Inhibition of post-replication repair by isonicotinic acid hydrazide

In the presence of the alkylating mutagen N-methyl-N-nitrosourea (MNU), the well-known tuberculostatic ionicotinic acid hydrazide (INH), even in otherwise ineffective doses, depressed cell number and mitotic index in peripheral human lymphocytes and inhibited the post-replication repair process in Chinese hamster cells (CHO). INH had no influence on unscheduled DNA synthesis (cut-and-patch repair), which was negligible in CHO cells under our conditions.     

A sensitive method to measure physical and chemical carcinogen-induced "unscheduled DNA synthesis" in rapidly dividing eukaryotic cells

A sensitive assay for quantitating ‘unscheduled DNA synthesis’ (repair synthesis) in transformed human amnion (AV₃) cells has been developed. The combined use of hydroxyurea and arginine-deficient culture medium enabled the detection of 10–20 fold increases in ‘unscheduled DNA synthesis’ after treatment with N-acetoxy-2-acetylaminofluorene or ultraviolet light. The technique allows the detection of ‘DNA repair synthesis’ following treatment with extremely low doses of mutagens and carcinogens.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.     

EHRLICH ASCITES TUMOR (EAT) CHALONE EFFECTS ON NASCENT DNA SYNTHESIS AND DNA POLYMERASE ALPHA AND BETA

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of ³H-thymidine (³H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50–200 μg/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude α and β-polymerase activities were inhibited. Crude DNA polymerase from C₃H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or ‘gapped’ DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting α- and β-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating that DNA ligase is not inhibited.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.     

Mechanism of action of interferon is linked to its ability to protect the cell from interstrand cross-linking of DNA, induced by 8-methoxypsoralen, activated by long-wave UV-light

Treatment of human lymphocytes in the G1 phase of mitotic cycle with human lymphoblastoid interferon (Ly-IFN) decreased the frequencies of chromosome aberrations induced by 8-methoxy-psoralen-induced interstrand cross-links. Anticlastogenic effect of Ly-IFN was accompanied by stimulation of unscheduled synthesis of DNA in the G2 phase of mitotic cycle, as shown by increased percent of labeled cells registered by 3H-thymidine autoradiography. The data obtained seem to indicate that the mechanism of Ly-IFN protection is connected with stimulation of postreplicative repair.     

Inhibition of unscheduled DNA synthesis and repair of potentially lethal X-ray damage by 2-deoxy-D-glucose in yeast.

The effects of the glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on DNA repair (assayed by unscheduled DNA synthesis) and on the repair of potentially-lethal damage (assayed by cell viability after irradiation) have been studied in X-irradiated respiratory-deficient yeast cells (auxotroph for 5'-thymidine-monophosphate). Experimental results show that: (a) both these phenomena can be inhibited by 2-DG; (b) the repair of potentially-lethal damage occurs after the unscheduled DNA synthesis is almost complete; and (c) the repair of potentially-lethal damage can be inhibited by 2-DG even after the completion of the unscheduled DNA synthesis.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G1 cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains — BALB/c, C3H/He and C57BL/10.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G₁ cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair process in human diploid fibroblasts

Formaldehyde treatment of human fibroblasts gave rise to DNA damage detected by a nick translation assay. This damage was not repaired by typical 'long-patch'-type excision repair as evidenced by the failure of DNA repair inhibitor post-treatment to elevate the amount of DNA strand breakage. In addition, the effects of formaldehyde on DNA repair were examined in light of a recent report suggesting that formaldehyde inhibited the repair of X-ray-induced strand breaks and UV- and benzo [a]pyrene diol epoxide-induced unscheduled DNA synthesis in human bronchial cells. We report that formaldehyde (1) was ineffective at inhibiting the sealing of X-ray- or bleomycin-induced DNA strand breaks, (2) did not inhibit the removal of pyrimidine dimers from cellular DNA at short treatment times, and (3) that the previously observed inhibition of unscheduled DNA synthesis was most likely due to the inhibition of uptake of labeled precursor into formaldehyde-treated cells. Thus, our findings are not consistent with the notion that formaldehyde inhibits the repair process in human fibroblasts. Finally, formaldehyde was shown to elevate the level of misincorporation of bases into synthetic polynucleotides catalyzed by E. coli DNA polymerase I, indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA bases.     

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl

BACKGROUND AND AIMS: The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. METHODS: Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. KEY RESULTS: Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. CONCLUSIONS: Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.