Transcriptomic profiling of gamma ray induced mutants from the CGL1 human hybrid cell system reveals novel insights into the mechanisms of radiation-induced carcinogenesis.

BACKGROUND: Somatic cell hybrid systems generated by combining cancerous with non-cancerous cells provide useful model systems to study neoplastic transformation. Combined with recent advances in omics-based technologies, novel molecular signatures that drive radiation-induced carcinogenesis can be analyzed at an exceptional global level. METHODS: Here, we present a complete whole-transcriptome analysis of gamma-induced mutants (GIM) and gamma irradiated control (CON) segregants isolated from the CGL1 (HeLa x normal fibroblast) human hybrid cell-system exposed to high doses of radiation. Using the Human Transcriptome Array 2.0 microarray technology and conservative discrimination parameters, we have elucidated 1067 differentially expressed genes (DEGs) between tumorigenic and non-tumorigenic cells. RESULTS: Gene ontology enrichment analysis revealed that tumorigenic cells demonstrated shifts in extracellular matrix (ECM) and cellular adhesion profiles, dysregulation of cyclic AMP (cAMP) signaling, and alterations in nutrient transport and cellular energetics. Furthermore, putative upstream master regulator analysis demonstrated that loss of TGFß1 signaling due to reduced SMAD3 expression is involved in radiation-induced carcinogenesis. CONCLUSIONS: Taken together, this study presents novel insights into specific gene expression and pathway level differences that contribute to radiation-induced carcinogenesis in a human cell-based model. This global transcriptomic analysis and our published tumor suppressor gene deletion loci analyses will allow us to identify and functionally test candidate nexus upstream tumor suppressor genes that are deleted or silenced after exposure to radiation.

MODELING STUDIES ON DICENTRICS INDUCTION AFTER SUB-MICROMETER FOCUSED ION BEAM GRID IRRADIATION.

The biophysical simulation tool PARTRAC contains modules for DNA damage response representing non-homologous end joining of DNA double-strand breaks (DSB) and the formation of chromosomal aberrations. Individual DNA ends from the induced DSB are followed regarding both their enzymatic processing and spatial mobility, as is needed for chromosome aberrations to arise via ligating broken ends from different chromosomes. In particular, by tracking the genomic locations of the ligated fragments and the positions of centromeres, the induction of dicentrics can be modeled. In recent experiments, the impact of spatial clustering of DNA damage on dicentric yields has been assessed in AL human-hamster hybrid cells: Defined numbers of 20 MeV protons (linear energy transfer, LET 2.6 keV/µm), 45 MeV Li ions (60 keV/µm) and 55 MeV C ions (310 keV/µm) focused to sub-µm spot sizes were applied with the ion microbeam SNAKE in diverse grid modes, keeping the absorbed dose constant. The impact of the µm-scaled spatial distribution of DSB (focusing effect) has thus been separated from nm-scaled DSB complexity (LET effect). The data provide a unique benchmark for the model calculations. Model and parameter refinements are described that enabled the simulations to largely reproduce both the LET-dependence and the focusing effect as well as the usual biphasic rejoining kinetics. The predictive power of the refined model has been benchmarked against dicentric yields for photon irradiation.

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB.

Cellular transformation assays have been utilized for many years as powerful in vitro methods for examining neoplastic transformation potential/frequency and mechanisms of carcinogenesis for both chemical and radiological carcinogens. These mouse and human cell based assays are labor intensive but do provide quantitative information on the numbers of neoplastically transformed foci produced after carcinogenic exposure and potential molecular mechanisms involved. Several mouse and human cell systems have been generated to undertake these studies, and they vary in experimental length and endpoint assessment. The CGL1 human cell hybrid neoplastic model is a non-tumorigenic pre-neoplastic cell that was derived from the fusion of HeLa cervical cancer cells and a normal human skin fibroblast. It has been utilized for the several decades to study the carcinogenic/neoplastic transformation potential of a variety of ionizing radiation doses, dose rates and radiation types, including UV, X ray, gamma ray, neutrons, protons and alpha particles. It is unique in that the CGL1 assay has a relatively short assay time of 18-21 days, and rather than relying on morphological endpoints to detect neoplastic transformation utilizes a simple staining method that detects the tumorigenic marker alkaline phosphatase on the neoplastically transformed cells cell surface. In addition to being of human origin, the CGL1 assay is able to detect and quantify the carcinogenic potential of very low doses of ionizing radiation (in the mGy range), and utilizes a neoplastic endpoint (re-expression of alkaline phosphatase) that can be detected on both viable and paraformaldehyde fixed cells. In this article, we review the history of the CGL1 neoplastic transformation model system from its initial development through the wide variety of studies examining the effects of all types of ionizing radiation on neoplastic transformation. In addition, we discuss the potential of the CGL1 model system to investigate the effects of near zero background radiation levels available within the radiation biology lab we have established in SNOLAB.

