DNA synthesis on mammalian cells and its modification by glyoxylic compounds

The interaction of glyoxal and four other glyoxylic compounds with semiconservative DNA synthesis and with unscheduled DNA synthesis induced by 25-G X-rays on TC-SV40 hamster cells has been studied. Both syntheses were evaluated by measuring the incorporation of [3H-methyl]-thymidine into the newly synthetized DNA. The unscheduled DNA synthesis amounts to 4% of semiconservative synthesis. The modification of both syntheses by the glyoxylic compounds was tested using the products at non-toxic concentrations for the cells. All the glyoxals inhibited semiconservative DNA synthesis and potentiated unscheduled DNA synthesis at rather similar levels. These effects have been compared with the radiosensitizing activity of these glyoxals in the same TC-SV40 cells and no relationship could be established.     

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.