WTK1 Cells Research Articles

Radiation-Induced Chromosome Instability in WTK1 and TK6 Human Lymphoblastoid Cells

The effects of ionizing radiation on single nucleotide polymorphism (SNP) copy number variations between TK6 and WTK1 cell lines are described herein. Specifically, the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) was analyzed in the presence and absence of ionizing radiation. WTK1 cells contain a p53 mutation, whereas the TK6 cell line has the native p53 tumor-suppressor gene. Each cell line was isolated post-irradiation for SNP analysis, which showed significant, genome-wide impacts on both cell lines; for the mutant WTK1 sample, there were a total of 48 gene deletions and no gene amplifications, whereas for the wild-type TK6 sample, there were 217 gene deletions and 9 gene amplifications. It appears that both cell lines are affected in the areas of cell-cycle control, but that other affected areas differ significantly between the two.

How Bulky of Base Adducts can be Responsible for Clastogenicity rather than Mutagenicity?

Two types of TK mutants are induced by genotoxic factors; normally growing (NG) TK mutants due to point mutations of targeted TK locus, and slowly growing (SG) mutants due to gross structural changes involving the growth-regulating gene outside targeted TK locus. In this study, human lymphoblstoid WTK1 cells were used to consider how bulky n- alkylated bases can induce SG mutants. For this purpose, n-alkyl methanesulfonates (AMS) having an n-alkyl group with 3-7 carbons [n-propy methanesulfonate (PMS), n-butyl methanesulfonate (BMS), n-pentyl methanesulfonate (PeMS), n- hexyl methanesulfonate (HexMS), and heptyl methanesulfonate(HepMS)] were synthesized. n-alkyl methanesulfonates having n-alkyl groups with 1-7 carbons induced NG mutants, but n-alkyl methanesulfonates having n-alkyl groups with ≥4 carbons but not with ≤3 carbons induced SG mutants. n-Alkyl methanesulfonates having n-Alkyl groups with ≥4 carbons have been shown to induce bulky adducts that cause disturbances to the helical DNA structure and are removed by nucleotide excision repair. It could be considered that n-alkyl groups with ≥4 carbons causing disturbances to the helical DNA structure induce SG mutants to result in clastogenicity rather than mutagenicity.

Genotoxicity-Suppressing Effect of Sophora japonica L. Aqueous Extract

The dry flower buds of Sophora japonica L. are used as a hemostatic agent in traditional Chinese medicine. In the comet assay, aqueous extracts of S. japonica decreased and increased the tail length significantly in cultured human lymphoblastoid WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors, respectively. The extract did not affect the tail length in methyl methanesulfonate-exposed cells. In the present study, the aqueous extract of the flower buds of S. japonica was separated by repeated column chromatography, yielding four types of flavonoid glycosides. Among them, only rutin, similar to the extract, decreased and increased the tail length significantly in WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors hydroxyurea (10 mM) and cytosine-1-β-D-arabinofuranoside (1.8 mM), respectively. The genotoxicity-suppressing effect of rutin was further studied using the micronucleus test. Rutin significantly decreased the frequency of micronucleated binucleate cells in UV-exposed WTK1 cells but did not affect this frequency in UV-exposed XPL3KA (Xeroderma pigmentosum group C) cells. These results suggest that the anti-genotoxic potential of rutin is due to an enhanced incision step of global genome repair (GGR) sub-pathways in nucleotide excision repair (NER). Herein, we show that S. japonica exhibits heretofore unknown anti-genotoxic potential against UV by enhancing the incision of GGR sub-pathways in NER, and that its anti-genotoxic component is rutin.

Incomplete Split-Dose Recovery and Induced Radioresistance During Fractionated Irradiation of a Human Glioblastoma Cell Line In Vitro

