Peter O'Neill

Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis

An important stage in tumorigenesis is the ability of a precancerous cell to escape natural anticancer signals imposed on it by neighboring cells and its microenvironment. We have previously characterized a system of intercellular induction of apoptosis whereby nontransformed cells selectively remove transformed cells from coculture via cytokine and reactive oxygen/nitrogen species (ROS/RNS) signaling. We report that irradiation of nontransformed cells with low doses of either high linear energy transfer (LET) alpha-particles or low-LET gamma-rays leads to stimulation of intercellular induction of apoptosis. The use of scavengers and inhibitors confirms the involvement of ROS/RNS signaling and of the importance of transformed cell NADPH oxidase in the selectivity of the system. Doses as low as 2-mGy gamma-rays and 0.29-mGy alpha-particles were sufficient to produce an observable increase in transformed cell apoptosis. This radiation-stimulated effect saturates at very low doses (50 mGy for gamma-rays and 25 mGy for alpha-particles). The use of transforming growth factor-beta (TGF-beta) neutralizing antibody confirms a role for the cytokine in the radiation-induced signaling. The system may represent a natural anticancer mechanism stimulated by extremely low doses of ionizing radiation.

Induction and rejoining of DNA double-strand breaks in V79-4 mammalian cells following gamma- and alpha-irradiation.

The induction and rejoining of DNA double-strand breaks (dsbs) in V79-4 mammalian cells following irradiation by 60Co gamma-rays and 238Pu alpha-particles (average LET 120 keV microns-1) under aerobic conditions have been determined using both the sucrose sedimentation and filter elution techniques under non-denaturing conditions. Cellular inactivation was also determined. The dependence of the initial yield of dsbs at 277 K on dose under aerobic conditions is linear with a relative biological effectiveness (RBE) for alpha-particles of 0.85 +/- 0.14 (sedimentation) and 0.68 +/- 0.12 (elution) compared with 60Co gamma-rays. The ability of the cells to rejoin dsbs at 310K is significantly reduced for alpha-irradiations with only 30-50% rejoined for a 3-h incubation period. With low LET radiation, > 90% of the dsbs are rejoined within 3 h at a dose of 20 Gy. The RBE for cellular inactivation was determined to be 4.0 at the 1% survival level. From the cellular dimensions and the D0-value for cellular inactivation by alpha-particles, it is determined that, on average, 4.7 tracks traverse the cell nucleus per lethal lesion. Under hypoxic conditions, the RBE values for induction of dsbs and cellular inactivation (10% level) by alpha-particles are approximately 3.0 and approximately 11.8 respectively. From these findings, it is suggested that the residual DNA damage and not the initial damage is reflected in the cellular inactivation. It is inferred that the difference in repair of the various lesions is a reflection of the differences in the complexity of the clustered damage produced by these radiations.

Induction and quantification of γ-H2AX foci following low and high LET-irradiation

Purpose: To investigate quantitatively the induction and rejoining of DNA double strand breaks (DSB) in V79-4 and xrs-5 Chinese hamster cells and HF19 human fibroblast cells, using the phosphorylation of the histone protein H2AX (γ-H2AX) as an indicator of DSB, exposed to low doses of either low linear energy transfer (LET) 60Co γ-rays or high LET α-particles. Materials and methods: Cells were irradiated with low or high LET (20 – 2000 mGy). The γ-H2AX foci were detected using immunohistochemistry and quantified by image analysis. Results: The number of DSB determined 30 min post γ-irradiation at 37°C is 12.2 (±1.5), 13.5 (±1.6) and 19.1 (±1.7) foci/cell/Gy for V79-4, xrs-5 and HF19 cells respectively, comparable with levels detected in V79-4 cells using pulse field gel electrophoresis. 6 h post γ-irradiation, γ-H2AX foci levels in V79-4 and HF19 cells approach control levels but remain higher in DSB repair deficient xrs-5 cells. γ-H2AX foci levels remain significantly higher than controls at 6 h in α-irradiated cells. Conclusions: γ-radiation and α-radiation induced the phosphorylation of H2AX in response to DSB at low doses; the variation in the rate of dephosphorylation of induced foci are dependent both on radiation quality and cell characteristics.

