Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.     

Use of Comet-FISH in the study of DNA damage and repair: review

The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest, including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA regions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene regions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the measured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.     

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.     

Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload--in vitro and ex vivo results with human and rat cells

Iron exposure enhances colorectal carcinogeneis, by producing reactive oxygen species, which damage lipids, proteins and DNA. We recently demonstrated that ferric-nitrilotriacetate (Fe-NTA) damages DNA of human colon cells in different stages of malignant transformation. Opposed to this, little is known on systemic effects of iron and it is still difficult to determine the border between essential iron supplementation and iron overload in humans. The aim of this study was to determine whether Fe-NTA causes global and specific DNA damage in peripheral leucocytes. Human leucocytes were treated in vitro with Fe-NTA for 30 min at 37 degrees C. Male Sprague Dawley rats were fed (6 weeks) with an iron-overload diet (9.9 g Fe/kg DM) and whole blood was collected. DNA damage was measured in human and rat blood cells using the alkaline version of the Comet Assay with repair specific enzymes. In human cells the distribution of TP53 in the comet images was detected using fluorescence in situ hybridization (Comet FISH) to measure DNA damage in the region of the TP53 gene. Fe-NTA (10-500 microM) was clearly genotoxic in human leucocytes in vitro, and also in leucocytes of rats fed the iron overload diet. The induced damage in human leucocytes was approximately two-fold that observed previously in human colon cells. Oxidized bases were induced by iron in rat leucocytes in vivo, while they were not induced in human leucocytes in vitro. Fe-NTA enhanced the migration of TP53 signals into the comet tail of human leucocytes, indicating a high susceptibility of this tumour-relevant gene towards DNA damage induced by iron overload. In conclusion, iron markedly induced DNA damage in human and rat leucocytes, which shows that these white blood cells are sufficiently sensitive to assess exposure to iron. The measurement of DNA damage in human leucocytes could be used as a sensitive biomarker to study iron overload in vivo in humans and thus to determine whether supplementation results in genotoxic risk.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

Danthron Induces DNA Damage and Inhibits DNA Repair Gene Expressions in GBM 8401 Human Brain Glioblastoma Multiforms Cells

Our primary studies had shown that danthron induced cytotoxic effects, including apoptosis and inhibition of migration and invasion. However, danthron-affected DNA damage and repair gene expressions are not clear. In this study, we investigated to examine whether or not danthron induced DNA damage and inhibited DNA repair gene expression in human brain glioblastoma multiforms (GBM 8401) cells. The results from Comet assay indicated that incubation of GBM 8401 cells with 0, 50, 100 and 150 μM of danthron led to a longer DNA migration smear based on the single cell electrophoresis (Comet tail). The results from real-time PCR assay demonstrated that 100 μM of danthron for 24 h treatment in GBM 8401 cells led to decrease all examined ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA-1), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK) and O ⁶ -methylguanine-DNA methyltransferase (MGMT) mRNA expressions. Taken together, the present study showed that danthron caused DNA damage and inhibited DNA repair genes, which may be the factors for danthron-inhibited cell growth in vitro.     

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.     

Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells.

Repair of DNA cross-links by mitomycin C (MMC) was studied in mammalian cells. Skin cells from a patient with Fanconi's anemia (FA9 cells) were about 6 times as sensitive to MMC killing as HeLa S3 cells with normal excision repair ability, while excision-reduced mouse L and human xeroderma pigmentosum (XP2OS) cells were more resistant to it than HeLa S3 cells. Alkaline sucrose sedimentation of DNA revealed that perhaps half-excision of cross-links and its repair occurred efficiently until 4 h of post-MMC time in L-cells and, though more slowly, in HeLa S3 cells. Thus, the excision repair pathway is the first step of the cross-link repair in mammalian cells, but it seems different from the $\text{uvrA}$-dependent pathway in $\text{E. coli}$, since XP2OS cells survived MMC almost normally. Contrarily, FA9 DNA sedimented much faster at 4 h of post-MMC time, suggesting a possible impairment in FA cell's ability to unhook cross-links.     

A common carcinogen benzo[a]pyrene causes p53 overexpression in mouse cervix via DNA damage

Benzo[a]pyrene (BaP) is cytotoxic and/or genotoxic to lung, stomach and skin tissue in the body. However, the effect of BaP on cervical tissue remains unclear. The present study detected DNA damage and the expression of the p53 gene in BaP-induced cervical tissue in female mice. Animals were intraperitoneally injected and orally gavaged with BaP at the doses of 2.5, 5, and 10mg/kg twice a week for 14 weeks. The single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to detect the expression of p53 protein and p53 mRNA, respectively. The results showed that BaP induced a significant and dose-dependent increase of the number of cells with DNA damaged and the tail length as well as Comet tail moment in cervical tissue. The expression level of p53 protein and mRNA was increased. The results demonstrate that BaP may show toxic effect on the cervix by increasing DNA damage and the expression of the p53 gene.     

Germ cell and dose-dependent DNA damage measured by the comet assay in murine spermatozoaa after testicular X-irradiation

The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25-4 Gy X-rays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.     

紫外辐射诱导烟草原生质体中DNA损伤的彗星电泳检测

以烟草原生质体为材料,采用彗星电泳检测用0.5W·m^-2紫外线以不同时间（0、5、10、30、60和120s）诱导的烟草原生质体中DNA的损伤。结果表明,在0～10s的时间内代表DNA损伤程度的尾矩、Olive尾矩等参数与紫外线照射时间具有良好的时间依赖关系。本文建立的烟草原生质体体系采用彗星电泳技术,可以快速而灵敏地检测紫外线对植物细胞的损伤程度。     

Effects of Embryonic Neural Stem Cell Transplantation on DNA Damage in the Brain and Spinal Cord Following Spinal Cord Injury

Embryonic neural stem cell (ENSC) transplantation is used experimentally for the improvement of spinal cord repair following spinal cord injury (SCI). However, the effects of such intervention on oxidative stress and cell death remain unknown. We used in vivo Comet assay in the acute and chronic SCI groups compared with the SCI+ENSC transplantation groups of experimental rats in order to evaluate DNA damage in the spinal cord. Chronic SCI resulted in the generation of oxidative DNA damage in the spinal cord brain and kidneys, as indicated by high Comet assay parameters, including the percentage of DNA in the tail (T%, or TD), tail moment (TM), and tail length (TL). The DNA damage levels significantly decreased after ENSC transplantation in the spinal cords of acute and chronic SCI groups within the lesion site and rostrally and caudally to the injury, and in the brains and kidneys of the chronic SCI group. Thus, ENSC transplantation is found to be an effective tool for limitation of DNA damage following spinal cord injury.     

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell ^1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Evaluation of the DNA repair host-mediated assay. II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VALr) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3-4-fold increase in VALr mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.