Fractionated radiotherapy is part of the standard treatment of glioblastoma but radioresistance contributes to local recurrence of these highly malignant tumors. Classical radiobiological models assume complete recovery of surviving cells ascribed to repair of sublethal damage (SLD) between fractions, implying that cell survival after the i’th fraction is determined by the surviving fraction (SF) after a single fraction of dose, d, repeated i times. The purpose was to test this hypothesis in vitro. The radioresistant U251 glioblastoma cell line (p53-mutated) was used. Clonogenic cell survival after irradiation with 6 MV X-rays was determined by the colony formation assay and survival curves were fitted by the linear-quadratic model. In order to test the effect of tumor suppressor protein p53 on survival curve shape and split-dose recovery, early-passage human skin fibroblasts and the human lymphoblastoid cells lines TK6 (p53-wild-type), TK6-E6 (p53-suppressed), and WTK1 (p53-mutated), were included. Mean values ± std.err. are shown and statistical significance was tested by the t-test. Single-dose survival curves of U251 cells showed radioresistance with downward curvature at higher doses (α=0.147±0.021 Gy-1; β=0.039±0.003 Gy-2; n=9). Split-dose recovery between two fractions of 4 Gy each was observed for 15 min to 9h intervals with maximal but incomplete recovery reached at 4h. The survival curve shape for graded second doses at 4h did not recapitulate the curve for single-dose irradiation but was steeper with less downward curvature (a=0.400±0.114 Gy-1, α=0.0005; β=0.020±0.017 Gy-2, p=0.03; n=3). Fractionated irradiation with daily doses of 2 Gy, did not produce the expected exponential decrease of SF(i×d)=SF(d)i. Instead, a downward curvature during the first 2-3 fractions was followed by a reduced slope indicating induced radioresistance. Early-passage fibroblasts with a nearly exponential survival curve showed no split-dose recovery in spite of being proficient for DNA double-strand break (DSB) repair. The association between downward curvature and split-dose recovery was confirmed in the relatively radioresistant WTK1 cell line whereas the sensitive TK6 and TK6-E6 cell lines showed essentially exponential survival curves with little or no split-dose recovery. The reduced recovery after the first few fractions and induced radioresistance after multiple fractions may support the use of highly hypofractionated or single-dose radiotherapy. Whereas SLD repair (detected as split-dose recovery) was associated with downward curvature of cell survival curves, this did not correlate with DSB repair proficiency or lack of functional p53. These results are conflicting with classical radiobiological models and imply that a better understanding of the SLD repair phenotype is needed.

A HYPOTHESIS OF RADIORESISTANCE AND CELL-SURVIVAL CURVE SHAPE BASED ON CELL-CYCLE PROGRESSION AND DAMAGE TOLERANCE.

Exponential survival curves of early-passage human fibroblasts challenge classic biophysical models of cell inactivation. Thus, X-ray doses of 2-4 Gy inactivate normal, human skin fibroblasts in spite of negligible residual double-strand breaks. By contrast, radioresistant p53-mutant U251 glioblastoma cells proliferate in spite of residual damage. Similarly, p53 wildtype TK6 lymphoblastoid cells show exponential survival curves while the related p53-mutant WTK1 cell line continued to proliferate and showed a shouldered survival curve. Here, we propose a model in which the radioresistant shoulder region is due to tolerance to certain types or amounts of residual damage that would otherwise inactivate normal cells. Thus, the steeper initial slope and absence of a shoulder in the survival curve of normal cells may not imply a higher number of residual lesions but rather non-tolerance to these lesions.

P53-Suppression by Overexpression of the HPV16 E6 Gene in Lymphoblastoid TK6 Cells Causes a Change in Cell-Death Mode but not Radiosensitivity