Extracellular Matrix Metalloproteinase Inducer (CD147) Confers Resistance of Breast Cancer Cells to Anoikis through Inhibition of Bim

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, and growth and survival of malignant cells and confers resistance to some chemotherapeutic drugs. However, the molecular mechanisms underlying the actions of EMMPRIN are not fully understood. In this study we sought to determine whether EMMPRIN contributes to the malignant phenotype of breast cancer by inhibiting anoikis, a form of apoptosis induced by loss or alteration of cell-cell or cell-matrix anchorage, and to explore the signaling pathways involved. We found that in the absence of attachment, human breast carcinoma cells expressing high levels of EMMPRIN formed less compact aggregates with larger surface area and less fibronectin matrix assembly, had higher viability, and were resistant to anoikis. Knockdown of EMMPRIN expression by RNA interference (small interfering RNA or short hairpin RNA) sensitized cancer cells to anoikis, as demonstrated by activation of caspase-3, increased DNA fragmentation, and decreased cellular viability. Furthermore, we observed that the accumulation of Bim, a proapoptotic BH3-only protein, was reduced in EMMPRIN-expressing cells and that silencing of EMMPRIN expression elevated Bim protein levels and enhanced cellular sensitivity to anoikis. Treatment of cells with a MEK inhibitor (U0126) or proteasome inhibitor (epoxomicin) also up-regulated Bim accumulation and rendered cells more sensitive to anoikis. These results indicated that expression of EMMPRIN protects cancer cells from anoikis and that this effect is mediated at least in part by a MAP kinase-dependent reduction of Bim. Because anoikis deficiency is a key feature of neoplastic transformation and invasive growth of epithelial cancer cells, our study on the role of EMMPRIN in anoikis resistance and the mechanism involved underscores the potential of EMMPRIN expression as a prognostic marker and novel target for cancer therapy.

The Effect of Dimethyl Sulfoxide on the Induction of DNA Double-Strand Breaks in V79-4 Mammalian Cells by Alpha Particles

The present study was undertaken to assess the protective effect of dimethyl sulfoxide (DMSO) against the induction and rejoining of DNA double-strand breaks (DSBs) and inactivation of V79-4 Chinese hamster cells by both high- and low-linear energy transfer (LET) radiations. The cells were exposed under aerobic conditions as monolayers to either low-LET photons (60Co gamma rays) or high-LET alpha particles (238Pu) at 277 K. The initial yield of DSBs, determined by elution under nondenaturing conditions, is linearly dependent on dose. When the irradiation was carried out in the presence of DMSO (0-0.6 mol dm-3), the initial yields of DSBs induced by both gamma and alpha-particle irradiation decrease. With gamma irradiation at [DMSO] > 0.6 mol dm-3, a further decrease in the yield of DSBs occurs. DMSO (0.5 mol dm-3) reduces the initial yield of DSBs by 50 +/- 5% and 32 +/- 4% for photons and alpha particles, respectively. DMSO protects more effectively against cellular inactivation and DSB induction at low LET compared with alpha-particle irradiation with protection factors of 1.7 and 1.4, respectively, for survival and 2.0 and 1.5, respectively, for DSBs. After incubation of the irradiated cells for 3 h at 310 K after high-LET irradiation, the residual yield of DSBs is reduced by < 13% when the irradiations were carried out in the presence of 0.5 mol dm-3 DMSO. With gamma irradiation in the presence of 0.5 mol dm-3 DMSO, 90% of the DSBs are rejoined by 3 h incubation at 310 K. Therefore, the nonscavengeable DSBs induced by alpha particles are not significantly rejoined within 3 h, in contrast to rejoining of the majority of the nonscavengeable DSBs induced by gamma irradiation. From comparison of the data on DSBs and survival for alpha-particle irradiation, it is inferred that the severity of damage is reduced by DMSO through minimizing the formation of OH-induced sugar/base modifications in the vicinity of nonscavengeable DSBs.

The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA base damage

The potential for genetic change arising from specific single types of DNA lesion has been thoroughly explored, but much less is known about the mutagenic effects of DNA lesions present in clustered damage sites. Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. We have investigated the potential of a non-mutagenic DNA base lesion, 5,6-dihydrothymine (DHT), to influence the mutagenicity of 8-oxo-7,8-dihydroguanine (8-oxoG) when the two lesions are closely opposed. Using a bacterial plasmid-based assay we present the first report of a significantly higher mutation frequency for the clustered DHT and 8-oxoG lesions than for single 8-oxoG in wild-type and in glycosylase-deficient strains. We propose that endonuclease III has an important role in the initial stages of processing DHT/8-oxoG clusters, removing DHT to give an intermediate with an abasic site or single-strand break opposing 8-oxoG. We suggest that this mutagenic intermediate is common to several different combinations of base lesions forming clustered DNA damage sites. The MutY glycosylase, acting post-replication, is most important for reducing mutation formation. Recovered plasmids commonly gave rise to both wild-type and mutant progeny, suggesting that there is differential replication of the two DNA strands carrying specific forms of base damage.

