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.     

The epidemiology of UV induced skin cancer.

There is persuasive evidence that each of the three main types of skin cancer, basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma, is caused by sun exposure. The incidence rate of each is higher in fairer skinned, sun-sensitive rather than darker skinned, less sun-sensitive people; risk increases with increasing ambient solar radiation; the highest densities are on the most sun exposed parts of the body and the lowest on the least exposed; and they are associated in individuals with total (mainly SCC), occupational (mainly SCC) and non-occupational or recreational sun exposure (mainly melanoma and BCC) and a history of sunburn and presence of benign sun damage in the skin. That UV radiation specifically causes these skin cancers depends on indirect inferences from the action spectrum of solar radiation for skin cancer from studies in animals and the action spectrum for dipyrimidine dimers and evidence that presumed causative mutations for skin cancer arise most commonly at dipyrimidine sites. Sun protection is essential if skin cancer incidence is to be reduced. The epidemiological data suggest that in implementing sun protection an increase in intermittency of exposure should be avoided, that sun protection will have the greatest impact if achieved as early as possible in life and that it will probably have an impact later in life, especially in those who had high childhood exposure to solar radiation.     

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.     

CORRELATION BETWEEN INACTIVATION OF HUMAN CELLS and NUMBERS OF PYRIMIDINE DIMERS INDUCED BY A SUN LAMP and 254 nm RADIATION

Abstract— Nondividing human fibroblasts are inactivated by radiation from a source (a Westinghouse sun lamp) that simulates the UV spectrum of sunlight. Survival curves determined for a DNA excision repair-proficient and a repair-deficient strain (XP12BE) are related to those determined using germicidal light (254 nm) by constant fluence modification factors. In addition, the same fraction of XP12BE cells are killed per pyrimidine dimer by 254 nm and sun lamp light. These results, when related to other survival and photoreactivation studies, suggest that the mechanism for inactivation of nondividing human cells by sun lamp light is the same as that by 254 nm and that pyrimidine dimers are the major responsible photolesion. Repair reverses some of the lethal effects of this light. We suggest that these conclusions apply to sunlight-irradiated skin cells in vivo.     

HOST CELL REACTIVATION BY FIBROBLASTS FROM PATIENTS WITH PIGMENTARY DEGENERATION OF THE RETINA

–Cockayne syndrome (CS) is an autosomal recessive disease characterized by numerous clinical abnormalities including acute sun sensitivity and primary pigmentary degeneration of the retina. Cultured fibroblasts from CS patients are hypersensitive to ultraviolet (UV) radiation. Since host cell reactivation of irradiated virus is a useful probe to evaluate repair in different host cells, we studied such host cell reactivation in CS and in other diseases with retinal degeneration. The survival of UV-irradiated Herpes simplex virus type 1 was determined in fibroblast lines from four normal donors. two patients with CS, one with both xeroderma pigmentosum (XP) and CS, and from several other patients with (Usher syndrome, olivopontocerebellar atrophy, retinitis pigmentosa) and without (XP, ataxia telangiectasia) primary pigmentary degeneration of the retina. The viral survival curves (log survival vs linear fluence) in all cell lines showed two components: a very sensitive initial component (not quantitated in this study) followed by an exponential, less sensitive component. The exponential component had greater sensitivity than normal in the case of the CS patients, the patient with both XP and CS. and the XP patient. We propose that patients with CS have defective repair of DNA which may be the cause of their retinal degeneration.     

UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer.

Repair of UV induced DNA damage is of key importance to UV-induced skin carcinogenesis. Specific signal transduction pathways that regulate cell cycling, differentiation and apoptosis are found to be corrupted in skin cancers, e.g., the epidermal growth-stimulating Hedgehog pathway in basal cell carcinomas (BCCs). Mutations in genes coding for proteins in these pathways lead to persistent disturbances that are passed along to daughter cells, e.g., mutations in the gene for the Patched (PTCH) protein in the Hedgehog pathway. Thus far only the point mutations in the P53 gene from squamous cell carcinomas and BCCs, and in PTCH gene from BCC of xeroderma pigmentosum (XP) patients appear to be unambiguously attributable to solar UV radiation. Solar UVB radiation is most effective in causing these point mutations. Other forms of UV-induced genetic changes (e.g., deletions) may, however, contribute to skin carcinogenesis with different wavelength dependencies.     

