The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage.

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation.

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.

Induction and repair of DNA cross-links in chinese hamster ovary cells treated with various platinum coordination compounds in relation to platinum binding to DNA, cytotoxicity, mutagenicity, and antitumor activity.

Several effects of four diamminechloroplatinum compounds (II and IV) in Chinese hamster ovary cells were studied. The two cis-compounds [cis-diamminedichloroplatinum(II) and cis-diamminetetrachloroplatinum(IV)] are known to possess antitumor activity, whereas the two trans-stereoisomers [trans-diamminedichloroplatinum(II) and trans-diamminetetrachloroplatinum(IV)] are inactive. When the effects of the cis-and trans-platinum compounds were compared after treatments that resulted in the binding of equal amounts of platinum to the DNA of the cells, the following differences were found: (a) the cis-platinum adducts gave a much higher cytotoxicity; (b) only the cis-platinum-DNA complexes were strongly mutagenic (forward mutations at the hypoxanthine-guanine phosphoribosyltransferase locus); (c) the cis-platinum adducts induced more sister chromatid exchanges; (d) the cis compounds initially induced fewer DNA-protein cross-links (Factors 5 to 8), but these cis-platinum cross-links were much more persistent; (e) for both cis complexes, the amount of DNA interstrand cross-links passed through a maximum between 6 and 12 hr after treatment, and the cross-links were repaired slowly. One trans-compound [trans-diamminetetrachloroplatinum(IV)] resembled the cis complexes with respect to the overall kinetics of formation and disappearance of this type of lesion, but the repair went faster. For the other trans compound [trans-diamminedichloroplatinum(II)], the highest number of cross-links was detected directly after the treatment of the cells, and they were rapidly eliminated. Neither the number of platinum-DNA lesions as such nor the initial amount of DNA interstrand cross-links could be related to the (geno)toxic effects of the compounds. However, as the slow repair of the cis-platinum-induced interstrand and DNA-protein cross-links leads to a certain persistency of these adducts, the unrepaired lesions might be responsible for cytotoxicity, mutagenicity, and antitumor activity. This indicates discriminating properties of the repair systems for certain cis-or trans-platinum-DNA adducts. The sister chromatid exchange induction seems to be related to the persistent DNA interstrand cross-links.

Formation and repair of DNA interstrand cross-links in relation to cytotoxicity and unscheduled DNA synthesis induced in control and mutant human cells treated with cis-diamminedichloroplatinum(II).

A comparative study was performed with a variety of human cell lines on the effects of treatments with cis-diamminedichloroplatinum(II) (cisplatin) on cell survival and the induction of unscheduled DNA synthesis. In addition to control fibroblasts (Han, MB), cell lines defective in DNA repair were used [xeroderma pigmentosum, XP(A) and XP(F), and Fanconi's anemia (FA)], as well as cells deficient in arylsulfatase A (mucolipidosis II, ML1 and ML2). Ultraviolet light and mitomycin C were included in this study as model DNA-damaging agents. Furthermore, induction of DNA interstrand cross-links by cisplatin and their repair were studied. As for survival, only XP cells were abnormally sensitive to ultraviolet light, and only FA cells were abnormally sensitive to mitomycin C. To cisplatin, however, all mutants tested were more sensitive (2 to 5 times) than were normal cells. Unscheduled DNA synthesis induction by ultraviolet light was strong in all but the XP cells; the other two agents did not induce unscheduled DNA synthesis. Induction of DNA interstrand cross-links by cisplatin was linear with dose. Formation continued for up to 18 to 24 h after treatment. During this period, all cells but the ML mutants responded similarly. In ML cells, much fewer cross-links were induced, which were repaired rapidly. In FA cells, accumulation continued for at least 96 h; in the other cells, most of the cross-links had been removed after that period. In the discussion, the cisplatin-induced DNA interstrand cross-links are proposed as an important potentially lethal lesion, in view of their persistence in the highly sensitive FA cells. Furthermore, the possible involvement of certain steps of the long-patch excision repair pathway in the removal of this lesion is considered. The sensitivity of ML cells to cisplatin is attributed to cytoplasmic effects, rather than to chromosomal damage.

