Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells.

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.

Induction of cyclobutane pyrimidine dimers, pyrimidine(6-4)pyrimidone photoproducts, and Dewar valence isomers by natural sunlight in normal human mononuclear cells.

Immunocytochemistry was used for the direct measurement of cyclobutane pyrimidine dimers, (6-4) photoproducts, and Dewar isomers in normal human mononuclear cells following irradiation by natural sunlight or by a FS20 broad spectrum UVB sunlamp. The induction of each type of photoproduct was detected following 30-60 min sunlight exposure or with FS20 fluences as low as 50-100 Jm-2. With increasing FS20 fluences, there was a dose-dependent increase in the binding of pyrimidine dimer, (6-4) photoproduct, and Dewar isomer-specific monoclonal antibodies. The relative ratio of Dewar isomer to (6-4) photoproduct antibody binding sites was much higher following exposure to natural sunlight than to broad spectrum UVB. With the (6-4) monoclonal antibody, a small increase in binding sites was evident after a 1-h exposure to natural sunlight. This remained relatively constant with further exposure. These results are consistent with the hypothesis that, following irradiation with natural sunlight, the majority of (6-4) photoproducts are converted into Dewar valence isomers.

The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect.

The DNA repair-deficient genetic disorders xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) can both result from mutations in the XPD gene, the sites of the mutations differing between the two disorders. The hallmarks of XP are multiple pigmentation changes in the skin and a greatly elevated frequency of skin cancers, characteristics that are not seen in TTD. XP-D and most TTD patients have reduced levels of DNA repair, but some recent reports have suggested that the repair deficiencies in TTD cells are milder than in XP-D cells. We reported recently that inhibition of intracellular adhesion molecule-1 (ICAM-1) expression by UVB irradiation was similar in normal and TTD cells but increased in XP-D cells, suggesting a correlation between ICAM-1 inhibition and cancer proneness. In the first part of the current work, we have extended these studies and found several other examples, including XP-G and Cockayne syndrome cells, in which increased ICAM-1 inhibition correlated with cancer proneness. However, we also discovered that a subset of TTD cells, in which arg112 in the NH2-terminal region of the XPD protein is mutated to histidine, had an ICAM-1 response similar to that of XP-D cells. In the second part of the work, we have shown that TTD cells with this specific NH2-terminal mutation are more sensitive to UV irradiation than other TTDs, most of which are mutated in the COOH-terminal region, and are indistinguishable from XP-D cells in cell killing, incision breaks, and repair of cyclobutane pyrimidine dimers. Because the clinical phenotypes of these patients do not obviously differ from those of TTDs with mutations at other sites, we conclude that the lack of skin abnormalities in TTD is independent of the defective cellular responses to UV. It is likely to result from a transcriptional defect, which prevents the skin abnormalities from being expressed.

Ultraviolet-B-induced apoptosis and cytokine release in xeroderma pigmentosum keratinocytes.

We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B.

Induction of interleukin-6 production by ultraviolet radiation in normal human epidermal keratinocytes and in a human keratinocyte cell line is mediated by DNA damage.

The sunburn reaction is the most common consequence of human exposure to ultraviolet radiation (UVR), and is mediated at least in part by interleukin-6 (IL-6). The aim of this study was to determine if DNA is a major chromophore involved in the induction of IL-6 following UV irradiation of a human epidermoid carcinoma cell line (KB), and of normal human epidermal keratinocytes. We first confirmed that IL-6 release was associated with enhanced levels of IL-6 mRNA transcripts. The wavelength dependence for IL-6 release was then investigated by irradiating the cells at defined wavelengths (254, 302, 313, 334, and 365 nm) with a monochromator. The maximum effect on IL-6 release was observed at 254 nm with only low levels of induction observed at wavelengths above 313 nm. The wavelength dependence for UV-induced IL-6 release was similar to that for DNA absorption or for the induction of cyclobutane pyrimidine dimers (CPD). To determine whether UV-induced DNA damage mediated IL-6 secretion, the role of CPD was investigated by treating keratinocytes with photosomes (photolyase encapsulated in liposomes) followed by photoreactivating light. This photoreversal procedure led to a reduction in the levels of the UVC-induced secretion of IL-6, which in normal human keratinocytes was unambiguously associated with repair of CPD. We conclude that the release of IL-6 from human keratinocytes following short-wave UVC and UVB irradiation is mediated by DNA damage and that CPD play an important role in this process.

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6-4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6-4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6-4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fluences, which induce equivalent yields of cyclobutane dimers, (6-4) photoproducts or (6-4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6-4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6-4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

Inhibition of RNA and DNA synthesis in UV-irradiated normal human fibroblasts is correlated with pyrimidine (6-4) pyrimidone photoproduct formation.