MOLECULAR MECHANISMS OF ULTRAVIOLET RADIATION CARCINOGENESIS

UV radiation is a potent DNA damaging agent and a known inducer of skin cancer in experimental animals. There is excellent scientific evidence to indicate that most non-melanoma human skin cancers are induced by repeated exposure to sunlight. UV radiation is unique in that it induces DNA damage that differs from the lesions induced by any other carcinogen. The prevalence of skin cancer on sun-exposed body sites in individuals with the inherited disorder XP suggests that defective repair of UV-induced DNA damage can lead to cancer induction. Carcinogenesis in the skin, as elsewhere, is a multistep process in which a series of genetic and epigenetic events leads to the emergence of a clone of cells that have escaped normal growth control mechanisms. The principal candidates that are involved in these events are oncogenes and tumor suppressor genes. Oncogenes display a positive effect on transformation, whereas tumor suppressor genes have an essentially negative effect, blocking transformation. Activated ras oncogenes have been identified in human skin cancers. In most cases, the mutations in the ras oncogenes have been localized to pyrimidine-rich sequences, which indicates that these sites are probably the targets for UV-induced DNA damage and subsequent mutation and transformation. The finding that activation of ras oncogenes in benign and self-regressing keratoacanthomas in both humans and in animals indicates that they play a role in the early stages of carcinogenesis (Corominas et al., 1989; Kumar et al., 1990). Since cancers do not arise immediately after exposure to physical or chemical carcinogens, ras oncogenes must remain latent for long periods of time. Tumor growth and progression into the more malignant stages may require additional events involving activation of other oncogenes or deletion of growth suppressor genes. In addition, amplification of proto-oncogenes or other genes may also be involved in tumor induction or progression. In contrast to the few studies that implicate the involvement of oncogenes in UV carcinogenesis, the role of tumor suppressor genes in UV carcinogenesis is unknown. Since cancer-prone individuals, particularly XP patients, lack one or more repair pathways, one can speculate that DNA repair enzymes would confer susceptibility to both spontaneous and environmentally induced cancers. Another potential candidate that can function as a tumor suppressor gene is the normal c-Ha-ras gene. Spandidos and Wilkie (1988) have shown that the normal c-Ha-ras gene can suppress transformation induced by the mutated ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

UV doses of young adults

Since 1986, people have been informed that they get about 80% of their lifetime ultraviolet (UV) dose by the age of 18. This belief originated from the mathematical conclusion that diligent use of sunscreens (sun protection factor 15 or higher) during the first 18 years of life would reduce the lifetime incidence of nonmelanoma skin cancers by 78%. These data were misconstrued to mean that individuals also got about 80% of their lifetime dose of UV by the age of 18 (linear relationship). However, these calculations were based on the incidence of nonmelanoma skin cancers being related to the square of the UV dose. Careful analysis of UV exposure data shows that Americans actually get less than 25% of their lifetime UV dose by the age of 18. This finding also appears to be true worldwide because Australia, UK and The Netherlands report a similar UV exposure pattern. UV-initiated damage early in life can be promoted by subsequent exposures to progress into tumors later in life. For example, the nonmelanoma skin cancer, squamous cell carcinoma, is dependent on the cumulative UV dose. Thus, a better educational approach for reducing skin cancers would be to instruct fair-skinned individuals to protect themselves throughout their lives from being exposed to too much UV radiation.     

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.     

Human Hair as a Natural Sun Protection Agent: A Quantitative Study

The rising incidence of skin cancers attributable to excessive sun exposure has become a major health concern worldwide. While numerous studies have analyzed the sun protective effect of sunscreens, clothing and antioxidants, none to date have measured the photoprotective effect of hair, despite clinical evidence that individuals with balding or thinning hair are at greater risk of skin lesions that can progress to cancer, hence the recommendation to use hats or umbrellas. We analyzed the level of protection offered by hair according to hair density, thickness and color using the spectral transmittance and corrected for relative erythema effectiveness. Our results show that hair provides a barrier against both UVB and UVA radiation which is significantly increased with respect to the hair density, thickness and the presence of melanins. This is the first study to quantify sun protection factor offered by hair, namely hair ultraviolet protection factor (HUPF). We believe that hair should be recognized as an important natural sun barrier in the prevention of UV‐induced skin cancers.     