Immortalization of carcinogen-treated Syrian hamster embryo cells occurs indirectly via an induced process.

The hypothesis that rodent cells can be immortalized by the direct induction of a single mutation-like event was tested by initiating cultures of benzo(a)pyrene treated Syrian hamster embryo cells with low inocula and expanding these few cells maximally until senescence prevented further culturing or immortalization took place. According to the mutation hypothesis immortalization is hardly to be expected under these conditions. However, immortalization was frequently observed. Therefore the induction of immortalization appears indirect. The progeny of benzo(a)pyrene treated cells immortalized with a rate of 3.9 x 10(-8)/cell/generation, which is 64 times higher than the spontaneous rate. The results are in line with the probabilistic theory developed in 1980 by both Fernandez et al. (Proc. Natl. Acad. Sci. USA, 77: 7272-7276, 1980) and Kennedy et al. (Proc. Natl. Acad. Sci. USA, 77: 7262-7266, 1980), which states that treatment of cells with a carcinogen can result in a so-called activated state of the treated cells which is transmitted to the progeny and which results in an enhanced rate of transforming events.

cis-Diamminedichloroplatinum(II)-induced DNA adducts in peripheral leukocytes from seven cancer patients: quantitative immunochemical detection of the adduct induction and removal after a single dose of cis-diamminedichloroplatinum(II).

The same four platinum-containing products identified in nucleolytic digests of DNA treated with cisplatin (cisDDP) in vitro now have been shown to be present in digested DNA originating from human cells after in vivo exposure. The immunochemical detection of these products at the fmol level became possible by the application of an existing and two newly raised rabbit antisera with specificities towards the various cisDDP-DNA derived products. In DNA isolated from white blood cells of a number of cancer patients treated with the drug for the first time, intrastrand cross-links on pGpG base sequences appeared to be the main adduct, followed by the intrastrand cross-links on pApG sequences, interstrand cross-links, and/or intrastrand cross-links on two guanines separated by one or more bases and a very low amount of monofunctionally bound cisDDP to guanine; typical proportions were 65, 22, 13, and less than 1%, respectively. The induction and removal of the main adduct, the intrastrand cross-link on pGpG sequences, have been studied in DNA from blood samples of six male patients after their first cisDDP treatment. The results indicate that the susceptibility of blood cells to cisDDP-DNA adduct formation can show strong individually determined differences. From the data it is also clear that a substantial part of the adducts is removed within the first 24 h after the cisDDP-infusion.

Molecular analysis of ethyl methanesulfonate-induced mutations at the hprt gene in the ethyl methanesulfonate-sensitive Chinese hamster cell line EM-C11 and its parental line CHO9.

The Chinese hamster cell line EM-C11 has been shown to be 5 times more sensitive than its parental line CHO9, but not hypermutable, after treatment with ethyl methanesulfonate. Ethyl methanesulfonate-induced mutational spectra were determined at the hprt locus to investigate whether the same adducts are responsible for mutation induction in both cell lines. The mutational spectra for EM-C11 and CHO9 show an important difference. GC-->AT transitions were found in both cell lines at similar frequencies; however, the spectrum of CHO9 contains a class of AT-->GC transitions, which seems to be replaced by a group of deletions in EM-C11. Since the ethyl methanesulfonate-induced mutation frequency for both lines is the same at equal exposure, it is hypothesized that the lesions leading to AT-->GC transitions in CHO9 are responsible for the deletions in EM-C11. This phenomenon might be explained if the responsible adduct(s) in CHO9 is bypassed resulting in replication errors, while blocking DNA synthesis in EM-C11 causing the observed increase in cell death. In surviving EM-C11 cells, DNA strand exchanges might have occurred at the position of stalled replication forks, leading to gross molecular changes. The adduct probably responsible for the AT-->GC transitions in CHO9 and the deletions in EM-C11 is 3-ethyladenine.

