Connexins and cyclooxygenase-2 crosstalk in the expression of radiation-induced bystander effects.

BACKGROUND: Signalling events mediated by connexins and cyclooxygenase-2 (COX-2) have important roles in bystander effects induced by ionising radiation. However, whether these proteins mediate bystander effects independently or cooperatively has not been investigated. METHODS: Bystander normal human fibroblasts were cocultured with irradiated adenocarcinoma HeLa cells in which specific connexins (Cx) are expressed in the absence of endogenous Cx, before and after COX-2 knockdown, to investigate DNA damage in bystander cells and their progeny. RESULTS: Inducible expression of gap junctions composed of connexin26 (Cx26) in irradiated HeLa cells enhanced the induction of micronuclei in bystander cells (P<0.01) and reduced the coculture time necessary for manifestation of the effect. In contrast, expression of connexin32 (Cx32) conferred protective effects. COX-2 knockdown in irradiated HeLa Cx26 cells attenuated the bystander response due to connexin expression. However, COX-2 knockdown resulted in enhanced micronucleus formation in the progeny of the bystander cells (P<0.001). COX-2 knockdown delayed junctional communication in HeLa Cx26 cells, and reduced, in the plasma membrane, the physical interaction of Cx26 with MAPKKK, a controller of the MAPK pathway that regulates COX-2 and connexin. CONCLUSIONS: Junctional communication and COX-2 cooperatively mediate the propagation of radiation-induced non-targeted effects. Characterising the mediating events affected by both mechanisms may lead to new approaches that mitigate secondary debilitating effects of cancer radiotherapy.

Radiation-induced non-targeted response in vivo: role of the TGFß-TGFBR1-COX-2 signalling pathway.

BACKGROUND: Previous studies from our group and others have shown that cyclooxygenase-2 (COX-2) has an essential role in radiation-induced non-targeted responses and genomic instability in vivo. However, the signalling pathways involved in such effects remain unclear. METHODS: A 1 cm(2) area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays. Nimesulide, a selective COX-2 inhibitor, was given to mice for five consecutive days before irradiation. Changes in transforming growth factor-beta (TGF-ß) and TGF-ß receptor type-1 (TGFBR1) mediated signalling pathways, in the out of radiation field lung and liver tissues were examined. RESULTS: While the plasma level of cytokines remained unchanged, the expression of TGF-ß and its receptors was elevated in non-targeted lung tissues after partial body irradiation. In contrast to the predominant expression of TGF-ß in stromal and alveolar cells, but not in bronchial epithelial cells, TGF-ß receptors, especially TGFBR1 were significantly elevated in non-targeted bronchial epithelial cells, which is consistent with the induction of COX-2. The different expression levels of TGFBR1 between liver and lung resulted in a tissue specific induction of COX-2 in these two non-targeted tissues. Multiple TGF-ß induced signalling pathways were activated in the non-targeted lung tissues. CONCLUSION: The TGFß-TGFBR1-COX-2 Signalling Pathway has a critical role in radiation-induced non-targeted response in vivo.

Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice.

BACKGROUND: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. METHODS: A 1-cm(2) area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. RESULTS: Compared with sham-treated controls, the Spi(-) mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. CONCLUSION: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis.

Dynamic equilibrium between cancer stem cells and non-stem cancer cells in human SW620 and MCF-7 cancer cell populations.

BACKGROUND: Cancer stem cells (CSCs) paradigm suggests that CSCs might have important clinical implications in cancer therapy. Previously, we reported that accumulation efficiency of CSCs is different post low- and high-LET irradiation in 48 h. METHODS: Cancer stem cells and non-stem cancer cells (NSCCs) were sorted and functionally identified through a variety of assays such as antigen profiles and sphere formation. Inter-conversion between CSCs and NSCCs were in situ visualised. Cancer stem cells proportions were assayed over multiple generations under normal and irradiation surroundings. Supplement and inhibition of TGF-ß1, as well as immunofluorescence assay of E-cadherin and Vimentin, were performed. RESULTS: Surface antigen markers of CSCs and NSCCs exist in an intrinsic homoeostasis state with spontaneous and in situ visualisable inter-conversions, irrespective of prior radiations. Supplement with TGF-ß1 accelerates the equilibrium, whereas inhibition of TGF-ß signalling disturbs the equilibrium and significantly decreases CSC proportion. Epithelial mesenchymal transition (EMT) might be activated during the process. CONCLUSION: Our results indicate that the intrinsic inter-conversion and dynamic equilibrium between CSCs and NSCCs exist under normal and irradiation surroundings, and TGF-ß might have important roles in the equilibrium through activating EMT.