Photoprotection in human skin--a multifaceted SOS response

Human skin has developed elaborate defense mechanisms for combating a wide variety of potentially damaging environmental factors; principal among these is UV light. Despite these defenses, short-term damage may include painful sunburn and long-term UV damage results in both accelerated skin aging and skin cancers such as basal cell carcinoma, squamous cell carcinoma and even malignant melanoma. While UV radiation damages many cellular constituents, its most lasting effects involve DNA alteration. The following sections briefly review UV-inducible protective responses in bacteria and in skin, thymidine dinucleotides (pTT) as a powerful probe of DNA damage responses, and potential means of harnessing these inducible responses therapeutically to reduce the now enormous burden of cutaneous photodamage in our society.     

Drivers for Sun Protection in Black South Africans

Sun protection behavior studies are almost exclusively carried out in populations with fair skin for the obvious reason that people with unpigmented skin are more susceptible to the health impacts of excess solar ultraviolet radiation exposure. In a dataset of 1271 Black South Africans, we analyzed factors related to sun protection applied when spending time outdoors including awareness of skin cancer, gender, age and Living Standards Measure (LSM) where 1–4 equate to the lowest, 5–7 intermediate and 8–10 the highest LSM status. The most important driver for Black South Africans to use sun protection was whether they were aware of skin cancer (OR: 2.6 for those who were aware versus those who were not, P < 0.0001). Sunscreen was preferred by respondents in LSM 8-10 whereas people in the lowest group (LSM 1-4) favored shade, umbrellas and hats. One in two respondents claimed to use some form of sun protection, which appears to be higher than deeply pigmented populations in other countries.     

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.     

Phototesting in lupus erythematosus tumidus--review of 60 patients

Photosensitivity is an important characteristic feature of several forms of lupus erythematosus (LE), and induction of skin lesions by UV-A and UV-B irradiation has been proved to be an optimal model for evaluating light sensitivity in patients with this disease. Because lupus erythematosus tumidus (LET) has rarely been documented in the literature and is often difficult to differentiate from other photodermatoses such as polymorphous light eruption, we performed photoprovocation tests in 60 patients with LET according to a standardized protocol. Areas of uninvolved skin on the upper back were irradiated with single doses of UV-A (100 J/cm2) and/or UV-B (1.5 minimal erythema dose) daily for three consecutive days. Interestingly, patients with LET are more photosensitive than those with subacute cutaneous lupus erythematosus, and in our study experimental phototesting revealed characteristic skin lesions in 43 patients (72%). Because of the latency period in developing positive phototest reactions, it might be difficult for these patients to link sun exposure with their skin lesions. Furthermore, our data revealed a positive correlation of antinuclear antibodies and positive provocative phototest reactions in these patients as seen for other forms of LE. In conclusion, the high incidence of positive phototest reactions in correlation with the clinical findings, history of photosensitivity and antinuclear antibodies enable the classification of LET as the most photosensitive type of LE.     

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells

The crucial role of DNA polymerase eta in protecting against sunlight‐induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP‐V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XP‐V cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP‐V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP‐V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and γH2AX signaling. Corroborating ATR participation in UVA‐DDR, a significant increase in Chk1 protein phosphorylation, as well as S‐phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N‐acetylcysteine (NAC), which significantly protects XP‐V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA‐induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells.     