The influence of reference radiation photon energy on high-LET RBE: comparison of human peripheral lymphocytes and human-hamster hybrid AL cells.

The relative biological effectiveness (RBE) based on the induction of dicentrics in any cell type is principally an important information for the increasing application of high-LET radiation in cancer therapy. Since the standard system of human lymphocytes for measuring dicentrics are not compatible with our microbeam irradiation setup where attaching cells are essential, we used human-hamster hybrid AL cells which do attach on foils and fulfil the special experimental requirement for microbeam irradiations. In this work, the dose-response of AL cells to photons of different energy, 70 and 200 kV X-rays and 60Co γ-rays, is characterized and compared to human lymphocytes. The total number of induced dicentrics in AL cells is approximately one order of magnitude smaller. Despite the smaller α and ß parameters of the measured linear-quadratic dose-response relationship, the α/ß-ratio versus photon energy dependence is identical within the accuracy of measurement for AL cells and human lymphocytes. Thus, the influence of the reference radiation used for RBE determination is the same. For therapy relevant doses of 2 Gy (60Co equivalent), the difference in RBE is around 20% only. These findings indicate that the biological effectiveness in AL cells can give important information for human cells, especially for studies where attaching cells are essential.

900 MHz radiation does not induce micronucleus formation in different cell types.

The exposure of the population to non-ionising electromagnetic radiation is still increasing, mainly due to mobile communication. Whether low-intensity electromagnetic fields can cause other effects apart from heating has been a subject of debate. One of the effects, which were proposed to be caused by mobile phone radiation, is the occurrence of mitotic disturbances. The aim of this study was to investigate possible consequences of these mitotic disturbances as manifest genomic damage, i.e. micronucleus induction. Cells were irradiated at a frequency of 900 MHz, which is located in one of the main frequency bands applied for mobile communication. Two cell types were used, HaCaT cells as human cells and A(L) cells (human-hamster hybrid cells), in which mitotic disturbances had been reported to occur. After different post-exposure incubation periods, cells were fixed and micronucleus frequencies were evaluated. Both cell types did not show any genomic damage after exposure. To adapt the protocol for the micronucleus test into the direction of the protocol for mitotic disturbances, the post-exposure incubation period was reduced and exposure time was extended to one cell cycle length. This did not result in any increase of the genomic damage. In conclusion, micronucleus induction was not observed as a consequence of exposure to non-ionising radiation, even though this agent was reported to cause mitotic disturbances under similar experimental conditions.

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells.

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

Spindle disturbances in human-hamster hybrid (A(L) ) cells induced by the electrical component of the mobile communication frequency range signal.

The production of spindle disturbances in a human-hamster hybrid (A(L) ) cell line by an electromagnetic field (EMF) with field strength of 90 V/m at a frequency of 900 MHz was studied in greater detail. The experimental setup presented allows investigating whether either the electrical (E) and/or the magnetic (H) field component of EMF can be associated with the effectiveness of the spindle-disturbing potential. Therefore, both field components of a transversal electromagnetic field (TEM) wave have been separated during exposure of the biological system. This procedure should give more insight on understanding the underlying mechanisms of non-thermal effects of EMF. A statistical comparison of the proportions of the fractions of ana- and telophases with spindle disturbances, obtained for five different exposure conditions with respect to unexposed controls (sham condition), showed that only cells exposed to the H-field component of the EMF were not different from the control. Therefore, the results of the present study indicate that an exposure of cells to EMF at E-field strengths of 45 and 90 V/m, as well as to the separated E component of the EMF, induces significant spindle disturbances in ana- and telophases of the cell cycle.

Terahertz radiation induces spindle disturbances in human-hamster hybrid cells.

The aim of this study was to investigate and quantify the production of spindle disturbances in A(L) cells, a human-hamster hybrid cell line, by 0.106 THz radiation (continuous wave). Monolayer cultures in petri dishes were exposed for 0.5 h to 0.106 THz radiation with power densities ranging from 0.043 mW/cm(2) to 4.3 mW/cm(2) or were kept under sham conditions (negative control) for the same period. As a positive control, 100 µg/ml of the insecticide trichlorfon, which is an aneuploidy-inducing agent, was used for an exposure period of 6 h. During exposure, the sample containers were kept at defined environmental conditions in a modified incubator as required by the cells. Based on a total of 6,365 analyzed mitotic cells, the results of two replicate experiments suggest that 0.106 THz radiation is a spindle-acting agent as predominately indicated by the appearance of spindle disturbances at the anaphase and telophase (especially lagging and non-disjunction of single chromosomes) of cell divisions. The findings in the present study do not necessarily imply disease or injury but may be important for evaluating possible underlying mechanisms.