Rejoining of gamma-radiation-induced single-strand breaks in plasmid DNA by human cell extracts: Dependence on the concentration of the hydroxyl radical scavenger, Tris

The rejoining of single-strand breaks induced by gamma irradiation in plasmid DNA under different scavenging conditions is described using human cell extracts. As the scavenging capacity of the irradiated solution increases from 1.5 x 10(7) to 3 x 10(8) s-1 using Tris-HCl as a scavenger, the ratio of single- to double-strand breaks is reduced from approximately 70:1 to 40:1. After irradiation, a proportion of DNA molecules have no initial strand breaks but contain damage that is converted to strand breaks when incubated either at 37 degrees C or in the presence of cellular extract. Repair of damage by the extracts is dependent upon the scavenging capacity of the irradiated solution. Optimal rejoining is observed when the scavenging capacity is < 1.5 x 10(7) s-1, and results in the repair of some initial strand breaks. As the scavenging capacity increases to 3 x 10(8) s-1 the proportion of breaks repaired is significantly reduced. The relative increase in the yield of double-strand breaks and reduced repairability of single-strand breaks at a scavenging capacity of 3 x 10(8) s-1 is consistent with the concept that the severity of damage increases upon increasing the scavenger concentration.

Participation of DNA-PKcs in DSB Repair after Exposure to High- and Low-LET Radiation

Abstract Cellular lesions (e.g. DSBs) are induced into DNA upon exposure to radiation, with DSB complexity increasing with radiation ionization density. Using M059K and M059J human glioblastoma cells (proficient and deficient in DNA-PKcs activity, respectively), we investigated the repair of DNA damage, including DSBs, induced by high- and low-LET radiation [γ rays, α particles and high-charge and energy (HZE) ions]. In the absence of DNA-PKcs activity, less DSB repair and increased recruitment of RAD51 was seen at 24 h. After exposure to 56Fe heavy ions, the number of cells with RAD51 tracks was less than the number of cells with γ-H2AX at 24 h with both cell lines. Using α particles, comparable numbers of cells with visible γ-H2AX and RAD51 were seen at 24 h in both cell lines. M059J cells irradiated with α particles accumulated in S phase, with a greater number of cyclin A and RAD51 co-stained cells seen at 24 h compared with M059K cells, where an S-phase block is absent. It is proposed that DNA-PKcs plays a role in the repair of some frank DSBs, which are longer-lived in NHEJ-deficient cells, and some non-DSB clustered damage sites that are converted into DSBs at replication as the cell cycles through to S phase.

Induction of DNA strand breaks by RSU-1069, a nitroimidazole-aziridine radiosensitizer: Role of binding of both unreduced and radiation-reduced forms to DNA, in vitro

[2-14C]-RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol], either as a parent (unreduced) or following radiation reduction, binds to calf thymus DNA in vitro. Radiation-reduced RSU-1069 binds to a greater extent and more rapidly than the parent compound. RSU-1137, a nonaziridino analogue of RSU-1069, binds following radiation reduction. Radiation-reduced misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) exhibits binding ratios a thousand-fold less than those of reduced RSU-1069. There is no evidence for binding of parent misonidazole. Both parent and reduced RSU-1069 cause single strand breaks (ssbs) in pSV2 gpt plasmid DNA with the reduced compound causing a greater number of breaks. Parent and reduced RSU-1137 and misonidazole do not cause ssbs. It is inferred that the aziridine moiety present in both parent and reduced RSU-1069 is required for ssb production. RSU-1069 reacts with inorganic phosphate probably via nucleophilic ring-opening of the aziridine fragment. Incubation of plasmid DNA with reduced RSU-1069 in the presence of either phosphate or deoxyribose-5-phosphate at concentrations greater than 0.35 mol dm-3 prevents strand breakage, whereas 1.2 mol dm-3 deoxyribose does not protect against strand breakage formation. From these findings it is proposed that the observed binding to DNA occurs via the aziridine and the reduced nitro group of RSU-1069 and that these two have different target sites. Binding to DNA via the reduced nitro group may serve to increase aziridine attack due to localization at or near its target.

Ionization of polynucleotides and DNA in aqueous solution by 193 nm pulsed laser light: identification of base-derived radicals.

Light of 193 nm wavelength ionizes polyA, polyC, polyG, and ss and ds DNA in aqueous solution in a monophotonic process giving hydrated electrons and radicals that result from the radical cations of the bases; there is spectroscopic evidence for positive charge migration in DNA from adenine to guanine moieties.