Performance Evaluation for Repair of HSGc-C5 Carcinoma Cell Using Geant4-DNA

Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for different cell lines. Thus, we extended the Geant4-DNA application with a TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA rejoining kinetics of the HSGc-C5 cells irradiated by 70 MeV protons at the cyclotron facility at the National Institutes for Quantum and Radiological Science and Technology (QST). Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. The optimized DNA rejoining speeds were reasonably agreed with the experimental DNA rejoining speeds. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5 cells.

Whole Transcriptome Analysis Revealed a Stress Response to Deep Underground Environment Conditions in Chinese Hamster V79 Lung Fibroblast Cells

Background: Prior studies have shown that the proliferation of V79 lung fibroblast cells could be inhibited by low background radiation (LBR) in deep underground laboratory (DUGL). In the current study, we revealed further molecular changes by performing whole transcriptome analysis on the expression profiles of long non-coding RNA (lncRNA), messenger RNA (mRNA), circular RNA (circRNA) and microRNA (miRNA) in V79 cells cultured for two days in a DUGL.Methods: Whole transcriptome analysis including lncRNA, mRNAs, circ RNA and miRNA was performed in V79 cells cultured for two days in DUGL and above ground laboratory (AGL), respectively. The differentially expressed (DE) lncRNA, mRNA, circRNA, and miRNA in V79 cells were identified by the comparison between DUGL and AGL groups. Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to verify the selected RNA sequencings. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed for the DE mRNAs which enabled to predict target genes of lncRNA and host genes of circRNA.Results: With |log2(Fold-change)| ≥ 1.0 and p < 0.05, a total of 1257 mRNAs (353 mRNAs up-regulated, 904 mRNAs down-regulated), 866 lncRNAs (145 lncRNAs up-regulated, 721 lncRNAs down-regulated), and 474 circRNAs (247 circRNAs up-regulated, 227 circRNAs down-regulated) were significantly altered between the two groups. There was no significant difference in miRNA between the two groups. The altered RNA profiles were mainly discovered in lncRNAs, mRNAs and circRNAs. DE RNAs were involved in many pathways including ECM-RI, PI3K-Akt signaling, RNA transport and the cell cycle under the LBR stress of the deep underground environment.Conclusion: Taken together, these results suggest that the LBR in the DUGL could induce transcriptional repression, thus reducing metabolic process and reprogramming the overall gene expression profile in V79 cells.

Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum—in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues—incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

Performance Evaluation for Repair of Hsgc-c5 Carcinoma Cell Using geant4-Dna

Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA-rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for the different cell lines. Thus, we extended the Geant4-DNA application with an updated TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA-rejoining kinetics of the HSGc-C5s cells. Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5s cells.

Structural Changes in HPRT Gene of V79 Cells After Irradiation With Heavy Ions—Immediate and Delayed Effects

The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11B, 18O, 20Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60Co gamma rays up to 136 keV/μm of 20Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

Proteomics provides insights into the inhibition of Chinese hamster V79 cell proliferation in the deep underground environment

As resources in the shallow depths of the earth exhausted, people will spend extended periods of time in the deep underground space. However, little is known about the deep underground environment affecting the health of organisms. Hence, we established both deep underground laboratory (DUGL) and above ground laboratory (AGL) to investigate the effect of environmental factors on organisms. Six environmental parameters were monitored in the DUGL and AGL. Growth curves were recorded and tandem mass tag (TMT) proteomics analysis were performed to explore the proliferative ability and differentially abundant proteins (DAPs) in V79 cells (a cell line widely used in biological study in DUGLs) cultured in the DUGL and AGL. Parallel Reaction Monitoring was conducted to verify the TMT results. γ ray dose rate showed the most detectable difference between the two laboratories, whereby γ ray dose rate was significantly lower in the DUGL compared to the AGL. V79 cell proliferation was slower in the DUGL. Quantitative proteomics detected 980 DAPs (absolute fold change ≥ 1.2, p < 0.05) between V79 cells cultured in the DUGL and AGL. Of these, 576 proteins were up-regulated and 404 proteins were down-regulated in V79 cells cultured in the DUGL. KEGG pathway analysis revealed that seven pathways (e.g. ribosome, RNA transport and oxidative phosphorylation) were significantly enriched. These data suggest that proliferation of V79 cells was inhibited in the DUGL, likely because cells were exposed to reduced background radiation. The apparent changes in the proteome profile may have induced cellular changes that delayed proliferation but enhanced survival, rendering V79 cells adaptable to the changing environment.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi)

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.