The human lymphoblastoid cell line TK6E6 with suppressed expression of tumor suppressor protein p53 shows similar high radiosensitivity as p53-wildtype TK6 cells with a linear survival curve but reduced/delayed apoptosis compared with TK6. By contrast, the related cell line WTK1 expresses high levels of mutated p53, is radioresistant with a shouldered survival curve and shows little apoptosis. The purpose was to test if the differences in radiosensitivity might be related to differences in cell-cycle progression and cell-death mode. The related human lymphoblastoid cell lines TK6 (p53-wildtype), TK6E6 (overexpressing the E6 gene of human papilloma virus 16), and WTK1 (p53-mutant) were irradiated with 6 MV X-rays. Cell survival was determined by the colony formation assay and apoptosis quantified by the sub-G1 fraction using flow cytometry. Cell-cycle analysis was performed by flow cytometry on BrdU pulse-labeled cells. The ratio of p-PLK1 to p-CHK1, which controls the release from the G2 phase into mitosis, was quantified by Western blotting using phospho-specific antibodies. After irradiation (IR) with a dose of 3 Gy, progression of BrdU-negative cells from G1 into S was arrested in TK6 showing an intact G1/S checkpoint which was absent in TK6E6 and WTK1. Normal cell-cycle progression was slower in TK6E6 and WTK1 (cell doubling time: 11.6h) than in TK6 (10.3h). However, progression of BrdU-positive cells from S into G2 showed a larger delay relative to unirradiated controls in TK6E6 (4h) compared with TK6 and WTK1 (2h). Notably, following irradiation of cultures enriched in S-phase, only 50% (1 Gy) to 30% (3 Gy) of the BrdU-labelled TK6E6 cells leaving G2 after release of the G2/M block entered G1 compared with 100% and 60% for TK6 cells. Together with the reduced levels of apoptosis in TK6E6 this strongly suggests a shift in cell death mode from apoptotic towards mitotic death. All three cell lines showed an intact G2/M checkpoint (blockage). A 15-25% decrease in the ratio p-PLK1p-CHK1 was seen after irradiation of TK6 and TK6E6 cells, respectively, which were restored to the original levels 7h and 12h after irradiation. By contrast, p-PLK1/p-CHK1 in unirradiated, normally cycling cells was 45% higher in WTK1 cells than in TK6 but continued to decrease up to 24h after irradiation although by this time WTK1 cells had started to progress through M into G1. Suppression of p53 had little influence on survival rates but caused a shift from apoptotic to mitotic cell death. The radioresistance of p53-mutated WTK1 cells was not associated with an inefficient G2/M block or premature release into mitosis but the cells progressed through G1 and S at the same rate as unirradiated cells. Since WTK1 are known to show genomic instability, their radioresistance might be due to tolerance to some type(s) damage that inactivate p53-wildtype cells, possibly mediated by a gain-of-function mutation in p53 or by a change in a different gene.

Whole genome and normalized mRNA sequencing reveal genetic status of TK6, WTK1, and NH32 human B-lymphoblastoid cell lines

Closely related TK6, WTK1, and NH32 human B-lymphoblastoid cell lines differ in their p53 functional status. These lines are used frequently in genotoxicity studies and in studies aimed at understanding the role of p53 in DNA repair. Despite their routine use, little is known about the genetic status of these cells. To provide insight into their genetic composition, we sequenced and analyzed the entire genome of TK6 cells, as well as the normalized transcriptomes of TK6, WTK1, and NH32 cells. Whole genome sequencing (WGS) identified 21,561 genes and 5.17×106 small variants. Within the small variants, 50.54% were naturally occurring single nucleotide polymorphisms (SNPs) and 49.46% were mutations. The mutations were comprised of 92.97% single base-pair substitutions and 7.03% insertions or deletions (indels). The number of predicted genes, SNPs, and small mutations are similar to frequencies observed in the human population in general. Normalized mRNA-seq analysis identified the expression of transcripts bearing SNPs or mutations for TK6, WTK1, and NH32 as 2.88%, 2.04%, and 1.71%, respectively, and several of the variant transcripts identified appear to have important implications in genetic toxicology. These include a single base deletion mutation in the ferritin heavy chain gene (FTH1) resulting in a frame shift and protein truncation in TK6 that impairs iron metabolism. SNPs in the thiopurine S-methyltransferase (TPMT) gene (TPMT*3A SNP), and in the xenobiotic metabolizing enzyme, NADPH quinine oxidoreductase 1 (NQO1) gene (NQO1*2 SNP), are both associated with decreased enzyme activity. The clinically relevant TPMT*3A and NQO1*2 SNPs can make these cell lines useful in pharmacogenetic studies aimed at improving or tailoring drug treatment regimens that minimize toxicity and enhance efficacy.

Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid

Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway.

DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5‐ethynyl‐2′‐deoxyuridine incorporated into DNA

The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

Expression Pattern of Small Nucleolar RNA Host Genes and Long Non-Coding RNA in X-rays-Treated Lymphoblastoid Cells

A wide variety of biological effects are induced in cells that are exposed to ionizing radiation. The expression changes of coding mRNA and non-coding micro-RNA have been implicated in irradiated cells. The involvement of other classes of non-coding RNAs (ncRNA), such as small nucleolar RNAs (snoRNAs), long ncRNAs (lncRNAs), and PIWI-interacting RNAs (piRNAs) in cells recovering from radiation-induced damage has not been examined. Thus, we investigated whether these ncRNA were undergoing changes in cells exposed to ionizing radiation. The modulation of ncRNAs expression was determined in human TK6 (p53 positive) and WTK1 (p53 negative) cells. The snoRNA host genes SNHG1, SNHG6, and SNHG11 were induced in TK6 cells. In WTK1 cells, SNHG1 was induced but SNHG6, and SNHG11 were repressed. SNHG7 was repressed in TK6 cells and was upregulated in WTK1 cells. The lncRNA MALAT1 and SOX2OT were induced in both TK6 and WTK1 cells and SRA1 was induced in TK6 cells only. Interestingly, the MIAT and PIWIL1 were not expressed in TK6 cells before or after the ionizing radiation treatment. The MIAT and PIWIL1 were upregulated in WTK1 cells. This data provides evidence that altered ncRNA expression is a part of the complex stress response operating in radiation-treated cells and this response depends on functional p53.