Overlapping pathways for repair of damage from ultraviolet light and chemical carcinogens in human fibroblasts.

DNA excision repair was measured in cultured human fibroblasts after single or dual treatments with ultraviolet radiation, 4-nitroquinoline 1-oxide, or N-acetoxy-2-acetylaminofluorene. Three approaches were used to monitor repair: unscheduled DNA synthesis, measured by autoradiography; repair replication, measured by the incorporation of a density-labeled DNA precursor into repaired regions; and excision of ultraviolet endonuclease-sensitive sites. When a single repair- saturating dose of one of the three carcinogens was administered, little stimulation of unscheduled DNA synthesis or repair replication could be observed by additional treatment with one of the other carcinogens. In no instance was total additivity of repair observed. These observations were confirmed by showing that the excision of endonuclease-sensitive sites produced by ultraviolet damage (i.e., pyrimidine dimers) was inhibited by exposure to 4-nitroquinoline 1-oxide and N-acetoxy-2-acetylaminofluorene. The data indicate that the repair of lesions induced by these substances may have common rate-limiting steps, a conclusion previously indicated by the repair deficiency in xeroderma pigmentosum cells in which a single mutation eliminates the repair of damage caused by each of these agents.

Immortalization of Syrian hamster embryo cells: probabilistic event or deterministic process.

Previous findings on the induction of immortalization in SHE cells have been explained with the activation/alteration hypothesis which postulates that treatment with a carcinogen results in the induction of a so-called "activated state" which enhances the rate of a probabilistic event in the progeny of the treated cells. This event is supposed to be a mutation. Because it has been recently indicated that in mammalian cells the switching on of signal transduction pathways by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or carcinogens can lead to genetic instability in the progeny of the treated cells, the possibility of an analogy between the induction of genetic instability and induction of immortalization after treatment with TPA was investigated. No effect of TPA was found on the rate of immortalization/cell/generation, not in otherwise untreated cells nor in cells treated with benzo(a)pyrene. TPA was found to enhance the life span of SHE cells. The life span of a culture correlated with its growth rate and its cell density at confluence both in the absence and presence of TPA. These correlations are supposed to reflect a regulation mechanism involved in the program of cellular senescence, and supposedly TPA can partly reverse this program. Treatment with benzo(a)pyrene also interferes with the life span resulting in premature senescence in most of the cells and extension of life span in a small fraction of the cells which subsequently can become immortal. Repeated switching from logarithmic growth to G0 also enhanced life span and rate of immortalization. The findings indicate that the activated state is a disturbance of a differentiation program affecting in SHE cells the program of cellular senescence and that, as an explanation for immortalization, epigenetic alterations causing a deterministic process of dedifferentiation in a subpopulation of the cells appear as plausible or perhaps even more plausible as a probabilistic mutation. This indicates that disturbance of differentiation might be among the causes of genetic instability.

Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts.

UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, e.g., (6-4) photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse.

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.

Strand-specific removal of cyclobutane pyrimidine dimers from the p53 gene in the epidermis of UVB-irradiated hairless mice.

Removal of UVB-induced cyclobutane pyrimidine dimers (CPD) from each of the two strands of the transcriptionally active p53 tumor suppressor gene and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was determined in the epidermis of the hairless mouse using the CPD-specific enzyme T4 endonuclease V. Mice were exposed to a single dose of UVB (2 kJ/m2) and kept in darkness for up to 24 h. About 80% of the CPD were removed from the transcribed strand of the p53 and HPRT genes within 24 h. Most rapid removal was observed during the first 4 h. In contrast, very little removal of CPD from the nontranscribed strand of the p53 and the HPRT genes was observed in 24 h. The same low level of repair was observed in the inactive c-mos proto-oncogene. The efficient repair of the transcribed strand compared to the nontranscribed strand of transcriptionally active genes in the epidermis of the hairless mouse resembles the repair of CPD in cultured rodent cells. Moreover, the selective removal of CPD from the transcribed strand of the p53 gene correlates well with the known strand bias of u.v.-induced mutations at dipyrimidine sites in the p53 gene of u.v.-induced mouse skin tumors.

