The regulation of DNA repair processes in mammalian cells. II. The repair of DNA radiation damage in NIH 3T3 murine cells transformed by the v-myc oncogene]

The kinetics of repair of the ionizing radiation-induced DNA single- and double-strand breaks in the normal NIH 3T3 mouse cells and in those transformed with virus oncogenes v-myc has been investigated. The incubation of non-transformed cells for 18 hours in serum-free medium results in significant decrease in the rate of the single-strand DNA breaks repair during the first minutes of post-irradiation incubation. This effect is absent in transformed cells. The DNA double-strand breaks repair is more efficient in transformed NIH 3T3 cells as compared to that in the non-transformed ones both after their incubation in the medium with 10% fetal bovine serum or without serum. However, more significant differences in the rate of elimination of these DNA lesions was found in the serum-free medium. Hence, the presence of v-myc sequences in the transformed cells prevented from a decrease in the efficiency of DNA repair due to incubation of cell culture in serum-free medium. These results agree with the assumption that c-myc gene product may be a mediator in regulation of DNA repair by the epidermal growth factor. These data also show that the c-myc gene expression in an important condition providing a high efficiency of the constitutive DNA repair process.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell.A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Somatomedin C/insulin-like growth factor 1: an intratesticular differentiative factor of Leydig cells?

By using immature porcine Sertoli cells cultured in serum-free defined medium, we report that medium conditioned by Sertoli cells contained immunoreactive somatomedin C/insulin-like growth factor 1 (SmC/IGF1) measured following acidic gel filtration. The release of this immunoreactive SmC/IGF1 was slightly increased following Sertoli cell treatment with fibroblast growth factor but not with follicle-stimulating hormone or growth hormone. On the other hand, human biosynthetic SmC/IGF1 exerts a potent stimulatory effect on Leydig cell differentiated functions such as LH/hCG-binding (greater than 4-fold) and hCG-stimulated testosterone secretion (greater than 15-fold). This effect was dose and time dependent and the maximal increase of Leydig cell function was observed following 48 h treatment with 50 ng/ml SmC/IGF1. The steroidogenic action of the peptide was not related to Leydig cell growth since both cell number and 3H-thymidine incorporation into DNA were not or slightly (approximately equal to 1.5-fold) increased in the optimal conditions with SmC/IGF1 treatment (100 ng/ml for 48 h). Moreover, the concomitant treatment of Leydig cells by both arabinoside C (10(-5) M), a DNA synthesis inhibitor, and SmC/IGF1 did not modify the stimulating effect of the peptide on LH/hCG-binding and hCG-stimulated testosterone production. Taken together, the present findings support the concept that Sertoli cell derived SmC/IGF1 could be a potent regulator of Leydig cell differentiated functions.     

Growth factor requirements for DNA synthesis by Leydig cells from the immature rat.

Puberty in the male is dependent upon the elevated production of testosterone by the Leydig cells. LH affects this increase in testosterone output by increasing the total number of Leydig cells in the testis and by stimulating the steroidogenic pathway in these cells. Since Leydig cell proliferation is a prerequisite for the onset of puberty, we have examined the ability of LH and growth factors known to be present in the testis to promote DNA synthesis. Leydig cells were isolated from 21-day-old rats, cultured in serum-free medium for 48 h to become quiescent, and then treated with LH and growth factors for 18 h. [3H]Thymidine incorporation into DNA was assessed over the subsequent 4-h incubation period. Cells in control cultures incorporated low levels of [3H]thymidine into DNA and were stimulated after treatment with LH (100 ng/ml). Insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), previously localized in Leydig cells by immunohistochemistry, also stimulated [2H]thymidine incorporation into DNA. The responses of the Leydig cells to maximum levels of insulin and TGF-alpha were dependent on the cell density. Insulin and TGF-alpha alone and in combination increased the number of cells labeled with [3H]thymidine, as assessed by autoradiography. TGF-beta, known to be secreted by Sertoli cells, also stimulated DNA synthesis under basal conditions, but the maximum response was significantly lower than that achieved in the presence of TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.     

Induction of gene mutations and the lethal action of ultraviolet rays in synchronized Chinese hamster cells

Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis.     