Mechanism of genotoxicity induced by targeted cytoplasmic irradiation.

BACKGROUND: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown. METHODS: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human-hamster hybrid (A(L)) cells. RESULTS: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in A(L) cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of A(L) cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway. CONCLUSION: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.

Recent progress on the Chinese space programme and radiation research.

Manned space exploration was initiated in China in 1992, and substantial progress has been made. The next step is to build the Chinese Space Station (CSS), which is planned to be launched in 2020. The CSS will provide an on-orbit laboratory for experimental studies including space radiation research. The health risk of space radiation, especially carcinogenesis, is a major concern for long-term space exploration. Establishing a risk assessment system suitable for Chinese astronauts and developing effective countermeasures are major tasks for Chinese space radiobiologists. The Institute of Space Life Sciences, Soochow University has focused on these topics for years. We established cancer models with low-dose-rate exposure of alpha particles, and elucidated a microRNA-TGFß network regulating bystander effects and a lncRNA-cytoskeleton network regulating genomic instability induced by ionising radiation. We also confirmed the radioresistance of quiescent cells, which inspires a potential strategy to improve individual radioresistance during long-term space travel. However, we believe that a multi-disciplinary strategy must be developed to protect astronauts from highly energised space radiation.

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells.

The initiation and propagation of the early processes of bystander signaling induced by low-dose alpha-particle irradiation are very important for understanding the underlying mechanism of the bystander process. Our previous investigation showed that the medium collected from cell culture exposed to low-dose alpha-particle rapidly induced phosphorylated form of H2AX protein foci formation among the non-irradiated medium receptor cells in a time-dependent manner. Using N(G)-methyl-L-arginine, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and N(omega)-nitro-L-arginine (L-NNA) treatment before exposure to 1 cGy alpha-particle, we showed in the present study that nitric oxide (NO(*)) produced in the irradiated cells was important and necessary for the DNA double strand break inducing activity (DIA) of conditioned medium and the generation of NO(*) in irradiated confluent AG1522 cells is in a time-dependent manner and that almost all NO(*) was generated within 15 min post-irradiation. Concurrently, the kinetics of NO(*) production in the medium of irradiated cells after irradiation was rapid and in a time-dependent manner as well, with a maximum yield observed at 10 min after irradiation with electron spin resonance analysis. Furthermore, our results that 7-Nitroindazole and L-NNA, but not aminoguanidine hemisulfate, treatment before exposure to 1 cGy alpha-particle significantly decrease the DIA of the conditioned medium suggested that constitutive NO(*) from the irradiated cells possibly acted as an intercellular signaling molecule to initiate and activate the early process (

Mitochondria-dependent signalling pathway are involved in the early process of radiation-induced bystander effects.

Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (rho(0)) A(L) or normal (rho(+)) A(L) cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal A(L) cells treated with mitochondrial respiratory chain function inhibitors had an attenuated gamma-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal A(L) donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased gamma-H2AX induction and that radiation could stimulate cellular NO and O(2)(*-) production in irradiated rho(+) A(L) cells, but not in rho(0) A(L) cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O(2)(*-) levels in irradiated rho(+) A(L) cells, suggest that radiation-induced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process.

Growth factor biomarkers associated with estrogen- and radiation-induced breast cancer progression.