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.

Enhancing radiation hybrid mapping through whole genome amplification.

Radiation hybrid (RH) mapping is limited by the inherent genomic instability of RH clones entailing both, limited DNA sample amounts and genomic heterogeneity of the clones. Here the instability of RH clones is quantified and the suitability of the multiple strand displacement whole genome amplification method (WGA) for radiation hybrid mapping is assessed. To quantify the instability of RH clones, eleven clones of a 10,000(Rad) rhesus macaque radiation hybrid panel were passaged ten times and analyzed by interspersed repeat sequence specific quantitative PCR and by genotyping of 46 macaque chromosome 5 STS markers. The quantitative PCR data indicate an average loss of 55% of the donor DNA over 10 passages. Over the same period, a dropout of 46.2% of the STS markers was observed. These data indicate a genome wide half-life of the donor DNA of 8.7 passages and of 10.6 passages for the chromosome 5 markers. The genotyping data of the genomic RH DNA were compared to three sets of WGA experiments: 1) single wgaDNA amplifications, 2) six WGA replicates, and 3) re-amplification of wga DNA. The assays demonstrated concordance rates of 97.6%, 98% and 99.3%, respectively, and indicated the marker specificity of some repeated WGA dropouts. The study confirms that WGA is suitable for RH mapping studies should enable the accurate analysis of almost an infinite numbers of markers. WGA will allow the analysis of earliest RH clone passages, thus limiting their heterogeneity and RH mapping artifacts.

Investigation of bystander effects in hybrid cells by means of cell fusion and premature chromosome condensation induction.

The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by premature chromosome condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

The RBE of 3.4 MeV alpha-particles and 0.565 MeV neutrons relative to 60Co gamma-rays for neoplastic transformation of human hybrid cells and the impact of culture conditions.

The neoplastic transformation of human hybrid CGL1 cells is affected by perturbations from external influences such as serum batch and concentration, the number of medium changes during the 21-day expression period and cell seeding density. Nevertheless, for doses up to 1.5 Gy, published transformation frequencies for low linear energy transfer (LET) radiations (gamma-rays, MeV electrons or photons) are in good agreement, whereas for higher doses larger variations are reported. The (60)Co gamma-ray data here for doses up to 1.5 Gy, using a low-yield serum batch and only one medium change, are in agreement with published frequencies of neoplastic transformation of human hybrid cells. For 3.4 MeV alpha-particles (LET = 124 keV/mum) and 0.565 MeV monoenergetic neutrons relative to low doses of (60)Co gamma-rays, a maximum relative biological effectiveness (RBE(M)) of 2.8 +/- 0.2 and 1.5 +/- 0.2, respectively, was calculated. Surprisingly, at higher doses of (60)Co gamma-rays lower frequencies of neoplastic transformation were observed. This non-monotonic dose relationship for neoplastic transformation by (60)Co gamma-rays is likely due to the lack of a G2/M arrest observed at low doses resulting in higher transformation frequencies per dose, whereas the lower frequencies per dose observed for higher doses are likely related to the induction of a G2/M arrest.

Contribution of radiation hybrids to genome mapping in domestic animals.

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described.

Comparative RH maps of the river buffalo and bovine Y chromosomes.

Radiation hybrid maps were constructed for river buffalo and cattle Y chromosomes. A total of 41 cattle-derived Y-chromosome molecular markers were selected and tested with 2 previously described 5,000-rad whole-genome radiation hybrid (RH) panels (river buffalo - BBURH(5000) and cattle - BTARH(5000)) for generation of maps. Among the initial 41 selected markers, a subset of 26 markers generated PCR products suitable for scoring with the BBURH(5000) panel. Of these, 19 markers (73%) were distributed in 1 linkage group spanning 341.3 cR. Retention frequencies (RF) for individual markers ranged from 17.8% for SMCY to 56.7% for BTY1, with an average RF of 37.6%. From the selected markers, 37 generated reliable scores using the BTARH(5000) panel. The newly constructed BTAY RH map contains 28 markers distributed within 1 linkage group. Twenty-four of these markers had been previously mapped on BTAY using a 7,000-rad cattle-hamster WG-RH panel and 4 markers were mapped for the first time (ZFY, SeqRep, RepSeqS4 and BTY1). The length of the BTAY RH map was estimated to be 602.4 cR. Retention frequencies for individual mapped markers ranged from 10% (INRA126) to 63.3% (SeqRep), with an average RF of 35.3%. RH marker positions along the Y chromosome were compared between BBUY and BTAY, which revealed differences in the order of some of the markers. The BBUY pseudoautosomal region (PAR) is delineated by 3 BTAY PAR markers (MAF45, TGLA325 and UMN2008). These markers are telomeric in both species but are not found in the same order. Here we have demonstrated the effective use of bovine Y chromosome markers for the development of the first BBUY RH map. Likewise, these set of markers can be used for comparative assessment of Y chromosomes in other members of the Bovidae family.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects.

