RELATIVE REPAIR OF ADENOVIRUS DAMAGED BY SUNLAMP, UV AND γ-IRRADIATION IN COCKAYNE SYNDROME FIBROBLASTS IS DIFFERENT FROM THAT IN XERODERMA PIGMENTOSUM FIBROBLASTS

The DNA repair capacities of three unrelated Cockayne syndrome (CS) fibroblast strains were compared to that of three unrelated xeroderma pigmentosum (XP) strains for three different DNA damaging agents using a sensitive host cell reactivation (HCR) technique. Adenovirus type 2 (Ad 2) was treated with either UV light, gamma-rays or sunlamp-irradiation and subsequently assayed for its ability to form viral structural antigens (Vag) in the CS and XP strains using immunofluorescent straining. D37 values for the survival of Ad 2 Vag synthesis in the CS and XP strains, expressed as a percentage of those obtained in normal strains, were used as a measure of DNA repair capacity. Percent HCR values in the XP strains XP25RO, XP2BE and XP5BE respectively were lowest for UV (6, 14 and 6%), intermediate for sunlamp-irradiation (18, 32 and 10%) and highest for gamma-irradiation (65, 61 and 60%), whereas for the CS strains CS1BE, CS3BE and CS278CTO respectively, percent HCR values were lowest for UV (26, 30 and 34%), intermediate for gamma-irradiation (61, 64 and 69%) and near normal for sunlamp-irradiation (82, 73 and 89%). These results suggest that the 'spectrum of lesions' which is defectively repaired in CS is not the same as that which is defectively repaired in XP.     

HOST CELL REACTIVATION IN MAMMALIAN CELLS-V. PHOTOREACTIVATION STUDIES WITH HERPES VIRUS IN MARSUPIAL AND HUMAN CELLS

Abstract— The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovan cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes virus in Potoroo cells indicated a high level of "dark" host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (Λ > 600 nm) and human cells with normal repair and with ceils deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreacti-vating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps.     

REDUCED REPAIR OF NON-DIMER PHOTOPRODUCTS IN A GENE TRANSFECTED INTO XERODERMA PIGMENTOSUM CELLS

Abstract— 4ells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m⁻² of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA.     

Characteristics of Xeroderma Pigmentosum in Japan: Lessons From Two Clinical Surveys and Measures for Patient Care

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease caused by deficiency in repair of DNA lesions generated by ultraviolet radiation and other compounds. Patients with XP display pigmentary change and numerous skin cancers in sun‐exposed sites, and some patients show exaggerated severe sunburns even upon minimum sun exposure as well as neurological symptoms. We conducted a nationwide survey for XP since 1980. In Japan, the frequency of the XP complementation group A is the highest, followed by the variant type; while in the Western countries, those of groups C or D are the highest. Regarding skin cancers in XP, basal cell carcinoma was the most frequent cancer that afflicted patients with XP, followed by squamous cell carcinoma, and malignant melanoma. The frequency of these skin cancers in patients with XP has decreased in these 20 years, and the age of onset of developing skin cancers is higher than those previously observed, owing to early diagnosis and education to patients and care takers on strict prevention from sunlight for patients with XP. On the other hand, the effective therapy for neurological XP has not been established yet, and this needs to be done urgently.     

HOST CELL REACTIVATION AND UV-ENHANCED REACTIVATION IN SYNCHRONIZED MAMMALIAN CELLS

Abstract— Does host cell reactivation (HCR) or UV-enhanced reactivation (UVER) of UV-irradiated Herpes simplex virus (UV-HSV) vary during the host mammalian cell cycle? The answer could be useful for interpreting UVER and/or the two-component nature of the UV-HSV survival curve. Procedures were developed for infection of mitotically-synchronized CV-1 monkey kidney cells. All virus survival curves determined at different cell cycle stages had two components with similar D₀'s and intercepts of the second components. Thus, no single stage of the host cell cycle was responsible for the second component of the virus survival curve. When the cells were UV-irradiated immediately prior to infection, enhanced survival of UV-HSV occurred for cell irradiation and virus infection initiated during late G₁/early S phase or late S/early G₂ phase but not during early G₁ phase. For infection delayed by 24 h after cell irradiation, UVER was found at all investigated times. These results indicate that: (1) HCR is similar at all stages of the host cell cycle; and (2) the “induction” of UVER is not as rapid for cell-irradiation in early G₁ phase. This latter observation may be one reason why normal, contact-inhibited cells do not express UVER as rapidly as faster growing, less contact-inhibited cells.     

