Patterns of unscheduled DNA synthesis in mouse embryo cells associated with in vitro aging and with spontaneous transformation to a continuous cell line

Monolayer cell cultures derived from B/C mouse embryos were examined for the ability to repair ultraviolet light-induced DNA damage (50–250 erg/mm²) during in vitro aging and subsequent alteration to a continuous cell line. Excision repair was measured by incubating the cultures with [³H]TdR and measuring DNA specific activity, and by performing quantitative autoradiography. DNA repair capacity declined during in vitro aging, and the level of repair correlated with the fraction of cells which retained the capacity to undergo scheduled DNA synthesis. This result indicates that mouse cells aged in vitro undergo a decline in their ability to repair UV-induced DNA damage comparable to that seen in cultured human fibroblasts. In cultures which spontaneously altered into continuously proliferating cell lines, DNA repair capacity increased to high levels, as did the fraction of cells capable of initiating scheduled DNA synthesis.     

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.     

The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress

Aging is associated with a reduction in the DNA repair capacity under oxidative stress. However, whether the DNA damage and repair capacity can be a biomarker of aging remains controversial. In this study, we demonstrated two cause-and-effect relationships, the one is between the DNA damage and repair capacity and the cellular age, another is between DNA damage and repair capacity and the level of oxidative stress in human embryonic lung fibroblasts (2BS) exposed to different doses of hydrogen peroxide (H₂O₂). To clarify the mechanisms of the age-related reduction in DNA damage and repair capacity, we preliminarily evaluated the expressions of six kinds of pivotal enzymes involved in the two classical DNA repair pathways. The DNA repair capacity was observed in human fibroblasts cells using the comet assay; the age-related DNA repair enzymes were selected by RT-PCR and then verified by Western blot in vitro. Results showed that the DNA repair capacity was negatively and linearly correlated with (i) cumulative population doubling (PD) levels only in the group of low concentration of hydrogen peroxide treatment, (ii) with the level of oxidative stress only in the group of young PD cells. The mRNA expression of DNA polymerase δ1 decreased substantially in senescent cells and showed negative linear-correlation with PD levels; the protein expression level was well consistent with the mRNA level. Taken together, DNA damage and repair capacity can be a biomarker of aging. Reduced expression of DNA polymerase δ1 may be responsible for the decrease of DNA repair capacity in senescent cells.     

Accumulation of oxidative DNA damage, 8-oxo-2'-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Effects of in vitro cellular aging on the content of 8-oxo-2'-deoxyguanosine, a typical oxidation product of DNA bases, were examined in cultured human skin fibroblasts. The 8-oxo-2'-deoxyguanosine content in the DNA of TIG-3S cells established from skin tissues of a fetal donor increased immediately before the cessation of proliferation. TIG-114 and TIG-104 cells established from skin tissues of adult and aged donors, respectively, showed similar changes in 8-oxo-2'-deoxyguanosine content during in vitro cellular aging. The accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells was dependent on the number of cell divisions, and not on the cultivation time. Increases in the activities of superoxide dismutase and glutathione peroxidase were observed prior to the increase in 8-oxo-2'-deoxyguanosine content, while the catalase activity decreased gradually during in vitro cellular aging at late-passage. Furthermore, the activities of 8-oxo-2'-deoxyguanosine endonuclease and DNA polymerases decreased with the progression of proliferation. These results indicate that defense systems against oxidative stress in late-passage cells remain sufficiently active before the cessation of cell division, but that repair systems against oxidative damage decay at late-passage. Oxidative stress beyond the antioxidant capacity and/or repair activity seems to result in an accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells.     

Developmental regulation and in vitro culture effects on expression of DNA repair and cell cycle checkpoint control genes in rhesus monkey oocytes and embryos

DNA repair is essential for maintaining genomic integrity, and may be required in the early embryo to correct damage inherited via the gametes, damage that arises during DNA replication, or damage that arises in response to exposure to genotoxic agents. The capacity of preimplantation stage mammalian embryos to repair damaged DNA has not been well characterized, particularly in primate embryos. In this study, we examined the expression of 48 mRNAs related to sensing different kinds of DNA damage, repairing that DNA damage, and controlling the cell cycle to provide an opportunity for DNA repair. The expression data reveal dynamic temporal changes, indicating a changing ability of the rhesus embryo to detect and repair different kinds of DNA damage. Low expression or overexpression of specific DNA repair genes may limit the ability of the embryo to respond to DNA damage at certain stages. Additionally, our data reveal that in vitro culture may lead to dysregulation of many such genes and a potentially impaired ability to repair DNA damage, thus affecting cellular viability and long-term embryo viability via effects on genome integrity. This effect of in vitro culture on nonhuman primate embryos may be relevant to assessing the potential advantages and disadvantages of prolonged in vitro culture of human embryos.     

Ultraviolet light-induced DNA damage in transcribed sequences: no change in repair with age

We studied the repair of a plasmid vector containing the chloramphenicol acetyltransferase (CAT) gene by treating the plasmid with UV light and then transfecting this plasmid into fibroblasts from human fetal lung (in vitro aging) and into primary cultured fibroblasts from rat lung and skin. This methodology allows us to examine the repair of specific transcribed DNA sequences. There was no age-related change in the repair of UV damage in these cells. Rat embryo fibroblasts at different passages transfected with the plasmid also revealed no significant alteration in UV repair as a function of passage number.     

Delayed kinetics of DNA double-strand break processing in normal and pathological aging

Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage-induced foci of phosphorylated histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ-H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ-H2AX foci than those taken from normal donors of comparable age. Further increases in γ-H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ-H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ-H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ-H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

Effect of age on endogenous DNA single-strand breakage, strand break induction and repair in the adult housefly, Musca domestica

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular senescence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by gamma-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results do not support the view that DNA single-strand breaks are a causal factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.     

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

Role of Claspin in regulation of nucleotide excision repair factor DDB2

The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.     

Benzyl chloride and 4-chloromethylbiphenyl induced DNA damage and its repair in excision-deficient (XP group A) or -proficient human cells

Benzyl chloride (BC) and 4-chloromethylbiphenyl (4CMB) induce a class of alkaline-stable DNA damage in human cells which, like UV-induced pyrimidine dimers, undergoes repair at a slow rate by an excision-repair pathway which can be inhibited by cytosine arabinoside (araC). In the present study, in an attempt to clarify whether BC and 4CMB are UV-like agents, the excision-deficient xeroderma pigmentosum complementation group A fibroblasts and excision-proficient human alveolar tumour cells (A549) were exposed to various doses of these compounds prior to monitoring the inhibition of cell growth, DNA damage and DNA repair. The data indicate that such XP fibroblasts repair BC- and 4CMB-induced DNA damage at a normal rate, which suggests that the alkaline-stable DNA adducts induced by these chloromethyl compounds and the UV-induced pyrimidine dimers are processed by distinct excision-repair mechanisms in human cells.     

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.     

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.     

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.     

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.