Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis.

BACKGROUND: The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. METHODS: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. RESULTS: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. CONCLUSION: We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.

Minimal ionizing radiation sensitivity in a large cohort of xeroderma pigmentosum fibroblasts.

We have examined our ionizing radiation survival data for 33 xeroderma pigmentosum (XP) primary fibroblast lines and compared the data to that of 53 normal fibroblast lines, 7 Cockayne syndrome (CS) lines, 4 combined XP/CS lines and 8 ataxia-telangiectasia fibroblast lines. Although there are differences in radiosensitivity between cell lines within each class, we have no convincing evidence that XP lines as a group are more sensitive to ionizing radiation than the general population. However, because the XP phenotype may lead to premature ageing, especially of sun-exposed tissues, we would still advocate caution when XP patients come to radiotherapy. Our results confirm the extreme ionizing radiation hypersensitivity of ataxia-telangiectasia; they are also consistent with a tendency for slight hypersensitivity in CS, but not (necessarily) in combined XP/CS.

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum.

XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.

Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene.

The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.

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.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Four radiation hypersensitivity cases and their implications for clinical radiotherapy.

BACKGROUND AND PURPOSE: Over a 20 year period, four out of 2000 paediatric radiotherapy patients, treated at St. Bartholomew's Hospital (three with lymphoma, one with angiosarcoma), have revealed extreme/fatal clinical hypersensitivity in normal tissues. PATIENTS AND METHODS: Cellular hypersensitivity was confirmed in vitro and attributed to the ataxia-telangiectasia (A-T) gene in cases I and II, a newly described defect in the DNA ligase 4 gene in case III, and a novel and as yet incompletely defined, molecular defect in case IV who presented with xeroderma pigmentosum (XP). RESULTS: The severe clinical hypersensitivity preceded the cellular and molecular analysis, but did not manifest as a clinically exaggerated normal tissue reaction until 3+ weeks after the start of a conventionally fractionated course of radiotherapy, by which time the latent damage had been inflicted. There were no clinical stigmata to alert the clinician to a predisposing syndrome in two patients (cases I and II). We point out that approximately 20% of A-T patients are classified as variants with delayed expression of clinical symptoms, and case II falls into this category. CONCLUSIONS: As lymphoma (incidence, one in 100000 children) constituted the majority of the diagnoses, questions arise as to: (1), the probability of other centres having experienced and being presented in the future with similar problems (particularly bearing in mind that other oncologically predisposing radiosensitivity syndromes have not been not represented in our experience); and (2), the appropriateness, efficiency and applicability of predictive assays. Unambiguous cellular radiosensitivity would have been apparent from clonal assays on fibroblast cultures from all four cases prior to treatment, but such assays take 4-6 weeks to produce results. While estimates of chromosome damage or clonal assays on pre-treatment blood derived cells would be faster, there is a health economics issue as to the general applicability of such 'screening' assays.

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.

Radiosensitivity in Nijmegen Breakage Syndrome cells is attributable to a repair defect and not cell cycle checkpoint defects.

Cells derived from Nijmegen Breakage Syndrome (NBS) patients display radiosensitivity and cell cycle checkpoint defects. Here, we examine whether the radiosensitivity of NBS cells is the result of a repair defect or whether it can be attributed to impaired checkpoint arrest. We report a small increased fraction of unrejoined double strand breaks and, more significantly, increased chromosome breaks in noncycling NBS cells at 24 h after irradiation. One of the NBS lines examined (347BR) was atypical in showing a nearly normal checkpoint response. In contrast to the mild checkpoint defect, 347BR displays marked y-ray sensitivity similar to that shown by other NBS lines. Thus, the gamma-ray sensitivity correlates with the repair defect rather than impaired checkpoint control. Taken together, the results provide direct evidence for a repair defect in NBS cells and are inconsistent with the suggestion that the radiosensitivity is attributable only to impaired checkpoint arrest. 347BR also displays elevated spontaneous damage that cannot be attributed to impaired G2-M arrest, suggesting a function of Nbsl in decreasing or limiting the impact of spontaneously arising double strand breaks.

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 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.

Co-cultivation of CD4+ and CD8+ human T-cells leads to the appearance of CD4 cells expressing CD8 through de novo synthesis of the CD8 alpha-subunit.