Partial complementation of the DNA repair defects in cells from xeroderma pigmentosum groups A, C, D and F but not G by the denV gene from bacteriophage T4

Endonuclease V (denV) from bacteriophage T4 was examined for its ability to complement the DNA repair defect in xeroderma pigmentosum (XP) cells from complementation groups A, C, D, F and G. The denV gene was introduced into SV40-transformed normal and XP cells using a retroviral vector. Expression of denV resulted in partial correction of UV sensitivity and increased host cell reactivation (HCR) of a UV-damaged reporter gene for XP cells from groups A, C and D, but not those from group G. Expression of denV in XP-F cells resulted in enhanced HCR of a UV-damaged reporter but did not affect UV sensitivity. The observed partial complementation is thought to reflect denV-mediated repair of cyclobutane-pyrimidine dimers (CPD), and is incomplete as denV does not recognize other UV-induced lesions, and may not even efficiently remove all CPD. As XP-F cells are believed to retain near-normal levels of CPD repair in the bulk of the genome, we believe that the disparity in the ability of denV to complement the repair deficiency in these cells results from an increased rate, but not level, of CPD repair. Furthermore, we suggest that the lack of correction in the XP-G cells examined results from an inability to process denV-incised CPD by the base excision repair pathway, as has been suggested for cells from the related genetic disorder, Cockayne syndrome. Expression of denV in repair proficient normal cells also resulted in increased HCR of the UV-damaged reporter construct, possibly arising from an increased rate of CPD repair in these cells.     

DAMAGE BY SOLAR RADIATION AT DEFINED WAVELENGTHS. INVOLVEMENT OF INDUCIBLE REPAIR SYSTEMS

Abstract The susceptibility of bacteriophage damaged by solar-ultraviolet (UV, 290-380 nm) radiations at denned wavelengths and by radiation at a visible wavelength (405 nm) to the Weigle reactivation system induced by far-UV (254 nm) irradiation of the host cell has been studied in a repair competent strain of Escherichia coli . The sector of inducible repair diminishes with wavelength, being very small after 313 nm irradiation and absent after irradiation at longer wavelengths. However, irradiation of bacteria at wavelengths as long as 313 nm induces a bacteriophage reactivation system as effectively as radiation at 254 nm in both the repair competent and an excision deficient host cell. At longer wavelengths pre-irradiation of the repair competent host cell enhances reactivation of 254 nm irradiated bacteriophage but the reactivation is smaller and the process quite distinct from that induced in the 254-313 nm region. We conclude that, with increasing wavelength, damage induced by solar UV radiations becomes increasingly less susceptible to repair systems induced by far-UV (pyrimidine dimers) and that this type of inducible repair system is no longer induced by wavelengths longer than 313 nm.     

UV-ENHANCED REACTIVATION OF MINUTE-VIRUS-OF-MICE: STIMULATION OF A LATE STEP IN THE VIRAL LIFE CYCLE

Abstract— UV-enhanced reactivation of minute-virus-of-mice (MVM), an autonomous parvovirus, was studied in parasynchronous mouse A9 cells. The survival of UV-irradiated MVM is increased in cells which have been UV-irradiated prior to infection. UV-enhanced reactivation can be explained neither by facilitated plaque detection on UV-treated indicator cells, nor by altered kinetics of virus production by UV-irradiated cells. No effect of the multiplicity of infection on virus survival was detected in unirra-diated or irradiated cells. The magnitude of UV-enhanced reactivation is a direct exponential function of the UV dose administered to the virus while virus survival is inversely proportional to the UV dosage. The expression of UV-enhanced reactivation can be activated in cells arrested in G₀, it requires de novo protein synthesis and it is maximal when cells are irradiated 30 h before the onset of viral DNA replication. Early phases of the viral cycle, such as adsorption to cellular receptors, migration to the nucleus and uncoating, were not affected by cell irradiation and are unlikely targets of the UV-enhanced reactivation function(s). These results, together with the single-strandedness of the viral genome, strongly suggest that the step stimulated in UV-irradiated cells functions concomitant with, or subsequent to, viral DNA replication.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

REPAIR OF THYMINE DIMERS AND (6–4) PHOTOPRODUCTS IN GROUP A XERODERMA PIGMENTOSUM CELL LINES HARBORING A TRANSFERRED NORMAL CHROMOSOME 9

Transfer of a normal chromosome 9 into a xeroderma pigmentosum (XP)-A cell line partially restored its DNA repair activity. XP-A cell lines harboring a transferred chromosome were much more UV-resistant than parental XP-A cells but still more UV-sensitive than normal cells. The amount of UV-induced unscheduled DNA synthesis was only one-third of that in normal cells. The repair of thymine dimers and (6-4) photoproducts in these cell lines was analyzed by using monoclonal antibodies raised against them. Although these XP-A cell lines carrying a normal chromosome 9 could repair (6-4) photoproduct with a little lower efficiency than normal cells, the repair of thymine dimers was completely absent in these cells. The present results suggest a gene-dosage effect in DNA excision repair mechanisms in human cells or a rather complicated mechanism which involves two or more pathways.     

