Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Loss of tumor-specific and idiotype-specific immunity with age.

The ultraviolet light-induced fibrosarcoma 1591 undergoes "first-set rejection" when transplanted into normal syngeneic mice. We found, however, that the primary resistance of normal mice decreases with age, beginning at 9--12 mo, equivalent to middle age for mice. Mice lose with age the capacity to mount both idiotypic and anti-idiotypic responses responsible for controlling the growth of tumor. This loss was correlated with quantitative as well as qualitative changes in the response, such as changes in specificity and clonotype. Normal young mice regularly expressed a dominant common anti-1591 "idiotype" as defined by an anti-idiotypic probe. The capability of normal mice to respond with lymphocytes of this dominant common idiotype began to decline at about 8 mo of age. At this time, animals still generated tumor-specific lymphocytes, but these lymphocytes appear to be idiotypically different lymphocyte clones. With further increase in age, animals responded with tumor-reactive lymphocytes that showed a marked cross-reactivity to other tumor target cell lines. Both in vivo and in vitro, the capability of normal mice to mount an immune response that was specific for the 1591 tumor cells decreased between 9 and 14 mo, which was the age individual mice became increasingly susceptible to a challenge with 1591 tumor cells. Thus, our data suggest that clones of tumor-specific T cells provide primary and early protection of young animals against challenge with malignant 1591 cells. However, the dominance of these tumor-specific T cell clones in a primary immune response is lost in middle-age. Because the ability of animals to mount anti-idiotypic immune response also declined in middle-aged animals, it is possible that the observed loss of clonal dominance of tumor-specific clones with increasing age is at least partially related to age-dependent changes in the anti-idiotypic compartment.

Immunoselection of tumor cell variants by mice suppressed with ultraviolet radiation.

It has previously been shown that mice exposed to ultraviolet radiation (UV) fail to reject highly immunogenic UV-induced tumors, which are regularly rejected by normal mice. The present study shows, however, that this immunosuppresion is incomplete, as UV-treated mice can still mount certain tumor-specific immune responses and reject smaller inocula of tumor cells that regularly grow progressively in athymic nude mice. Furthermore, all tumor cell lines that were reisolated from the tumor mass resulting from one tumor passage through UV-treated recipients heritably lost a tumor-specific determinant present on the parental tumor cells used for transplantation, and a large percentage of these reisolated variant tumors had changed to progressively growing tumors, in that they were no longer rejected by normal mice. In contrast, none of the tumors reisolated from passage through athymic nude mice or anti-idiotypically suppressed mice showed this change in antigenicity and progressive growth behavior. Thus, it appears that the phenotypic change in tumors reisolated from UV-treated mice was caused by immunoselection, and that the tumor-specific immunity in these mice apparently restrained the outgrowth of the parental tumor cells despite the partial immunosuppression. Because of the regularity at which tumor variants arose in the UV-treated mice after tumor transplantation, it appears that the partial immunosuppression caused by UV-treatment may have favored the outgrowth of antigenic variants from the parental tumor cell population, possibly by allowing more time for the generation of tumor variants. A similar immunoselection process might be part of tumor progression during tumor development and preferentially occur in cancer-bearing individuals showing concomitant tumor immunity.

Identification of the molecular target for the suppression of contact hypersensitivity by ultraviolet radiation.

This study was conducted to explore the involvement of DNA damage in the suppression of contact hypersensitivity (CHS) by UV irradiation. The opossum, Monodelphis domestica, was used because cells of these marsupials have an enzyme that is activated by visible light (photoreactivating enzyme) and repairs ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA. A single dose of 1,500 J/m2 of UVB (280-320 nm) radiation, representing 2 minimal erythema doses, was administered to the dorsal skin of opossums. This treatment prevented the opossums from developing a CHS response to dinitrofluorobenze (DNFB) applied either at the site of irradiation or an unirradiated site. In addition, this dose of UVR decreased the number of ATPase+ epidermal Langerhans cells in the dorsal epidermis to approximately 3% of that in unirradiated skin at the time of DNFB application. Treatment of the animals with wavelengths that activate the repair enzyme (320-500 nm, photoreactivating light, PRL) for 120 min immediately after UV irradiation inhibited the UVR-induced suppression of CHS almost completely. Exposure to PRL before UVR did not prevent UVR-induced suppression of CHS. PRL treatment after UV irradiation also prevented the decrease in the number of ATPase+ Langerhans cells. Measurements of lesions in DNA indicated that PRL treatment removed around 85% of the UVR-induced pyrimidine dimers. These data provide direct evidence that DNA, and most likely, the pyrimidine dimer, is the primary molecular target for the UVB-induced suppression of contact hypersensitivity to haptens applied to irradiated or unexposed skin.

