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Fas Ligand: A Sensor for DNA Damage Critical in Skin Cancer Etiology

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Science  06 Aug 1999:
Vol. 285, Issue 5429, pp. 898-900
DOI: 10.1126/science.285.5429.898

Abstract

DNA-damaged cells can either repair the DNA or be eliminated through a homeostatic control mechanism termed “cellular proofreading.” Elimination of DNA-damaged cells after ultraviolet radiation (UVR) through sunburn cell (apoptotic keratinocyte) formation is thought to be pivotal for the removal of precancerous skin cells. Sunburn cell formation was found to be dependent on Fas ligand (FasL), a pro-apoptotic protein induced by DNA damage. Chronic exposure to UVR caused 14 of 20 (70 percent) FasL-deficient mice and 1 of 20 (5 percent) wild-type mice to accumulate p53 mutations in the epidermis. Thus, FasL-mediated apoptosis is important for skin homeostasis, suggesting that the dysregulation of Fas-FasL interactions may be central to the development of skin cancer.

In the United States, nearly 1,000,000 persons will develop nonmelanoma skin cancer (NMSC) (basal and squamous cell carcinoma) this year. Although death from NMSC is rare, these patients experience a substantially increased mortality from other cancers (20 to 30% higher), suggesting that skin cancer susceptibility may be linked to the development of noncutaneous malignancies (1). UVR in sunlight is the principal carcinogen, serving as initiator and promoter for most skin tumors (2). UVR elicits p53-dependent apoptosis in DNA-damaged keratinocytes (sunburn cells), presumably as a “guardian of the tissue” response to eradicate precancerous cells in the skin (3). This p53-driven response, termed “cellular proofreading” (3), erases rather than repairs DNA damage. Mice deficient in p53 (p53-null) have reduced sunburn cell formation and increased susceptibility to UVR-induced skin carcinogenesis (2, 4), implicating apoptosis as a critical event in skin carcinogenesis. Fas and FasL are complementary receptor-ligand proteins eliciting apoptosis (5). Although Fas and FasL are induced by nuclear factor kappa B activation (6–8), wild-type p53 is essential for the transcriptional up-regulation of Fas after DNA damage (9–11). Thus, Fas and FasL may serve as external pro-apoptotic sensors of DNA damage–mediated cellular proofreading.

To investigate Fas and FasL expression in normal skin after UVR, we shaved C3H/HeJ wild-type mice, exposed them to a single dose of ultraviolet (UV) light (5 kJ/m2) (12), and harvested epidermal skin sections for immunohistochemistry (13). Nonirradiated skin had little Fas and FasL expression (Fig. 1). Both Fas and FasL expression were potently induced in the epidermis by UVR as early as 3 and 6 hours after irradiation, with maximal induction of both proteins occurring at 12 hours. By 24 hours, UVR-induced Fas and FasL expression in the epidermis was similar to that observed before treatment. Dermal Fas expression (hair follicles and sebaceous glands), in contrast, remained elevated at this time point. These results indicate that both Fas and FasL are transiently up-regulated only in the normal epidermis by UVR exposure, as previously reported in human skin (8).

Figure 1

Fas and FasL in skin after UVR exposure. Wild-type mice were acutely exposed to UV-B light (5 kJ/m2), skin samples were harvested at indicated times (hours) after UVR exposure, and immunohistochemistry was performed (13). Sections are representative of a minimum of four mice examined at each time point. Magnification, ×10.

Because the proapoptotic proteins Fas and FasL are coexpressed in the UVR-exposed epidermis, we examined C3H/HeJ wild-type and FasL-deficient (C3H/HeJ gld/gld) mice 24 hours after exposure to UVR (5 kJ/m2) to determine if the number of apoptotic keratinocytes was reduced (14). Typically, sunburn cells display a characteristic morphology of pycnotic nuclei and intensely eosinophilic cytoplasm detectable by light microscopy in hematoxylin-eosin–stained epidermal sections. Epidermal sections from UVR-treated wild-type mice had an average of 65.2 ± 17.3 (mean ± SE) sunburn cells per linear centimeter (Table 1), whereas epidermal sections from UVR-exposed FasL-deficient mice showed only 18.0 ± 9.3 sunburn cells per linear centimeter (P < 0.001, student'st test). The apoptotic nature of the sunburn cells identified by morphologic evaluation was confirmed by in situ TdT-mediated deoxyuridine triphosphate nick end labeling (TUNEL) (15). Few apoptotic cells were observed in the nonirradiated skin of either wild-type or FasL-deficient (gld/gld) mice (Fig. 2). In concert with our results using hematoxylin-eosin–stained epidermal sections, numerous apoptotic cells were detectable in wild-type, but not FasL-deficient, mice after UVR exposure. The reduction in sunburn cell formation in FasL-deficient mice was not attributable to kinetic differences in keratinocyte apoptosis because gld/gld mice had similar incidences in sunburn cell formation at 12, 24, and 48 hours after UVR exposure (16). Proliferative cell nuclear antigen expression in the epidermis was indistinguishable in wild-type and gld/gld mice 24 hours after UV-B exposure, making it unlikely that differences in UVR-induced keratinocyte apoptosis were attributable to differences in the epidermal proliferative responses (17). The contribution of T cells to this process also appears to be negligible as T cell–deficient mice (RAG 2−/−) demonstrate UVR-induced sunburn cell responses similar to those of their wild-type counterparts (18). Furthermore, p53 localized to the nucleus and induced the downstream effector p21 (waf1/cip1) in both wild-type and FasL-deficient mice treated with UVR (16), suggesting that repression of p53 activation and function is not likely to contribute to the observed reduction in sunburn cells. Thus, UVR-induced sunburn cell formation requires Fas and FasL interactions, and p53 activation alone is insufficient to induce wild-type levels of apoptosis in UVR-damaged keratinocytes.