Epigenetic changes in human cells exposed to X‐rays

An understanding of cellular processes that determine the response to ionizing radiation (IR) treatment is essential to improve radiotherapy. We investigated the DNA methylation changes in X‐ray‐irradiated TK6 and WTK1 cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an ELISA showed hypomethylation. Using AP‐PCR we observed time dependent dynamic changes in the regional genomic DNA methylation patterns. The AP‐PCR profiles were different between these cells indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells. TET1, involved in the conversion of 5‐methylcytosine to 5‐hydroxymethylcytosine was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns are changed at different genomic regions and are dependent upon time after irradiation and the genetic background of the cell.

Different responses to 5-fluoraouracil in mutagenicity and gene expression between two human lymphoblastoid cell lines with or without TP53 mutation.

Human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. TK6 cells have wild-type TP53 alleles, while WTK-1 cells have one allele of mutated TP53. Both cells were treated with 5-fluorouracil (5-FU), and gene mutation assay and micronucleus assay were performed to clarify the differential response related to the TP53 gene status. The effects of 5-FU on gene expression were assessed by microarray and quantitative RT-PCR analyses. In WTK-1 cells, 5-FU increased the frequency of cells with micronucleus and mutation. In TK6 cells, frequency of cells with micronucleus was increased but the mutation frequency was not. The cytotoxicity induced by 5-FU was more prominent in TK6 cells than in WTK-1 cells. Analysis of gene expression showed that the genes involved in the TP53 pathway were up-regulated in TK6 cells but not in WTK-1 cells. The differential responses to 5-FU between these cell lines appeared to be due to the difference in the TP53 gene status, thus providing a molecular basis for the bioassays using these cell lines in the toxicology field. Our results indicate that the clinical efficacy of 5-FU chemotherapy may depend on the TP53 genotype.

Expression of p53-Regulated Proteins in Human Cultured Lymphoblastoid TSCE5 and WTK1 Cell Lines during Spaceflight

The aim of this study was to determine the biological effects of space radiations, microgravity, and the interaction of them on the expression of p53-regulated proteins. Space experiments were performed with two human cultured lymphoblastoid cell lines: one line (TSCE5) bears a wild-type p53 gene status, and another line (WTK1) bears a mutated p53 gene status. Under 1 gravity or microgravity conditions, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples were simultaneously cultured for 8 days in the CBEF on the ground for 8 days. After spaceflight, protein expression was analyzed using a Panorama(TM) Ab MicroArray protein chips. It was found that p53-dependent up-regulated proteins in response to space radiations and space environment were MeCP2 (methyl CpG binding protein 2), and Notch1 (Notch homolog 1), respectively. On the other hand, p53-dependent down-regulated proteins were TGF-β, TWEAKR (tumor necrosis factor-like weak inducer of apoptosis receptor), phosho-Pyk2 (Proline-rich tyrosine kinase 2), and 14-3-3θ/τ which were affected by microgravity, and DR4 (death receptor 4), PRMT1 (protein arginine methyltransferase 1) and ROCK-2 (Rho-associated, coiled-coil containing protein kinase 2) in response to space radiations. ROCK-2 was also suppressed in response to the space environment. The data provides the p53-dependent regulated proteins by exposure to space radiations and/or microgravity during spaceflight. Our expression data revealed proteins that might help to advance the basic space radiation biology.

Differential DNA Methylation Alterations in Radiation-Sensitive and -Resistant Cells

An understanding of cellular processes that determine the response to ionizing radiation (IR) exposure is essential to improve radiotherapy and to assess risks to human health after accidental radiation exposure. Exposure to IR induces a multitude of biological effects. Recent studies have indicated the involvement of epigenetic events in regulating the responses of irradiated cells. DNA methylation, where the cytosine bases in CpG dimers are converted to 5-methyl cytosine, is an epigenetic event that has been shown to regulate a variety of biological processes. We investigated the DNA methylation changes in irradiated TK6 and WTK1 human cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an enzyme-linked immunosorbent assay-based assay showed hypomethylation in both type of cells. Using an arbitrarily primed polymerase chain reaction (AP-PCR) approach, we observed time-dependent dynamic changes in the regional genomic DNA methylation patterns in both cell lines. The AP-PCR DNA methylation profiles were different between TK6 and WTK1 cells, indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells after irradiation but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells after radiation treatment. TET1, involved in the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns, and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns change at different genomic regions and are dependent on time after irradiation and the genetic background of the cell.