UV-ENHANCED VIRUS REACTIVATION IN MAMMALIAN CELLS: EFFECTS OF METABOLIC INHIBITORS*

Abstract—The induction process of UV-enhanced reactivation of UV-irradiated herpes simplex virus was investigated in CV-1 monkey kidney cells. A protein synthesis inhibitor, cycloheximide (0.5–5 μg/m/), present in the culture medium For 24 h between cell irradiation and virus infection decreased the enhanced virus survival normally found in UV-irradiated cultures. The enhanced virus reactivation became essentially resistant to the addition of cycloheximide by 6–8 h after cell irradiation, indicating that the cycloheximide-sensitive process necessary for enhanced reactivation was complete by that time. Since cycloheximide not only inhibits protein synthesis, but DNA synthesis as well, we investigated the effect of a DNA synthesis inhibitor, hydroxyurea. Hydroxyurea did not decrease UV-enhanced virus survival, but resulted in enhanced virus survival even in unirradiated cells. Therefore, the cycloheximide-caused inhibition of UV-enhanced reactivation did not arise from inhibition of DNA synthesis. The combined results indicate that (1) UV-enhanced virus reactivation in monkey kidney cells requires de novo protein synthesis during the first 6–8 h after cell irradiation and that (2) DNA synthesis inhibition may be the initiating event.     

XERODERMA PIGMENTOSUM FIBROBLASTS INCLUDING CELLS FROM XP VARIANTS ARE ABNORMALLY SENSITIVE TO THE MUTAGENIC AND CYTOTOXIC ACTION OF BROAD SPECTRUM SIMULATED SUNLIGHT

Abstract— The cytotoxic and mutagenic effects of broad spectrum simulated sunlight, as delivered by a Westinghouse Sun Lamp FS 20 filtered to eliminate wavelengths below 290 nm, were determined in diploid human skin fibroblasts which differ in their ability to repair pyrimidine dimers, and compared with results obtained with UV 254 nm radiation. The cell strains tested included normal fibroblasts; excision repair-deficient xeroderma pigmentosum (XP) cells from patients XP12BE (complementation group A). XP7BE (group D). and XP2BI (group G): and an XP variant patient (XP4BE) whose cells excise pyrimidinc dimers at a normal rate, but exhibit abnormal replication of DNA containing unexcised lesions. Cytotoxicity was assayed from loss of colony-forming ability. The group A cells were most sensitive to the killing effect of the Sun Lamp; the group D and G cells were slightly less sensitive; the XP variant cells showed intermediate sensitivity; and normal cells were most resistant. When the Sun Lamp survival curves for the group A, group D, the XP variant and normal cells were compared with their respective UV 254 nm survival curves, the relationships between the strains were virtually identical (i. e. the curves were related by a constant fluence modification factor). suggesting a common lesion for cell killing. The marker for mutagenesis was resistance to 6-thioguanine. The group A XP cells proved most sensitive to mutations induced by the simulated sunlight: the variant cells were intermediate; and the normal cells were the most resistant. Again, when the curves for mutations induced in these cell strains by simulated sunlight were compared with their respective 254 nm UV mutation curves, these were related by a constant fluence modification factor. suggesting a common lesion for mutagenesis. These results. taken together with published data indicating that at equicytotoxic levels of UV254 nm radiation and the filtered Sun Lamp. the number of pyrimidine dimers in the DNA of XP12BE cells was equal. support the hypothesis that the dimer is the lesion principally involved in both effects. Our data also support the hypothesis that mutations are involved in the sunlight-induced skin cancer of XP patients.     