Breast cancer is the most common cancer in women worldwide. Transformation of a normal cell to a malignant one results from mutations in genes that encode key regulatory proteins. Growth factors are proteins secreted by a variety of transformed cells and tumors and function as autocrine regulators of growth. Biomarkers associated with cancer were examined in human breast epithelial cells transformed by high-LET radiation in the presence of 17beta-estradiol. An established cancer model was used in these studies. The MCF-10F cells that were irradiated with double doses of alpha-particles in the presence of estrogen (60 cGy + E/60 cGy + E, named Alpha 5) showed gradual phenotypic changes relative to control, including tumorigenicity in heterologous animals. Protein expression was determined by quantification of immunofluorescence staining coupled with confocal microscopy. The transforming growth factor alpha, epidermal growth factor, ERK1 and fibroblast growth factor-1 (Int2) protein expression was analyzed. Increased protein expression was observed in non-tumorigenic and tumorigenic alpha-irradiated and estrogen-treated cells. However, Stat-1alpha and pS2 protein expression was only increased in the tumorigenic Alpha 5 and Tumor 2 cell lines. It can be concluded that high-LET radiation in the presence of estrogen-induced changes in the proteins associated with growth factors and their overexpression may be a critical step in the cascade of events that characterize progression in breast cancer.

Allelic imbalance at 11q23-q24 chromosome associated with estrogen and radiation-induced breast cancer progression.

Multiple genetic alterations are common in cancers including those of the breast. The mechanisms leading to these alterations such as point mutations, gene amplifications, deletions and replication error are often associated with frequent and consistent loss of heterozygosity (LOH) or microsatellite instability (MSI). Several cytological and molecular studies have shown high frequency loss of genetic information on the long arm of chromosome 11 (i.e., 11q) in various primary breast cancers. In the present study allelic alterations in a refined position on the long arm of chromosome 11 were studied to identify the spectrum of induced damage at different stages of malignant transformation of MCF-10F cell lines after exposure to high-LET radiation using alpha-particles and exposure to estradiol by using PCR-single strand conformation polymorphism (SSCP) and fluorescence in situ hybridization (FISH) analysis. Microsatellite markers were selected from chromosome 11 (11q23-q24 loci) and it was found that frequency of allelic imbalance occurs at different stages of tumor progression with a range of 15-45% depending on the marker studied. These results strongly suggested the presence of several tumor suppressor genes in this critical region of chromosome 11 (11q23-q24). It also represents the first indication of allele loss at these loci in human breast epithelial cells induced by radiation and estrogen treatment suggesting a potential interventional target in breast carcinogenesis.

Taxol, radiation, and oncogenic transformation.

The novel antineoplastic drug taxol has been shown to be active clinically against several types of human tumors. With improvement in treatment strategy and the number of long-term survivors increasing, a question that needs to be addressed is the potential carcinogenic effect of the treatment in the induction of second malignancies. We show here that when tested using an in vitro assay for oncogenic transforming potential, taxol is ineffective in focus induction at doses significantly higher than those used in the clinic. However, taxol enhances the oncogenic potential of gamma-rays in a synergistic fashion. The fact that taxol blocks cells at the G2/M phases of the cell cycle may account for this interaction since G2/M are relatively radiosensitive phases of the cell cycle.

Effects of a high-sucrose diet on the development of enzyme-altered foci in chemical hepatocarcinogenesis in rats.

Dietary factors can modify metabolic events involved in the initiation, promotion, or progression of tumors. To determine whether a high-sucrose diet has any effect on the development of enzyme-altered foci during the promotion step of chemical hepatocarcinogenesis in rats, 1-day-old Sprague-Dawley rats were given a single i.p. dose of diethylnitrosamine; controls received an equivalent i.p. volume of 0.9% NaCl solution. At 21 days of age, the rats were weaned, segregated by sex, separated in groups, and fed modified AIN76A diets containing either 65% glucose or 65% sucrose, with or without 0.05% phenobarbital. At the end of a 4-week treatment period, the sucrose-fed control rats of either sex had significantly heavier livers than did those on the glucose diet. Enlarged livers were found also in the sucrose-fed diethylnitrosamine-treated female rats, which developed twice as many gamma-glutamyltranspeptidase-positive foci per sq cm of liver section than did those on the glucose diet. Addition of phenobarbital augmented the number of foci 3-fold in the sucrose group and 5-fold in the glucose group. Focus count per sq cm was similar in animals on the two phenobarbital-supplemented diets. Despite the absence of statistically significant liver enlargement, results analogous to those in females were obtained in carcinogen-treated males. Differences between treatments, however, were smaller. In both female and male rats, the DNA-synthesizing activity of hepatocytes in enzyme-altered foci was significantly higher than in the surrounding normal parenchymal cells, as determined by autoradiography. These studies indicate that a high-sucrose diet has a promoting effect during hepatocarcinogenesis induced in the rat by diethylnitrosamine and that this effect is weaker than that of 0.05% phenobarbital.