Quantitative and in situ Detection of Oxidatively Generated DNA Damage 8,5′‐Cyclo‐2′‐Deoxyadenosine Using an Immunoassay with a Novel Monoclonal Antibody

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix‐distorting types of DNA damage including sunlight‐induced pyrimidine dimers. In addition to skin disease, approximately 30% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of a particular type of oxidatively generated DNA damage called purine 8,5′‐cyclo‐2′‐deoxynucleosides (purine cyclonucleosides). However, there are no currently available methods to detect purine cyclonucleosides in DNA without the need for DNA hydrolysis. In this study, we generated a novel monoclonal antibody (CdA‐1) specific for purine cyclonucleosides in single‐stranded DNA that recognizes 8,5′‐cyclo‐2′‐deoxyadenosine (cyclo‐dA). An immunoassay using CdA‐1 revealed a linear dose response between known amounts of cyclo‐dA in oligonucleotides and the antibody binding to them. The quantitative immunoassay revealed that treatment with Fenton‐type reagents (CuCl₂/H₂O₂/ascorbate) efficiently produces cyclo‐dA in DNA in a dose‐dependent manner. Moreover, immunofluorescent analysis using CdA‐1 enabled the visualization of cyclo‐dA in human osteosarcoma cells, which had been transfected with oligonucleotides containing cyclo‐dA. Thus, the CdA‐1 antibody is a valuable tool for the detection and quantification of cyclo‐dA in DNA, and may be useful for characterizing the mechanism(s) underlying the development of XP neurological disease.     

Sun-reactive Skin Type in 4912 French adults participating in the SU.VI.MAX study

Phototype classifications were initially developed in an attempt to predict the skin reactions of patients to phototherapy and are now widely used to advise individuals with regard to sun protection. A transversal study was conducted on the SU.VI.MAX cohort to estimate the frequency of sun-reactive skin features in a large, general adult population-based sample, and to describe the associations between these features. The data were collected 3 years after the beginning of the SU.VI.MAX nutritional intervention study on 4912 volunteers (2868 women aged 35-60 years and 2044 men aged 45-60 years). A multiple correspondence analysis was performed to study the associations between the features. The results showed that these features correspond to a one-dimensional phenomenon, which allowed us to establish a score to summarize skin sensitivity to sun exposure. Furthermore, we found a link between gender and phototype using the Césarini classification (phototype > or = IV: 37% of women, 47% of men). The analysis of the relationship with sun-reactive skin features and the score revealed the same trend. Phenotypic evaluation appears to be a good estimator of skin sensitivity to sun exposure for clinical screening or for use in research, and is easy to collect at a lower cost. Moreover, the sun sensitivity difference between gender should be considered in education about photoprotection.     

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations

—Action spectra for UV-induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254-365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254-313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.     

Photoemission study of praseodymia in its highest oxidation state: the necessity of in situ plasma treatment

A cold radio frequency oxygen plasma treatment is demonstrated as a successful route to prepare clean, well-ordered, and stoichiometric PrO(2) layers on silicon. High structural quality of these layers is shown by x-ray diffraction. So far unobserved spectral characteristics in Pr 3d x-ray photoelectron (XP) spectra of PrO(2) are presented as a fingerprint for praseodymia in its highest oxidized state. They provide insight in the electronic ground state and the special role of praseodymia among the rare earth oxides. They also reveal that former XP studies suffered from a significant reduction at the surface.     

Anatomical distribution of solar ultraviolet exposures among cyclists

Exposure to solar ultraviolet (UV) radiation is the major environmental factor implicated in the development of melanoma and other skin cancers, as well as eye damage and skin photoaging. Outdoor recreational activities such as cycling are increasingly pursued for health benefits, however little information is available regarding potential adverse effects of excessive sun exposure in this setting, nor about the anatomical distribution of solar dose. Polysulphone badges (UV dosimeters) were attached to the head, backs of hands and ankles of 22 cyclists during a seven-day charity bicycle ride in Queensland, Australia. Average daily exposures exceeded one minimal erythemal dose (MED) at all body sites except the ankle. Significant differences in UV dose among the various body sites were noted, with highest exposures recorded on the top of the head. Mean doses received at the ankle (0.94 MED), back of the hand (1.28 MED) and side of the head (1.14 MED) were 51%, 71% and 63% of those received at the top of the head (1.80 MED), respectively. These data indicate that cycling exposes adherents to substantial doses of UV radiation. Moreover, our observations suggest that even vertically-oriented, potentially shaded sites such as the lower leg typically receive doses of solar radiation no less than half of maximally exposed sites.