INHIBITION OF DNA REPAIR SYNTHESIS BY SUNLIGHT

Abstract— DNA repair synthesis as determined by thymidine incorporation in the presence of hydroxyurea reached a much lower maximum level after solar compared with UVC exposure in five human melanoma cell lines, in HeLa cells, and in two human fibroblast strains. This finding was confirmed by determination of unscheduled DNA synthesis where both the number of labelled nuclei and grain count per nucleus were lower in sun-exposed cells. In a cloned human melanoma line (MM253cl), glass-filtered sunlight inhibited UVC repair synthesis, and solar UVB alone induced a higher level of repair synthesis than either complete sun or solar UVA plus solar UVB. The fluence response of filtered sunlight for inhibition of UVB (sunlamp) and UVC showed that most inhibition was obtained at low fluences (5-10 min), further exposure giving a plateau at 40% of the original level. Ultraviolet C and sunlight inactivated adenovirus 5 giving F ₀ values for virus survival 40-fold higher than for cell survival. Replication of either UVC- or solar-irradiated virus was not affected by prior irradiation of cells with glass-filtered sunlight. Stathmokinetic analysis of cell cycle progression by DNA flow cytometry showed that UVC and sunlamp UVB retarded cell movement from the G1 and S phases whereas equitoxic sunlight and glass-filtered sunlight (nontoxic) had no effect. These results indicate that solar UVA at low environmental fluences partially inhibits UVB repair synthesis in a range of human cell types but does not affect the replication of a UVB- or UVC-damaged virus when applied to the genome alone or to the host cell.     

Forty Years of Research on Xeroderma Pigmentosum at the US National Institutes of Health

In 1968, Dr. James Cleaver reported defective DNA repair in cultured cells from patients with xeroderma pigmentosum. This link between clinical disease and molecular pathophysiology has sparked interest in understanding not only the clinical characteristics of sun sensitivity, damage and cancer that occurred in XP patients but also the mechanisms underlying the damage and repair. While affected patients are rare, their exaggerated UV damage provides a window into the workings of DNA repair. These studies have clarified the importance of a functioning DNA repair system to the maintenance of skin and neurologic health in the general population. Understanding the role of damage in causing cancer, neurologic degeneration, hearing loss and internal cancers provides an opportunity for prevention and treatment. Characterizing complementation groups pointed to the importance of different underlying genes. Studying differences in cancer age of onset and underlying molecular signatures in cancers occurring either in XP patients or the general population has led to insights into differences in carcinogenic mechanisms. The accelerated development of cancers in XP has been used as a model to discover new cancer chemopreventive agents. An astute insight can be a “tipping point” triggering decades of productive inquiry.     

Development of Effective Skin Cancer Treatment and Prevention in Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is a rare, recessively transmitted genetic disease characterized by increasingly marked dyspigmentation and xerosis (dryness) of sun‐exposed tissues, especially skin. Skin cancers characteristically develop in sun‐exposed sites at very much earlier ages than in the general population; these are often multiple and hundreds or even thousands may develop. Eight complementation groups have been identified. Seven groups, XP‐A…G, are associated with defective genes encoding proteins involved in the nucleotide excision DNA repair (NER) pathway that recognizes and excises mutagenic changes induced in DNA by sunlight; the eighth group, XP‐V, is associated with defective translesion synthesis (TLS) bypassing such alterations. The dyspigmentation, xerosis and eventually carcinogenesis in XP patients appear to be due to their cells’ failure to respond properly to these mutagenic DNA alterations, leading to mutations in skin cells. A subset of cases, especially those in some complementation groups, may develop neurological degeneration, which may be severe. However, in most XP patients, in the past the multiple skin cancers have led to death at an early age due to either metastases or sepsis. Using either topical 5‐fluorouracil or imiquimod, we have developed a protocol that effectively prevents most skin cancer development in XP patients.     

Sunlight-induced killing of nondividing human cells in culture

Nondividing populations of human diploid fibroblasts that are DNA excision repair proficient (WS-1, KD. SSCW) and repair deficient (XP12BE) were exposed to mid-day summer sunlight for a determination of survival based on an ability of cells to remain attached to a culture vessel surface. Whereas mid- and far-UV wavelengths and radiation emitted from a sunlamp cause a gradual degeneraton and detachment of cells in a dose-dependent manner, sunlight does not promote cell killing that is evidenced by these criteria in repair proficient cells. Detachment of repair deficient cells is promoted to a limited extent but only at sunlight exposure times that are low with respect to the amount of DNA damage (pyrimidine dimers) induced. Repair proficient and deficient cells exposed to sunlight for longer times do not detach but are incapable of excuding a viable stain several days after exposure and appear as histologically fixed cells. Pyrimidine dimer levels in these sunlight irradiated cells were great enough to have promoted detachment had these levels been induced by UV (254 nm) alone. Other photodamage induced by these exposures evidently inhibits the dimer-induced cell degeneration that leads to cell detachment. We conclude that pyrimidine dimers are responsible for cell killing at short sunlight exposure times (< 40 min) but that at longer exposures (> 80 min) cells arc killed by a different mechanism that is independent of dimer-caused death.     

High Resolution MALDI Imaging Mass Spectrometry of Retinal Tissue Lipids

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism’s surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4 ^–/– knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.

REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.