Living with DNA Breaks is an Everyday Reality for Cells Adapted to High NaCl

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.     

Living with DNA breaks is an everyday reality for cells adapted to high NaCl

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.     

Response of renal inner medullary epithelial cells to osmotic stress

As part of the urinary concentrating mechanism, renal inner medullary epithelial (IME) cells are normally exposed to variable and often very high interstitial levels of NaCl and urea, yet they survive and function. We have been studying the mechanisms involved, using an established cell line (mIMCD3). Acute increase of NaCl or urea from 300 to >500 mOsmol/kg causes cell cycle delay and apoptosis. High NaCl, but not high urea, causes DNA double strand breaks. At 500-600 mOsmol/kg inhibition of DNA replication following high NaCl depends on activation of the tumor suppressor protein, p53, and provides time for DNA repair. If p53 expression is suppressed, cells continue to replicate DNA, and many of those cells die. At higher levels of NaCl (>650 mOsmol/kg) the mitochondria rapidly depolarize and most cells die within a few hours despite a high level of p53 protein (which, however, is less phosphorylated than at 500 mOsmol/kg). Since the levels of NaCl and urea that kill mIMCD3 cells are much lower than those that exist in vivo, we investigated the difference, using early passage mouse IME cells under various conditions. Passage 2 IME cells survive higher levels of NaCl and urea than do mIMCD3 cells, but still not levels as high as in vivo. However, when the osmolality is increased linearly over 20 h, as occurs in vivo, rather than as a single step, cell survival increases to levels close to those found in vivo. We conclude that a more gradual increase in osmolality provides time for accumulation of organic osmolytes and activation of heat shock protein, previously known to be important for cell survival.     

Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of the eubacterial family Deinococcaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. For example, after irradiation, D. radiodurans can repair > 100 DNA double-strand breaks per chromosome without lethality or mutagenesis, while most other organisms can survive no more than 2 or 3 double-strand breaks. The unusual resistance of D. radiodurans is recA dependent, but the repair pathway(s) is not understood. Recently, we described how a plasmid present in D. radiodurans (plasmid copy number, approximately 6 per cell; chromosome copy number, approximately 4 per cell) during high-dose irradiation undergoes extreme damage like the chromosome and is retained by the cell without selection and fully repaired with the same efficiency as the chromosome. In the current work, we have investigated the repair of two similar plasmids within the same cell. These two plasmids were designed to provide both restriction fragment polymorphisms and a drug selection indicator of recombination. This study presents a novel system of analysis of in vivo damage and recombinational repair, exploiting the unique ability of D. radiodurans to survive extraordinarily high levels of DNA damage. We report that homologous recombination among plasmids following irradiation is extensive. For example, 2% of Tcs plasmids become Tcr as a result of productive recombination within a 929-bp region of the plasmids after repair. Our results suggest that each plasmid may participate in as many as 6.7 recombinational events during repair, a value that extrapolates to > 700 events per chromosome undergoing repair simultaneously. These results indicate that the study of plasmid recombination within D. radiodurans may serve as an accurate model system for simultaneously occurring repair in the chromosome.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

High NaCl Promotes Cellular Senescence

High extracellular NaCl was previously shown to increase the number of DNA breaks in mammalian cells in tissue culture, renal medullary cells in vivo, C. elegans, and marine invertebrates. It was also shown to increase reactive oxygen species in renal cells, resulting in oxidation of proteins and DNA. Cellular senescence is a common response to such damage. Therefore, in the present studies we looked for signs of senescence in cells exposed to high NaCl. We find that (1) The rate of proliferation of HeLa cells exposed to high NaCl decreases gradually to the point of arrest, and the cells display signs of senescence, including hypertrophy and increased auto fluorescence. (2) High NaCl accelerates the appearance of senescence in primary mouse embryonic fibroblasts, as measured by β-galactosidase activity (SA-β-gal). (3) High NaCl retards growth and markedly decreases the life span of C. elegans, accompanied by features of accelerated aging, such as decreased locomotion and increased number of SA-β-gal positive cells. (4) Mouse renal medullary cells, which are normally continuously exposed to high NaCl, express increased p16^INK4 (another indicator of senescence) much earlier than do cells in the renal cortex, which has the same level of NaCl as peripheral blood. We conclude that high NaCl accelerates cellular senescence and aging, most likely secondary to the DNA breaks and oxidative damage that it causes.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.     

UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

The effect of deoxyadenosine plus deoxycoformycin on replicative and repair synthesis of DNA in human lymphoblasts and isolated nuclei

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Gadd45 in stress signaling

Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome.     

A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.     

Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis.     

X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here, we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase beta, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks, to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally regulated phenotypes, such as mitotic index.     

Antimetabolic activity of L-ascorbic acid in human and animal tumors

1. The effect of high concentrations of L-ascorbic acid on the growth of some human and animal transformed and non-transformed cell lines has been investigated. Directly implemented into culture of transformed cell lines it decreased [3H]thymidine, [3H]uridine and [3H]leucine incorporation into cells. Vitamin C inhibited DNA synthesis by transformed cells 3-4 times more efficiently than by normal cells. 2. In vivo treatment of athymic nude mice bearing human mammary carcinoma with 500 mg/kg L-ascorbic acid for the first 15 days markedly inhibited the growth of tumor cells. 3. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in mammary carcinoma cells treated with vitamin C. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase K assay. Removal of vitamin C caused an immediate onset of spontaneous repair of single or double stranded DNA breaks. If, however, vitamin was reintroduced into cell culture, this spontaneous repair was reversed. 4. Our results indicate an antimetabolic activity of L-ascorbic acid in human and animal transformed cells, probably due to lethal damages in DNA.     

Eumelanin causes DNA strand breaks and kills cells

Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gadd45-induced cell cycle G2/M arrest for improved transient gene expression in Chinese hamster ovary cells

Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.