Asbestos and DNA double strand breaks.

A radiosensitive DNA repair-deficient xrs-5 cell line was used to study asbestos cytotoxicity and DNA double strand breaks (DSBs). Although xrs-5 cells did not show any increase in sensitivity to chrysotile fibers in short-term (4-h) treatment when compared with wild-type CHO cells, longer-term exposure (24 h) gave significantly lower cell survival accompanied by a cell growth delay as well as a higher DNA DSB induction in this mutant cell line. These results suggest an important role played by DNA DSBs at the initial stage of asbestos injury.

Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells.

Crocidolite, one of the most carcinogenic forms of asbestos, is mutagenic in cultured mammalian cells when assayed using a system that can detect multilocus deletions. In the present study, we examined the effect of buthionine sulfoximine (BSO) on mutation frequency and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in human-hamster hybrid (A(L)) cells induced by crocidolite fibers in an attempt to determine the role of oxyradicals in mediating fiber mutagenesis. BSO, a competitive inhibitor of the enzyme gamma-glutamyl cysteine synthetase, depleted nonprotein sulfhydryls to <5% of control within 24 h at a nonmutagenic dose of 25 microM. In cells pretreated with BSO for 24 h, the mutation yield at the CD59 locus induced by a 4 microg/cm2 dose of crocidolite fibers was increased by more than 3-fold (P < 0.05). Using immunoperoxidase staining with a monoclonal antibody specific for 8-OHdG, we demonstrated that crocidolite fibers induced a dose-dependent increase in oxidative DNA damage in A(L) cells. Furthermore, addition of DMSO, a well-established hydroxyl radical (OH*) scavenger, dramatically suppressed 8-OHdG induction (P < 0.005). Our results definitely demonstrate that reactive oxygen species mediate fiber-induced DNA damage mutagenesis in A(L) cells in a concentration-dependent manner.

Chrysotile fiber is a strong mutagen in mammalian cells.

Although chrysotile asbestos is a proven human carcinogen, several studies have concluded that these fibers are not mutagenic to cultured mammalian cells. We show here, on the other hand, that when tested using the AL cell system that detects both intragenic and multilocus mutations, chrysotile is indeed mutagenic and comparable in strength to that of gamma-rays. Southern analysis of the induced mutants shows that the majority contains large deletions ranging in size from a few thousand to several million base pairs. Results of our study demonstrate that, while chrysotile may be less durable in vivo than the amphibole fibers such as crocidolites and amosites, it can effectively create genetic damage involved in the cancer process.

Analysis of p16 and p21(Cip1) expression in tumorigenic human bronchial epithelial cells induced by asbestos.

Although asbestos is carcinogenic to humans, the mechanism(s) by which it induces cancer is unknown. Using tumor cell lines generated previously by asbestos treatment of immortalized human bronchial epithelial (BEP2D) cells, we examined alterations in p16 and p21(Cip1) genes together with their protein levels. Results were compared with untreated BEP2D cells, normal human bronchial epithelial cells (NHBE), as well as non-tumorigenic fusion cell lines generated by fusing tumor cells with BEP2D cells. No deletion in the p16 gene was found in any of the tumor cell lines examined. Although p16 protein was expressed at a similar level in tumor and BEP2D cells, there was a fourfold decrease in its expression among NHBE cells. In contrast, both the protein and mRNA expression levels of p21(Cip1) were decreased by about threefold in tumor cell lines when compared with either BEP2D or NHBE cells, which had a similar expression level. Expression of p21(Cip1) mRNA was restored to the control level in all the fusion cell lines examined. The results suggested that down regulation of p21(Cip1) expression is linked to the tumorigenic conversion of BEP2D cells by asbestos.