Differences in pyrimidine dimer removal between rat skin cells in vitro and in vivo.

Pyrimidine dimers, the most abundant type of DNA lesions induced by ultraviolet light (UV), are rapidly repaired in human skin fibroblasts in vitro. In the same cell type from rats, however, there is hardly any removal of such dimers. To investigate whether this low capacity of rat skin cells to repair lesions in their DNA is an inherent characteristic of this species or an artifact due to cell culturing, we measured the removal of UV-induced pyrimidine dimers from rat epidermal keratinocytes both in vitro and in vivo. Epidermal keratinocytes in vitro were unable to remove any dimers over the first 3 h after UV-irradiation, while only about 20% was removed during a repair period of 24 h. In this respect, these cells were not different from cultured rat fibroblasts. In contrast to the results obtained with keratinocytes in vitro, we observed a rapid repair of pyrimidine dimers in UV-irradiated keratinocytes in vivo over the first 3 h; this rapid repair phase was followed by a much slower repair phase between 3 and 24 h. These results are discussed in terms of the possibility that mammalian cells are able to switch from one DNA repair pathway to another.

Detection of thymine dimers in suprabasal and basal cells of chronically UV-B exposed hairless mice.

An immunocytochemical method was developed to study induction and removal of DNA damage in specific cell populations in the epidermis of hairless mice during chronic ultraviolet (UV) exposure. Identification of mouse suprabasal cells was performed with an immunoperoxidase stain. This stain was shown not to affect the fluorescent nuclear stains, used to reveal DNA and DNA damage. In skin cells from hairless mice irradiated daily with 1500 J/m2 UV-B for 11 consecutive days, cyclobutane thymine dimers accumulated in epidermal cells and reached a maximum level after 3 d. Thereafter dimer levels dropped to a lower, more constant level. So epidermal cells in vivo, both suprabasal and basal cells, remove dimers effectively, in contrast to cultured rodent cells, which display hardly any repair in genomic DNA. Dimer content in suprabasal cells was higher than that in basal cells, but initially the patterns of induction and removal of dimers in both cell types were rather similar. At days 4-11, however, after the drop in dimer content, the amount of dimers in basal cells prior to UV exposure was almost as low as that in non-exposed cells. The results presented here suggest important roles for both UV-induced DNA repair and cell proliferation in protecting epidermal cells against the mutagenic and carcinogenic effects of UV.

The removal of UV-induced pyrimidine dimers from DNA of rat skin cells in vitro and in vivo in relation to aging.

Young and old rats were compared with respect to the capacity of their skin fibroblasts and epidermal keratinocytes to remove low levels of ultraviolet light (UV) induced UV-endonuclease sensitive sites (pyrimidime dimers) from their DNA, in vitro and in vivo, respectively. In vitro, over a 24-h time period, fibroblasts from both young and old rats were found to remove about 20% of the pyrimidine dimers originally induced by 4.6 J/m2 of UV-C. In vivo, after 2.6 kJ/m2 of UV-B hardly any UV lesions were found to be present in fibroblasts, as demonstrated by immunohistochemistry using an anti-thymine dimer antibody. As reported earlier (Mullaart et al., J. Invest. Dermatol., 90 (1988) 346-349) cultured epidermal keratinocytes do not differ from cultured fibroblasts in UV repair kinetics, whereas in vivo they remove at least 50% of the pyrimidine dimers induced by 4 kJ/m2 of UV-B within 3 h. We now show that epidermal keratinocytes from old rats are not deficient in their in vivo repair characteristics upon this low UV-B dose. However, since a considerable fraction of the pyrimidine dimers appeared to be persistent in fibroblasts and keratinocytes, demonstrated by both enzymatic and immunochemical assays, the possibility is discussed that long-term exposure of skin cells to UV may lead to an accumulation of DNA damage with age.

Immunochemical detection of DNA in alkaline sucrose gradient fractions.

A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.