Expression level of miR‐34a rather than P53 gene status correlates with mutability in related human lymphoblast cell lines

The P53 gene is a tumor suppressor gene and can prevent mutation and tumor induction though apoptosis and DNA repair when it is activated by genotoxic stress. miR-34a expression is regulated by the P53 gene and might be required for cell response to DNA damage. TK6 cells are human lymphoblast cells with normal P53 function while WTK1 and NH32 cells derived from the same progenitor as TK6 cells are P53-deficient. Previous mutation research showed an unexpected result that NH32 cells were much less mutable than WTK1 cells, although the P53 gene in both the cell lines is not functional. To explore the possible mechanisms involved in the different mutability of the cell lines and relationship between P53 and miR-34a, we investigated the expression levels of miR-34a in the cells. The basal and X-ray-induced expression levels of miR-34a in TK6 and NH32 cells were much higher than those in WTK1 cells. The miR-34a was also able to be up-regulated to respond to X-ray exposure without a functional P53 gene in both of the NH32 and WTK1 cells. In addition, the expression levels of miR-34a in these three cell lines are inversely correlated well with their mutability: higher levels of miR-34a correspond with less mutable cells. These results suggest that alteration of miR-34a expression is at least partially independent of P53 regulation and its expression levels are closely related to cells' mutability regardless of P53 status.

Genotoxicity-suppressing Effect of Aqueous Extract of Connarus ruber Cortex

The anti-genotoxicity eSect of aqueous extracts of Connarus ruber cortex was studied in cultured human cells and mice. Connarus extract decreased bi-nuclei cells with micronuclei (MNBNC) signiˆcantly in NER-proˆcient WTK1 cells that were exposed to MNU, MMC, or UVC and in NER-deˆcient XPL3KA cells (that is, in XP-C) that were exposed to MNU or MMC, but not UVC. The genotoxicitysuppressing eSect was further studied by the comet assay. Connarus extract decreased DNA migration signiˆcantly in WTK1 cells that were exposed to MNU or UVC and in XPL3KA cells that were exposed to MNU but not UVC. In WTK1 cells, in contrast, DNA migration increased with the extract in the presence of DNA repair inhibitors (araC and HU), suggesting that the anti-genotoxic potential is due to an enhanced incision step of global genome repair (GGR) subpathways in NER. Chemical analysis revealed that the extract contains epicatechine, one of the anti-mutagenic components contained in green tea. Connarus extract fractions that decreased UVC-induced DNA migration were those not to contain epicatechine and they were diSerent from those that decreased MNU-induced DNA migration, suggesting that some anti-mutagenic components other than epicatechine might be contained in Connarus extract and that a number of anti-genotoxic components with diSerent modes of anti-genotoxicity are contained in Connarus extract. The anti-clastogenic eSect of Connarus extracts was examined in mice using a micronucleus assay. When mice received ≦2000 mg/kg Connarus extract by oral gavage at the same time as intraperitoneal injection of MMC, a decrease in the frequency of micronucleated reticulocytes was observed. This decrease was not due to a delay in the maturation of micronucleated reticulocytes.

Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.

Comparison of in vitro micronucleus and gene mutation assay results for p53‐competent versus p53‐deficient human lymphoblastoid cells

The high frequency of false or irrelevant positive results in in vitro mammalian cell genotoxicity tests is a critical concern for regulators. Here, we tested whether such results may be due to the mammalian cells used in the tests being deficient in p53, which is involved in the maintenance of genomic stability. We compared the in vitro responses of two human lymphoblastoid cell lines derived from the same progenitor cell-p53-competent (TK6) and p53-deficient (WTK-1) cells-in a micronucleus (MN) test and a thymidine kinase gene (TK) mutation assay. We tested 14 chemicals including three mutagens and 11 clastogens and spindle poisons. The three mutagens evoked clear positive responses in both assays in both cell lines. The responses to the clastogens and spindle poisons, on the other hand, depended on the assay endpoint and/or the cell line. Most of clastogens and spindle poisons were positive in the MN test in both cell lines. In the TK mutation assay, on the other hand, WTK-1 cells but not TK6 cells detected spindle poisons, which may have been due to the disturbance of the spindle checkpoint and lack of apoptosis in the p53-deficient cells. Some chemicals that induced chromosome aberrations in rodent cells were negative in both TK6 and WTK-1 cells, indicating that a species-specific factor rather than p53 status was associated with the response. In conclusion, the p53 status did not seriously influence the MN test results but it did influence the TK mutation assay results.

