Heggert Rebel

Powerful Skin Cancer Protection by a CPD-Photolyase Transgene

BACKGROUNDnThe high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway.nnnRESULTSnTo assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis.nnnCONCLUSIONSnOur data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.

Early events in UV carcinogenesis--DNA damage, target cells and mutant p53 foci.

Skin carcinomas are the most common cancers in fair‐skinned populations of North West European descent. The risk is closely related to sun (UV) exposure and susceptibility to sunburn. Induction of squamous cell carcinomas (SCCs) in the skin of hairless mice by daily UVB exposure appears to emulate the genesis of these tumors in humans quite well. The carcinomas, and the UVB signature mutations that they carry in their p53 genes, can be linked most specifically to the induction of cyclobutane pyrimidine dimers (CPDs). The wavelength dependence of the induction of carcinomas parallels that of CPD induction over the UVB and UVA2 spectral regions. Microscopic clusters of cells overexpressing p53 with UVB signature mutations (“p53 patches”) can be detected in the interfollicular epidermis long before the skin tumors arise. DNA repair—more precisely nucleotide excision repair—is a crucial line of defense against UV‐induced p53 patches and skin carcinomas. Although chemoprevention of UV carcinogenesis, e.g. with difluoromethylornithine, may be successful by inhibiting the outgrowth of tumors, it may be better to counter the initial steps in tumor development. As the p53 patches appear to be potential precursors of SCCs, regression of p53 patches in unexposed skin should lower subsequent development of SCCs. However, “holoclonal” p53 patches might persist. Ablation of the interfollicular epidermis would be expected to abrogate development of SCC, and negation of this expectation [Faurschou A. et al., Exp. Dermatol. 2007;16:485–489] would indicate that SCCs stem from deep‐seated cells in the hair follicles. Careful examination of archival material showed that although most small p53 patches arise interfollicularly, some may actually arise high up in a follicle, in the infundibulum.

Enhanced DDB2 Expression Protects Mice from Carcinogenic Effects of Chronic UV-B Irradiation

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.

Ingenol Mebutate Field-Directed Treatment of UVB-Damaged Skin Reduces Lesion Formation and Removes Mutant p53 Patches

Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.

More epidermal p53 patches adjacent to skin carcinomas in renal transplant recipients than in immunocompetent patients: the role of azathioprine

Abstract:u2002 Immunosuppressive medication in renal transplant recipients (RTR) strongly increases the risk of cancers on sun‐exposed skin. This increased risk was considered an inevitable collateral effect of immunosuppression, because UV‐induced carcinomas in mice were found to be highly antigenic. Here, we posed the question whether immunosuppression also increases the frequency of p53‐mutant foci (‘p53 patches’), putative microscopic precursors of squamous cell carcinomas. As the majority of RTR was kept on azathioprine for most of the time, we investigated whether this drug could increase UV‐induced p53 patches by immunosuppression. As azathioprine can impair UV‐damaged DNA repair under certain conditions, we also investigated whether DNA repair was affected. Archive material of RTR and immunocompetent patients (ICP), as well as azathioprine‐administered hairless mice were examined for p53 patches. DNA repair was investigated by ascertaining the effect of azathioprine on unscheduled DNA synthesis (UDS) in UV‐irradiated human keratinocytes. P53 patches were more prevalent in RTR than in ICP in normal skin adjacent to carcinomas (Pu2003=u20030.02), in spite of a lower mean age in the RTR (52 vs 63u2003years, Pu2003=u20030.001), but we found no increase in UV‐induced p53 patches in mice that were immunosuppressed by azathioprine. We found a significant reduction in DNA repair activity in keratinocytes treated with azathioprine (Pu2003=u20030.011). UV‐induced UDS in humans is dominated by repair of cyclobutane pyrimidine dimers, and these DNA lesions can lead to ‘UV‐signature’ mutations in the P53 gene, giving rise to p53 patches.

Early and late effects of the immunosuppressants rapamycin and mycophenolate mofetil on UV carcinogenesis

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub‐sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (≥4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa‐fed group showed a reduction in UV‐signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf‐positive in the Rapa‐fed groups, but a correspondingly reduced expression of Hif1α target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV‐induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

UV exposure inhibits intestinal tumor growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet

Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV‐induced vitamin D. FabplCre;Apc15lox/+ mice, which develop intestinal tumors, and their parents were put on a vitamin D‐deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: meanu2009±u2009SD from 7.7u2009±u20091.9 in controls to 75u2009±u200915 nmol/l with vitamin D supplementation (no gender difference), and to 31u2009±u200913 nmol/l in males and 85u2009±u200917 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D‐supplemented group (130u2009±u200925 mm2, pu2009=u20090.018) and the UV‐exposed group (88u2009±u20099 mm2, pu2009<u20090.0005; no gender differences) compared to the control group (202u2009±u200923 mm2). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (pu2009=u20090.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.

UV-induced ablation of the epidermal basal layer including p53-mutant clones resets UV carcinogenesis showing squamous cell carcinomas to originate from interfollicular epidermis

Chronic ultraviolet (UV) exposure induces clones of cells overexpressing mutant p53 in the interfollicular (IF) epidermis and subsequently squamous cell carcinomas (SCCs) with similar p53 mutations. Mutated p53 may give cells growth advantage over neighbouring cells by impaired apoptosis. We tested this by UV overexposure of skin laden with p53-mutant clones and assessed the impact on subsequent tumour development. P53-mutant clones were induced in two groups of hairless SKH1 mice by daily exposures (500 J/m(2) UV from TL12 lamps) for 28 days. On day 29, one group was overexposed (to 10 kJ/m(2) UV), whereas the control group received the regular daily dose. After 1 week of recovery, the daily exposures were resumed in both groups to induce SCCs. UV overexposure forced the entire IF basal layer into caspase-3-driven apoptosis while leaving overlying layers with sunburn cells intact. No apparent regions were spared from apoptosis. Pulse-chase BrdU labelling showed the IF epidermis to be repopulated from the hair follicles (remaining p63 positive). One week after overexposure, the p53-mutant clones had virtually disappeared (0.6, 95% confidence interval 0.5-0.8 per mouse versus 102, 59-179, without overexposure). Tumour development was significantly delayed after UV overexposure (P < 0.0001) by an average of 27 days (standard error of the mean 3); a period matching that of daily exposures preceding the overexposure. Thus, we found that UV-induced ablation of the IF epidermal basal layer eliminates p53-mutant clones and resets UV carcinogenesis. Furthermore, and in contrast with earlier reports, our data show that UV-induced p53-mutant clones and SCCs originate from the IF epidermis.

Error-prone translesion replication of damaged DNA suppresses skin carcinogenesis by controlling inflammatory hyperplasia

The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.

A novel mouse model for Sézary Syndrome using xenotransplantation of Sézary cells into immunodeficient RAG2−/− γc−/− mice

Sézary syndrome (SS) is an aggressive cutaneous T‐cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2−/− γc−/− mice that are completely devoid of T‐, B‐ and NK‐cell activity. Injection of the SS cell line SeAx led to long‐term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT‐78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2−/− γc−/− mice results in long‐term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.