The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells

The mutagenicity of MNU, EMS, BMS and UV light was compared by analyzing the dose-response curve just before and after the replicative process of the HGPRT gene in synchronized V79 Chinese hamster cells. This system makes it possible to compare a 10-h period for repair of different mutagenic lesions with no time for repair. Additional time for repair in synchronized V79 cells resulted in a reduced response for MNU and UV, but not for EMS and BMS. This result suggests that an error-free repair process operates on mutagenic lesions in methylated DNA and on thymine dimers, but not on ethylated and butylated DNA. Based on these results, it is concluded that the repair capacity of V79 cells to remove mutagenic lesions is characterized as low for UV, moderate for MNU and not detectable for the mutagenic lesions induced by EMS and BMS.     

The citrus flavonoid naringenin stimulates DNA repair in prostate cancer cells

As part of a systematic study of the effects of phytochemicals beyond antioxidation on cancer prevention, we investigated whether naringenin (NR), a citrus flavonoid, stimulates DNA repair following oxidative damage in LNCaP human prostate cancer cells. The 8-hydroxydeoxyguanosine (8-OH-dG) to deoxyguanosine (dG) ratio was measured after cells were treated with 200 micromol/L of ferrous sulfate in serum-free medium followed by NR exposure for 24 h in growth medium. The results demonstrated that exposure to 10-80 micromol/L of NR led to a significant decrease in the ratio of 8-OH-dG to 10(6) dG. Because cells were treated with NR after ferrous sulfate was removed, we conclude that we demonstrated an effect on DNA repair beyond antioxidation. In support of this conclusion, we determined the induction of mRNA expression over time after oxidative stress followed by NR administration of three major enzymes in the DNA base excision repair (BER) pathway: 8-oxoguanine-DNA glycosylase 1 (hOGG1), apurinic/apyrimidinic endonuclease and DNA polymerase beta (DNA poly beta). hOGG1 and DNA poly beta mRNA expression in cells after 24-h exposure to NR was increased significantly compared with control cells without NR. The intracellular concentration of NR after exposure to 80 micromol/L was 3 pmol/mg protein, which is physiologically achievable in tissues. In conclusion, the cancer-preventive effects of citrus fruits demonstrated in epidemiological studies may be due in part to stimulation of DNA repair by NR, which by stimulating BER processes may prevent mutagenic changes in prostate cancer cells.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary—or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

DNA polymerase β and PARP activities in base excision repair in living cells

To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14 h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair capacity for uracil-DNA in wild type mouse fibroblasts was very strong, whereas the repair capacity for THF-DNA, although strong, was slightly weaker. Repair capacity in DNA polymerase β (Pol β) null cells for uracil-DNA and THF-DNA was reduced by approximately 15% and 20%, respectively, compared to that in wild type cells. In both cases, the repair deficiency was fully complemented in Pol β null cells expressing recombinant Pol β. The effect of inhibition of poly(ADP-ribose) polymerase (PARP) activity on repair capacity was examined by treatment of cells with the inhibitor 4-amino-1,8-naphthalimide (4-AN). PARP inhibition decreased the repair capacity for both lesions in wild type cells, and this reduction was to the same level as that seen in Pol β null cells. In contrast, 4-AN had no effect on repair in Pol β null cells. The results highlight that Pol β and PARP function in the same repair pathway, but also suggest that there is repair independent of both Pol β and PARP activities. Thus, before the BER capacity of a cell can be predicted or modulated, a better understanding of Pol β and PARP activity-independent BER pathways is required.     

Host cell reactivation by excision repair is error-free in human cells

Do host cell repair processes affect the mutagenesis of UV-irradiated virus in human cells? The answer was obtained by investigating the mutagenesis of UV-irradiated herpes simplex virus after the irradiated virus was grown in human cells that possess normal repair capacity (normal) or lack excision repair (XPA) or post-replication repair (XP var). Evidence is presented which indicate that XPA cells express no host cell reactivation, while XP var cells express the normal level. Viral mutagenesis was measured as the fraction of the progeny of the surviving virus capable of plaque formation in the presence of iododeoxycytidine. In the normal and XPA cells mutagenesis of the irradiated virus increased linearly with UV exposure. The UV exposure needed to yield a given mutagenesis level for virus grown in XPA cells was much lower than that for virus grown in normal cells. However, when the mutation frequencies were compared at similar virus survival levels, the data from virus grown in normal cells and in XPA cells were indistinguishable. Mutagenesis in XP var cells increased as dose squared and was similar in magnitude to that in normal cells. Thus the excision repair of normal cells which provided host cell reactivation by removing lethal UV damage also removed mutagenic lesions from the virus with the same efficiency, while the repair deficiency of XP var cells had a minor role in host cell reactivation and in mutagenesis. This demonstrates that in human cells host cell reactivation by excision repair is primarily an error-free process.     

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.     

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.