Transcriptional modulation of micro-RNA in human cells differing in radiation sensitivity

Purpose: The molecular basis of gene regulation in cells exposed to ionising radiation is not fully understood. Gene regulation occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) plays a significant role at the post-transcriptional gene regulation. miRNA are a recently identified class of RNA molecules 18–24 nucleotides in length that have been shown to negatively regulate the stability or translation of target messenger RNA. We hypothesised that ionising radiation induced stress response is controlled in part by miRNA and that a difference in tumour protein 53 (p53) status corresponds with altered expression in miRNA responses to ionising radiation.Materials and methods: To test this hypothesis, we investigated the relative expression of several miRNA by quantitative real-time polymerase chain reaction (QPCR) in human cell lines TK6 and WTK1 that differ in p53 status and radiosensitivity after exposure to high and low doses of X-radiation.Results: The suitability of several endogenous miRNA controls was tested for relative quantification by QPCR. The baseline expression of 21 miRNA targets in TK6 and WTK1 cells indicated a wide range of modulation between the two cell lines without exposure to ionising radiation. Differences in the relative expression of miRNA were observed among the two cell lines after radiation treatment. The expression patterns of many miRNA markedly differed within the same cell line after exposure to either 0.5 Gy or 2 Gy doses of X-rays. The expression of eight miRNA belonging to the lethal-7 (let-7) family, which are negative regulators of the rat sarcoma, RAS oncogene, was upregulated in irradiated TK6 cells but was downregulated in WTK1 cells. Alterations in the myelocytomatosis oncogene, c-MYC induced cluster of miRNA were also observed. The micro RNA, miR-15a and miR-16 were upregulated in 0.5 Gy-irradiated TK6 cells but were downregulated after a 2 Gy dose of X-rays. In contrast miR-15 and miR-16 were repressed in 0.5 Gy-exposed WTK1. The miR-21 was upregulated in 0.5 Gy-treated TK6 cells and its target genes programmed cell death factor 4 (hPDCD4) phosphatase and tensin homolog (hPTEN), and sprouty homolog 2 (hSPRY2) were found to be downregulated in these cells. The miR-21 was downregulated in 2 Gy-irradiated TK6 cells, and all three of its target genes were upregulated in 2 Gy-exposed TK6 cells.Conclusion: Taken together, these results establish the involvement of miRNA in radiation response and may potentially help explain the mechanisms of gene regulation in the cellular response to ionising radiation exposure.

Does p53 Status Affect the Sensitivity of the Comet Assay?

It has been shown that cells having mutant p53 are more sensitive to genotoxic effects than cells having wild-type p53. To determine whether p53 status affects the sensitivity of the comet assay, we compared the results of the comet assay in WTK1 cells having mutant p53 and TK6 cells having wild-type p53 in the presence and absence of DNA repair inhibitors. Cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and bleomycin (BLM) for 2 h in the presence or absence of araC and hydroxyurea (araC/HU) or irradiated to UVC. UVC irradiated cells were cultured for 2 h with or without araC/HU. Immediately after the exposure to chemical mutagens or 2 h after UVC irradiation, slides for the comet assay were prepared. In the absence of araC/HU, the lowest genotoxic doses (LGDs) were higher in TK6 than in WTK1 cells, showing that wild-type p53 reduces comet assay sensitivity. On the other hand, in the presence of araC/HU, the LGDs were almost the same in WTK1 and TK6 cells. In the presence of araC/HU, positive responses to UVC irradiation were observed earlier in TK6 than in WTK1 cells. Therefore, SSB formation followed by SSB rejoining during the nucleotide excision repair process is considered to occur earlier in TK6 than in WTK1 cells, which results in a lower comet assay sensitivity in cells having wild-type p53. Comet assay response to mutagens inducing DNA damages that are repaired by the excision repair system tended to be affected by p53 status.