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander gamma-H2AX induction. In this paper, we used normal (rho(+)) and mtDNA-depleted (rho(0)) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with gamma-H2AX, we found that the fraction of gamma-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated rho(+) cells at 10 min post-irradiation (rho(+) ICCM(10 min)) caused larger increases of bystander gamma-H2AX induction comparing to rho(0) ICCM(10 min), which only caused a slight increase of bystander gamma-H2AX induction. The rho(+) ICCM(10 min) could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with rho(0) ICCM(10 min). We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched gamma-H2AX induction by rho(+) ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59(-) gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of rho(+) ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

Neoplastic transformation induced by carbon ions.

PURPOSE: The objective of this experiment was to compare the oncogenic potential of carbon ion beams and conventional photon beams for use in radiotherapy. METHODS AND MATERIALS: The HeLa X human skin fibroblast cell line CGL1 was irradiated with carbon ions of three different energies (270, 100, and 11.4 MeV/u). Inactivation and transformation data were compared with those for 15 MeV photons. RESULTS: Inactivation and transformation frequencies for the 270 MeV/u carbon ions were similar to those for 15-MeV photons. The maximal relative biologic effectiveness (RBE(alpha)) values for 100MeV/u and 11.4 MeV/u carbon ions, respectively, were as follows: inactivation, 1.6 +/- 0.2 and 6.7 +/- 0.7; and transformation per surviving cell, 2.5 +/- 0.6 and 12 +/- 3. The curve for dose-transformation per cell at risk exhibited a maximum that was shifted toward lower doses at lower energies. CONCLUSIONS: Transformation induction per cell at risk for carbon ions in the entrance channel was comparable to that for photons, whereas for the lower energies, 100 MeV/u and 11 MeV/u, which are representative of the energies delivered to the tumor margins and volume, respectively, the probability of transformation in a single cell was greater than it was for photons. In addition, at isoeffective doses with respect to cell killing, the 11.4-MeV/u beam was more oncogenic than were photons.

Mapping the BDA20 gene to BTAX.

The lipocalin family is a large group of proteins that exhibits great structural and functional variation both within and among species, including a significant number of animal-derived aeroallergens, such as the bovine BDA20 (major cow dander allergen). This protein is classified as an occupational allergen causing asthma and other work-related allergic disorders among dairy farmers. Using a somatic cell panel the BDA20 gene was assigned to the bovine X chromosome (BTAX) with a significant concordant value of 97% to the previously mapped reference marker MAF45. A radiation hybrid (RH) mapping approach confirmed the assignment of BDA20 to BTAX. Two-point LOD scores showed that BDA20 is linked to XBM451 with a LOD score of 22.1 for a theta value of 0.03.

A computational model for radiation-induced cellular transformation to in vitro irradiation of cells by acute doses of X-rays.

This research incorporates new biological concepts to improve the predictive ability of a state-vector model with respect to dose-response data on in vitro oncogenic transformation, including mechanisms of DNA damage, DNA repair, cell death, cell proliferation and intercellular communication. Experimentally recognized biological processes, including background transformation, compensatory proliferation and bystander cell-killing effect were formulated mathematically and included as model parameters. These were then adjusted with an optimization method to reproduce in vitro transformation frequency data from C3H10T1/2 mouse cells exposed to acute doses of X-rays. A plateau observed in the data at low doses is reproduced well and a dose-dependent increase above 1 Gy is predicted almost precisely. Extension of the model predictions to the dose range 0-100 mGy indicates that transformation frequencies are practically constant over this low dose region. Results suggest a protective, rather than detrimental, bystander cell-killing effect. Further analysis of model sensitivity to this bystander parameter, though, revealed uncertainties with respect to its biological plausibility in the model.

Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.