Chromosome end-to-end associations and telomerase activity during cancer progression in human cells after treatment with alpha-particles simulating radon progeny.

Chromosome end-to-end associations seen at metaphase involve telomeres and are commonly observed in cells derived from individuals with ataxia telangiectasia and most types of human tumors. The associations may arise because of short telomeres and/or alterations of chromatin structure. There is a growing consensus that telomere length is stabilized by the activity of telomerase in immortal cells; however, it is not clear why some immortal cells display chromosome end-to-end associations. In the present study we evaluated chromosome end-to-end associations, telomere length and telomerase activity with the tumorigenic status of human bronchial epithelial cells immortalized with human papillomavirus. Oncogenic transformation was initiated using radon simulated alpha-particles and cells evaluated as primary, secondary and metastatic transformants. The fewest chromosome end associations and lowest telomerase activity were observed in the parental immortalized cells. However, increased levels of telomerase activity were detected in alpha-particle survivors while robust telomerase activity was seen in the tumorigenic cell lines. The tumorigenic cells that were telomerase positive and had the highest frequency of cells with chromosome end-to-end associations were also metastatic. No correlation was found between telomere length and the different stages of carcinogenicity.

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

In situ visualization of DSBs to assess the extranuclear/extracellular effects induced by low-dose alpha-particle irradiation.

Extranuclear/extracellular effects may have a significant effect on low-dose radiation risk assessment as well as on the shape of the dose-response relationship. Numerous studies using different end points such as sister chromatid exchanges, micronuclei and mutation have shown that this phenomenon exists in many cell types. However, these end points mostly reflect the late events after radiation damage, and little is known about the early response in this phenomenon. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using gamma-H2AX immunofluorescence staining, and there is evidence that the number of gamma-H2AX foci can be closely correlated with DSBs induced. Here we used gamma-H2AX as a biomarker to assess the extranuclear/extracellular effects induced by low-dose alpha particles in situ. The results show that a greater fraction of positive cells with DSBs (48.6%) was observed than the number of cells whose nuclei were actually traversed by the 1-cGy dose of alpha particles (9.2%). The fraction of DSB-positive cells was greatly reduced after treatment with either lindane or DMSO. These results suggest that in situ visualization of DSBs can be used to assess radiation-induced extranuclear/extracellular effects soon after irradiation. Moreover, the in situ DSB assay may provide a means to evaluate the spatial effect on unirradiated cells that are located in the neighboring region of cells irradiated by alpha particles.

Oncoprotein expression and morphological phenotypes of human breast epithelial cells transformed by the c-Ha-ras oncogene.

Activated oncogenes have been detected in a variety of malignant tumors and altered expressions of certain genes are known to play a functional role in the cancer process. The chemical carcinogen, BP, and the insertion of c-Ha-ras, induced characteristics of transformed phenotypes in a suitable human breast epithelial cell line. Carcinogen-treated and Ha-ras-transfected cells showed a progression of changes in the morphology, anchorage independent growth, invasiveness and tumorigenicity in SCID mice. Tumor growth occurs after a series of molecular events that parallel morphological changes. The aim of this work was to determine the neoplastic phenotypes following treatment with benzo(a)pyrene (BP) and transfection with c-Ha-ras oncogene changes and PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in MCF-10F cells, a spontaneously immortalized human breast epithelial cell line. Protein expression was determined by immunofluorescent staining coupled with confocal microscopy. An increased oncoprotein expression in comparison to MCF-10F cells was observed in PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in breast epithelial cells transformed with a chemical carcinogen and/or oncogene transfected that are not present in the MCF-10F. This in vitro cancer model can be used as a valuable model in the study of breast carcinogenesis.