UV-irradiation of living cells results in an inhibition of RNA and DNA synthesis. The purpose of this study was to determine whether specific photoproducts or the total combined yield of lesions were responsible for these effects. Asynchronously dividing human fibroblasts from normal donors were irradiated with UVC (254 nm), broad spectrum UVB (290-320 + nm, Westinghouse FS20 lamp) or narrow spectrum UVB (310-315 nm, Philips TL01 lamp) at fluences which induce known yields of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts or Dewar isomers. DNA synthesis was approximately 3-4 times more sensitive to both UVC and UVB irradiation than RNA synthesis. The immediate inhibition of RNA and DNA synthesis was correlated with (6-4) rather than overall photoproduct formation suggesting that the (6-4) photoproduct is the mediator of these inhibitory effects. In support of this suggestion we found that photoreactivation of cells cultured from the marsupial, mouse Sminthopsis crassicaudata, resulted in removal of 70% of pyrimidine dimers from the overall genome, but had only a slight effect on the recovery of RNA synthesis.

Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells.

Non-cycling human T-lymphocytes from normal subjects show a 10-fold greater sensitivity than fibroblasts to UV-B (280-315 nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides, each at a concentration of 10(-5) M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241; Green et al. (1994) Mutation Res., 315, 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cycling human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of read excisable damage, but should show normal resistance to agents which highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic procedures. Conditions favouring selective killing of non-dividing T-lymphocytes but sparing stem cells may be of value in bone marrow transplantation. Conditions favouring selective killing of dividing cancer cells but sparing non-dividing normal tissue may be of value in cancer therapy.

Measurement of DNA damage and repair capacity as a function of age using the comet assay.

A large number of studies indicate that DNA damage and mutation increase with age in human cells and tissues (1). Age-related degenerative disorders in which DNA damage has been invoked include heart disease and neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, or Parkinson's disease (2, 3). Patients with deficiencies in DNA repair, including xeroderma pigmentosum (XP) (4) and ataxia-telangiectasia (A-T) (5) show characteristic patterns of neurodegeneration (as opposed to a failure of normal development). The implication is that failure of repair can lead to accumulation of damage and degenerative disease. XPs and A-Ts are hypersensitive to specific types of DNA damage, and the degenerative damage in patients is tissue specific. DNA in every tissue, however, is under attack from a range of endogenously formed mutagens, including reactive oxygen species, nitric oxide, reactive metabolites, and breakdown products such as malondialdehyde. A series of repair enzymes recognize and remove these types of DNA damage from the genome. Failure to repair DNA may cause the synthesis of defective proteins, which will repair DNA less efficiently, and in turn lead to propagation of further errors (6). Alternatively, oxidative damage to mitochondrial proteins might cause less efficient processing of oxygen, release of higher levels of reactive oxygen species and increased levels of background DNA damage.

Histone H2AX phosphorylation as a molecular pharmacological marker for DNA interstrand crosslink cancer chemotherapy.

The aims of this study were to investigate mechanisms of action involved in H2AX phosphorylation by DNA interstrand crosslinking (ICL) agents and determine whether gammaH2AX could be a suitable pharmacological marker for identifying potential ICL cellular chemosensitivity. In normal human fibroblasts, after treatment with nitrogen mustard (HN2) or cisplatin, the peak gammaH2AX response was detected 2-3 h after the peak of DNA ICLs measured using the comet assay, a validated method for detecting ICLs in vitro or in clinical samples. Detection of gammaH2AX foci by immunofluorescence microscopy could be routinely detected with 6-10 times lower concentrations of both drugs compared to detection of ICLs using the comet assay. A major pathway for repairing DNA ICLs is the initial unhooking of the ICL by the ERCC1-XPF endonuclease followed by homologous recombination. HN2 or cisplatin-induced gammaH2AX foci persisted significantly longer in both, ERCC1 or XRCC3 (homologous recombination) defective Chinese hamster cells that are highly sensitive to cell killing by ICL agents compared to wild type or ionising radiation sensitive XRCC5 cells. An advantage of using gammaH2AX immunofluorescence over the comet assay is that it appears to detect ICL chemosensitivity in both ERCC1 and HR defective cells. With HN2 and cisplatin, gammaH2AX foci also persisted in chemosensitive human ovarian cancer cells (A2780) compared to chemoresistant (A2780cisR) cells. These results show that gammaH2AX can act as a highly sensitive and general marker of DNA damage induced by HN2 or cisplatin and shows promise for predicting potential cellular chemosensitivity to ICL agents.

Induction of mutagenic DNA damage in human fibroblasts after exposure to artificial tanning lamps.