The appearance of a population of dual-staining CD4+CD8+ cells in human T-lymphocyte cultures has been reported by various authors, including our own observation that they are always seen in simple phytohaemagglutinin-stimulated cultures from several different donors. The purpose of the present study was to investigate factors involved in the dual-staining (DS) phenotype, and to clarify some apparent inconsistencies between published observations. Our findings can be summarised as follows. 1. A population of DS CD4+CD8+ cells always appears in PHA-stimulated T-cell cultures if they contain both CD4 and CD8 subsets. The incidence of DS cells is related to PHA concentration, but other factors are involved since DS cells are not seen in PHA-stimulated cultures of purified CD4+ or CD8+ cells. Stimulation with PHA is not a prerequisite since very similar results are seen with ConA. 2. Direct physical contact between CD4+ and CD8+ cells is required for the appearance of the DS phenotype; soluble factors alone, including IL-4, appear nor to be responsible. 3. The DS phenotype in these conditions is always CD4+ cells weakly expressing CD8 and is a consequence of de novo synthesis of the CD8alpha molecule by the CD4+ cells.

Characterization of photosensitivity in the Smith-Lemli-Opitz syndrome: a new congenital photosensitivity syndrome.

Photosensitivity has recently been reported as a feature of the Smith-Lemli-Opitz syndrome (SLO). The aim of this study was to establish the photobiological features of this disorder and to examine the hypothesis that the photosensitivity is caused by the high levels of 7-dehydrocholesterol found in SLO. All known cases of SLO in the U.K. were reviewed and clinical details of photosensitivity were recorded in detail. The action spectrum of the photosensitive eruption was defined by monochromator light testing. Thirteen of the 23 subjects (57%) had severe photosensitivity, and in 10 there was no photosensitivity. No correlation was identified between levels of 7-dehydrocholesterol and severity of photosensitivity, suggesting that the photosensitivity in SLO is not caused by a direct phototoxic effect mediated by 7-dehydrocholesterol. A novel pattern of photosensitivity was observed, with onset of a sunburn-like erythema on sun-exposed skin within minutes of sun exposure, which persisted in most cases for up to 24-48 h before fading. Monochromator light testing in three subjects showed an ultraviolet (UV) A-mediated photosensitivity eruption with greatest photosensitivity at 350 nm. Photosensitivity is a common and prominent feature of SLO and appears to be UVA-mediated. Elucidation of its biochemical basis may provide insight into normal cutaneous protective mechanisms against UVA-induced photodamage, and also sun sensitivity in general.

Frequencies of hprt(-) mutations and bcl-2 translocations in circulating human lymphocytes are correlated with United Kingdom sunlight records.

Between 1983 and 1995 we have monitored human populations for evidence of exposure to environmental mutagens, taking blood samples to measure hprt(-) mutant frequency in T cells and more recently bcl-2 t(14:18) translocation frequency in B cells. We have now analysed data from 785 assays on 448 blood samples from 308 normal subjects and find that there is a highly significant statistical correlation between hprt(-) mutant frequency and the sunlight record for the 3 weeks prior to taking the blood sample. We discuss the weaknesses in retrospective studies of this nature and the possibility of spurious epidemiological correlations that may result. More controlled experiments can be envisaged that would give a firmer basis to the statistical associations observed. hprt(-) mutations in T cells show little evidence of a UV fingerprint, so that the correlation may be due to immunomodulation rather than mutation. We also find a correlation between the sunlight record and bcl-2 translocation. This translocation is found at a low frequency in the B cells of many normal subjects and is the commonest translocation observed in non-Hodgkin's lymphoma. Our results strengthen the case for a link between sunlight and this increasingly common cancer.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.

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.

Risk of breast cancer and other cancers in heterozygotes for ataxia-telangiectasia.

Mortality from cancer among 178 parents and 236 grandparents of 95 British patients with ataxia-telangiectasia was examined. For neither parents nor grandparents was mortality from all causes or from cancer appreciably elevated over that of the national population. Among mothers, three deaths from breast cancer gave rise to a standardized mortality ratio of 3.37 (95% confidence interval (CI): 0.69-9.84). In contrast, there was no excess of breast cancer in grandmothers, the standardized mortality ratio being 0.89 (95% CI: 0.18-2.59), based on three deaths. This is the largest study of families of ataxia-telangiectasia patients conducted in Britain but, nonetheless, the study is small and CIs are wide. However, taken together with data from other countries, an increased risk of breast cancer among female heterozygotes is still apparent, though lower than previously thought.

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

PURPOSE: To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. MATERIALS AND METHODS: The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Blöcher model can be used to express data as DSB after irradiation at 4 degrees C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4 degrees C followed by incubation at 37 degrees C) in untransformed human fibroblasts. RESULTS: The DSB induction rate after irradiation at 4 degrees C was 39.1+/-2.0 Gy(-1). The DSB repair rate obtained after doses of 10 to 80 Gy followed by repair times of 0 to 24 h was expressed as unrepaired DSB calculated from the Blöcher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. CONCLUSION: The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Blöcher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.

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.