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.     

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.     

ENHANCEMENT OF ULTRAVIOLET-DNA REPAIR IN den V GENE TRANSFECTANTS and T4 ENDONUCLEASE V-LIPOSOME RECIPIENTS

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.     

XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP F: MORE ASSIGNMENTS AND REPAIR CHARACTERISTICS

Abstract— The specific heterodikaryon complementation method enabled us to assign three patients with mild xeroderma pigmentosum (XP) symptoms (XP25KO, XP27KO, XP28KO) to complementation group F. UV-induced unscheduled DNA synthesis (UDS) remained unnormalized in the heterodikaryons between either of the above three XP strains and the reference group F XP3YO. All these particular XP strains as well as XP3YO exhibited an equally low level of10–15% UDS by a 3 h [³H]-thymidine labeling following 10 J/m² 254 nm UV, while they attained 60% UDS of normal at an extended time of 25 h. The present group F strains were 3 and 1.5 times as sensitive to the lethal effect of UV as normal and XP group E cells, respectively, based on the mean lethal dose ( Do ) comparison. Normal cells had the biphasic time-UDS kinetics of early rapid and late slow repair. Characteristically, however, all of the present group F strains were defective in only early rapid repair, but normally proficient in slow repair.     

DNA EXCISION REPAIR IN THE VERY UV-SENSITIVE XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP A STRAIN XP4LO

Abstract— XP4L0, a xeroderma pigmentosum complementation group A strain, exhibits very limited DNA repair activity. It has extreme sensitivity to UV (254 nm) as determined by colony forming ability. The rate of loss of UV (1 J/m²)-induced pyrimidine dimers from populations of quiescent, nondividing XP4LO cells was determined and found to be slower than that observed for other group A strains (XP25R0, XP12BE, XP8LO). The extreme UV-sensitivity is also exhibited by the nondividing cells in a survival assay that employs nondividing cell populations and does not involve cell reproduction. This result suggests that the extreme sensitivity measured previously by colony-forming ability (a cell-reproduction assay) is due to the excision repair defect alone and not to an additional post-replication repair defect. The very limited excision allows for an accurate definition of target size for inactivation of nondividing cells, about 1 pyrimidine dimer per 10⁵ base pairs, and when compared to results observed for other XP-A strains, provides further evidence that even though excision repair in group A is severely limited, it has biological significance.     

INCREASED LEVELS OF UNSCHEDULED DNA SYNTHESIS IN UV-IRRADIATED HUMAN FIBROBLASTS PRETREATED WITH SODIUM BUTYRATE

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 m M results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m². The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.     

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.     

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.     

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

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.     

Reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro: a model to study hypersensitivity to UV light

Xeroderma pigmentosum (XP) is a rare, recessive, photosensitive and cancer-prone syndrome, the biochemical hallmark of which is a defect in nucleotide excision repair of ultraviolet (UV)-induced mutagenic lesions. After isolation and amplification of several strains of XP-C keratinocytes and fibroblasts, a three-dimensional skin model in vitro comprising both epidermis and a dermal equivalent could be obtained. XP dermal tissues and XP epidermis displayed specific morphological and biochemical characteristics compared with tissues obtained with normal cells. One of the major features was the formation of epidermal invaginations into the dermal equivalent. After UV-B exposure, and contrary to repair of DNA lesions in normal cells, the XP model displayed repair deficiency with long-lasting persistence of UV-induced DNA damage and p53 positive nuclei. Recent data obtained after genetic correction leading to functional XPC gene in keratinocytes and fibroblasts revealed that several abnormal features could be normalized. In conclusion, reconstruction of XP skin in vitro provides a very promising system to study genetic hyperphotosensitivity and opens a rational perspective to XP tissue therapy.     

CYCLOBUTANE DIMERS AND (6-4)PHOTOPRODUCTS IN HUMAN CELLS ARE MENDED WITH THE SAME PATCH SIZES

The size of excision repair patches corresponding to excision of (6-4) pyrimidine-pyrimidone photoproducts and (5-5, 6-6) cyclobutane dimers have been independently determined by using bromodeoxyuridine substitution and density increases in isopycnic gradients of small DNA fragments. The two classes of photoproducts were distinguished by using (a) a xeroderma pigmentosum (XP) revertant cell line that excises (6-4) photoproducts normally, but does not excise cyclobutane dimers from bulk DNA or from an actively transcribed sequence; (b) an XP cell line containing the denV gene of bacteriophage T4, which repairs only cyclobutane dimers by a unique glycosylase mechanism, and (c) normal cells analyzed during time intervals in which cyclobutane dimer repair is the main repair process in action. The patch sizes for the two lesions were similar under all conditions and were estimated to be approximately 30-40 bases. These values are slightly large than corresponding estimates for Escherichia coli and Saccharomyces cerevisiae but close to estimates from in vitro experiments with human cell extracts. The size of 30 bases may consequently be very close to the actual distance between cleavage sites made on either side of a photoproduct during repair.     

UVB‐Induced Inflammatory Cytokine Release,DNA Damage and Apoptosis of Human Oral Compared with Skin Tissue Equivalents

People can get oral cancers from UV (290–400 nm) exposures. Besides high outdoor UV exposures, high indoor UV exposures to oral tissues can occur when consumers use UV‐emitting tanning devices to either tan or whiten their teeth. We compared the carcinogenic risks of skin to oral tissue cells after UVB (290–320 nm) exposures using commercially available 3D‐engineered models for human skin (EpiDerm?), gingival (EpiGing?) and oral (EpiOral?) tissues. To compare the relative carcinogenic risks, we investigated the release of cytokines, initial DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), repair of CPDs and apoptotic cell numbers. We measured cytokine release using cytometric beads with flow cytometry and previously developed a fluorescent immunohistochemical assay to quantify simultaneously CPD repair rates and apoptotic cell numbers. We found that interleukin‐8 (IL‐8) release and the initial CPDs are significantly higher, whereas the CPD repair rates and apoptotic cell numbers are significantly lower for oral compared with skin tissue cells. Thus, the increased release of the inflammatory cytokine IL‐8 along with inefficient CPD repair and decreased death rates for oral compared with skin tissue cells suggests that mutations are accumulating in the surviving population of oral cells increasing people's risks for getting oral cancers.     

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.