There is increasing concern about the adverse health effects associated with the use of sunbeds, particularly with respect to skin photocarcinogenesis. The induction of mutagenic DNA damage is a prerequisite for the development of skin tumours, and it is well established that direct types of damage such as cyclobutane pyrimidine dimers (CPDs) give rise to mutations in tumour suppressor genes and oncogenes. In addition, ultraviolet radiation may induce indirect types of DNA damage, including oxidative products, which are also potentially mutagenic. By using specific DNA repair enzymes (T4 endonuclease V and endonuclease III) and the comet assay we have been able to detect the induction of CPDs, oxidized or hydrated pyrimidine bases and single-strand breaks in cultured human fibroblasts (MRC-5) after exposure for between 15 s and 20 min on two different commercial sunbeds containing Philips 'Performance' 100W-R or Philips TL80W/10R lamps. The ratio of endonuclease III to T4 endonuclease V sensitive sites varied substantially between the two lamps and was 3.3% and 18%, respectively. The sunbed containing the 'Performance' 100W-R lamps was as potent at inducing CPDs as was natural sunlight in fine weather. These results establish that commercial tanning lamps produce the types of DNA damage associated with photocarcinogenesis in human cells, and complement epidemiological evidence indicating the potential risk of using sunbeds.

Repair of cyclobutane pyrimidine dimers in unstimulated human mononuclear cells is deficient at very low fluences of ultraviolet B and is not enhanced by addition of deoxyribonucleosides.

Unstimulated human T lymphocytes are exquisitely sensitive to UVB irradiation. This hypersensitivity appears to relate to low deoxyribonucleotide pool sizes. They have also been reported to be defective in global excision of cyclobutane pyrimidine dimers, but such experiments may have been carried out at supralethal doses, where unrepaired excision breaks persist indefinitely. We use a T4 endonuclease Comet assay to show that removal of cyclobutane pyrimidine dimers is defective in the unstimulated mononuclear cell fraction (mainly T lymphocytes) even at sublethal fluences from an FS20 broad spectrum UVB lamp. Moreover, removal is not enhanced by addition of deoxyribonucleosides to the medium. Cells which are failing to remove cyclobutane pyrimidine dimers readily form fresh incision breaks in response to a second UVB fluence, indicating that they retain repair capacity and suggesting that removal of types of damage other than cyclobutane pyrimidine dimers is effective.

Possible effects of sunlight on human lymphocytes.

The human population is exposed to both the ultraviolet A (UVA) and B (UVB) regions of the solar spectrum. UVB induces mainly dipyrimidine photoproducts in DNA by a direct photochemical mechanism, whereas UVA is absorbed by other cellular constituents and induces mainly oxidative damage indirectly. The proportions of the different dipyrimidine photoproducts, and the ratio of dipyrimidine to oxidative damage depend on the exact spectral output of a UV source. Irradiation of human epidermal keratinocytes induces release of cytokines, with cyclobutane pyrimidine dimers playing a significant role in the process. These cytokines may then modulate the activity of cells of the immune system. Freshly isolated human lymphocytes are exquisitely sensitive to UVB irradiation, because of their low deoxyribonucleotide pools. They also have a separate defect in removal of cyclobutane pyrimidine dimers from their DNA. We have observed that frequencies of mutations at the hprt locus in human T-lymphocytes and translocations involving the bcl2 locus in B-lymphocytes appear to be associated with sunlight levels over the period before the blood sample was taken. This may be an indirect cytokine-mediated effect, and may be relevant to the possible link between non-Hodgkin's lymphoma and sunlight. On the other hand, sunlight can have beneficial effects, and may protect against autoimmune diseases including type I diabetes and multiple sclerosis.

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes.

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

The 0.8% ultraviolet B content of an ultraviolet A sunlamp induces 75% of cyclobutane pyrimidine dimers in human keratinocytes in vitro.

Tanning lamps, emitting predominantly ultraviolet (UV) A, are used widely throughout the U.K. and other countries, but little is known about the long-term risks associated with their use, especially with respect to skin cancer. We have exposed normal human epidermal keratinocytes to a commercial tanning lamp and used the comet assay in association with DNA repair enzymes T4 endonuclease V and endonuclease III to investigate the relative yields of directly formed cyclobutane pyrimidine dimers (CPDs) and indirectly formed types of oxidative DNA damage. To put the risk of using tanning lamps into perspective, the sunbed used in this study (five Philips Performance 80W-R UVA tubes at a distance of 35 cm) was found to be approximately 0.7 times as potent at inducing CPDs as U.K. natural sunlight around noon on a fine summer day. This compares with a relative risk for CPD induction and erythema of 0.8 and 0.7 times, respectively, calculated from the relevant action spectra of tanning lamps and British noontime sunlight. To determine the relative contribution of UVB and UVA to the induction of CPDs and oxidative DNA damage, we modified the spectral output of the tanning lamps with a series of Schott WG UVB filters. The induction of CPDs was more dependent on the UVB component of the sunbed than oxidative types of damage. Schott WG UVB filters with 50% transmission at 305 nm reduced the yield of T4 endonuclease V sites by 42% while there was only a 17% decrease in the yield of endonuclease III sites. CPD induction was not completely abolished after irradiation through WG335 and WG345 nm filters despite there being no detectable UVB. From these data, it was estimated that, although the tanning lamps emitted only 0.8% of their total output in the UVB range, these wavelengths were responsible for the induction of over 75% of CPDs and 50% of the oxidative damage to DNA.

Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-alpha and intercellular adhesion molecule-1 expression.

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.