Figure 2

Sunburn cell induction in wild-type and FasL-deficient (gld/gld) mice after UVR. Mice were acutely exposed to UV-B light (5 kJ/m2), and skin sections were harvested for TUNEL analysis at 0 (NR) and 24 (UV) hours (15). A minimum of four mice (nonirradiated and irradiated) were examined; sections from two individual mice are shown. NR, nonirradiated. Magnification, ×10.

Table 1

Sunburn cell formation in wild-type and gldmice.

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The elimination of cells sustaining extensive DNA damage has been proposed as a mechanism to avoid propagation of cells constituting a neoplastic risk (3). In the event that cellular proofreading is hindered, mutations in critical genes regulating cellular proliferation, adhesion, and apoptosis would be expected to accumulate upon exposure to DNA-damaging agents. To test the premise that FasL-induced apoptosis restricts the accumulation of genetic mutations after DNA damage, we examined epidermal sheets fromgld/gld and wild-type mice for mutations in p53, a gene known to play a central role in UVR-induced, nonmelanoma skin carcinogenesis (Fig. 3). In contrast to the high frequency of mutant p53 alleles in tumor tissues, the frequency of p53 mutations in UV-irradiated skin is low because only a small fraction of the cells in the epidermis are mutated (19). Thus, detection of p53 mutations in UVR-exposed skin by conventional procedures is difficult. We used a sensitive allele-specific polymerase chain reaction (PCR) technique to detect p53 mutations (C → T transitions) at codons 270 and 275 because these codons are hotspots in UVR-induced mouse skin tumors (20, 21). Mice were chronically irradiated on shaved dorsal skin for 1 and 2 weeks, representing five and ten exposures to UV-B (5 kJ/m2), respectively (22). Two days after the final UVR exposure, epidermal sheets were harvested from 10 mice per group (with the exception of nonirradiated gld/gld mice, of which only nine mice were analyzed), and DNA was extracted. Mice were individually analyzed for p53 mutations in both codons 270 and 275, and the relative incidence was determined (Fig. 3B). Although only 1 of 20 wild-type mice showed evidence of p53 mutation after 1 or 2 weeks of chronic UVR exposure (codon 275), abundant p53 mutations were observed in FasL-deficient mice after only 1 week of UVR exposure (Fig. 3A). p53 mutations at codons 270 and 275 were detectable in 3 of 10 and 8 of 10, respectively, of the gld/gld animals analyzed after a single week of UV-B irradiation. FasL-deficient animals exposed to chronic UV-B irradiation for 2 weeks had p53 mutations in 4 of 10 and 6 of 10 in codons 270 and 275, respectively. No p53 mutations were detectable in nonirradiated wild-type or FasL-deficient mice. The infrequent p53 mutations in codons 270 and 275 in the epidermis of wild-type mice are in accordance with our previous report that such mutations are generally undetectable until 4 to 5 weeks after chronic UV-B exposure (23). The increase in p53 mutations in gld/gldmice exposed to UVR supports the hypothesis that FasL-induced apoptosis is required to efficiently eliminate DNA-damaged keratinocytes in the skin.

Figure 3

p53 mutations in the epidermis of wild-type and gld/gld mice after chronic UVR exposure. For each group, 10 individual mice were examined, with the exception of nonirradiatedgld/gld mice, of which only nine mice were examined. (A) Autoradiographs for codon 275 at the indicated type points. Each lane represents the analysis of an individual mouse. Bands indicate the presence of the C → T mutation in the codon. Mutations are present in wild-type mice only in lane 3 at week 1;gld/gld mice show mutations in lanes 1, 3 to 5, and 7 to 10 at week 1 and lanes 4 to 9 at week 2. (B) Graphic representation of mutation incidence. An unconditional exact two-sidedP value was computed with the unconditional exact confidence interval of 95% for the difference of proportions (StatXact-3, V3.0.2; Cytel Software, Cambridge, MA) to determine statistical significance between wild-type and gld/gld mice at each time point for each codon. **, P = 0.0046; *, P = 0.014.

Cellular proofreading has been defined in p53-null mice where p53-dependent apoptotic pathways and DNA repair mechanisms are similarly compromised, rendering the relative importance of apoptosis for the elimination of DNA damage suspect if typical DNA repair pathways are intact (2, 3). This work defines a critical role for apoptosis in cellular proofreading in the skin through FasL-Fas interactions. Circumvention of this apoptotic pathway, perhaps through a DNA damage threshold effect, may be essential for the survival of UVR-damaged cells containing mutations in critical regulatory genes. It is interesting to speculate that in surviving sun-damaged keratinocytes containing p53 mutations, consequent UVR-mediated DNA damage may not induce Fas expression (9,10) whereas FasL up-regulation would be likely retained (7). FasL expression in the absence of Fas-mediated apoptosis may confer a substantial advantage by immunologic privilege (24) allowing for the additional accumulation of DNA damage–induced mutations prerequisite for clonal expansion of the malignant cell.

UVR-induced FasL is the effector molecule mediating UVR-induced immune suppression (25), a process believed to be critical for the successful outgrowth of UVR-induced skin cancer in mice (26), and Fas loss of function results in the increased lung metastasis of UVR-induced murine melanomas (27). Taken together with the current findings, Fas and FasL interactions are likely pivotal in the etiology, growth, and metastasis of UVR-induced skin tumors.

  • * To whom correspondence should be addressed. E-mail: lowensch{at}mdanderson.org

REFERENCES AND NOTES

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