A critical role for Fas ligand in the active suppression of systemic immune responses by ultraviolet radiation.

Induction of antigen-specific suppression elicited by environmental insults, such as ultraviolet (UV)-B radiation in sunlight, can inhibit an effective immune response in vivo and may contribute to the outgrowth of UV-induced skin cancer. Although UV-induced DNA damage is known to be an initiating event in the immune suppression of most antigen responses, the underlying mechanism(s) of such suppression remain undefined. In this report, we document that Fas ligand (FasL) is critical for UV-induced systemic immune suppression. Normal mice acutely exposed to UV exhibit a profound suppression of both contact hypersensitivity and delayed type hypersensitivity (DTH) reactions and the development of transferable antigen-specific suppressor cells. FasL-deficient mice exposed to UV lack both transferable suppressor cell activity and primary suppression to all antigens tested, with the exception of the DTH response to allogeneic spleen cells. Interestingly, suppression of this response is also known to occur independently of UV-induced DNA damage. Delivery of alloantigen as protein, rather than intact cells, restored the requirement for FasL in UV-induced immune suppression of this response. These results substantiate that FasL/Fas interactions are essential for systemic UV-induced suppression of immune responses that involve host antigen presentation and suggest an interrelationship between UV-induced DNA damage and FasL in this phenomenon. Collectively, our results suggest a model whereby UV-induced DNA damage disarms the immune system in a manner similar to that observed in immunologically privileged sites.

Mechanisms of syngeneic tumor rejection. Susceptibility of host-selected progressor variants to various immunological effector cells.

The ultraviolet radiation-induced fibrosarcoma 1591 generally is rejected by normal syngeneic mice and only rarely exhibits progressive growth. We isolated five of these rare progressor tumors from normal animals to determine the selective pressures that had been exerted upon the parental tumor by normal immunocompetent hosts. We found that the variant tumor cell lines could neither induce nor be killed by tumor-specific lymphocytes, suggesting that selection had been exerted against tumor cells expressing the tumor-specific antigen. In contrast, no selection against natural killer cell activity or against nonspecific T cell-mediated immunity seems to have occurred because progressor tumor cells were highly sensitive to these types of effector cells and in fact induced these effector cells more effectively than did the parental tumor. Nude mice were found to be as capable as normal mice in generating natural killer activity in response to a challenge with progressor tumor cells, but they were unable to mount a nonspecific T lymphocyte response. This may account for the fact that the progressor tumors grew at a significantly faster rate in nude animals than in normal mice. Thus, our study shows that in this tumor system nonspecific T cell-mediated immunity may play a role in retarding tumor growth, but the absolute resistance of normal animals to progressive tumor growth critically depends upon the presence of T cell-mediated tumor-specific immunity. Furthermore, neither NK cells nor nonspecific cytotoxic T lymphocytes appear to play a role in immunoselection against this tumor in normal immunocompetent hosts.

CD40-CD40 ligand interactions in vivo regulate migration of antigen-bearing dendritic cells from the skin to draining lymph nodes.

Whereas CD40-CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand -/- mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand -/- mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand -/- mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-alpha production and was corrected by injecting recombinant TNF-alpha or an agonistic anti-CD40 monoclonal antibody. Thus, CD40-CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-alpha production and play a vital role in the initiation of acquired T cell-mediated immunity.

Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice.

Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.

Suppressor T lymphocytes control the development of primary skin cancers in ultraviolet-irradiated mice.

Exposure of mice to ultraviolet radiation results in the development of suppressor T lymphocytes in lymphoid organs, followed by the appearance of primary skin cancers. The presence or absence of these suppressor lymphocytes determines whether or not primary cancers will develop in the ultraviolet-irradiated skin. This demonstrates the importance of immunological regulatory pathways in carcinogenesis and provides an example of immunological surveillance.

Metastasis results from preexisting variant cells within a malignant tumor.

Clones derived in vitro from a parent culture of murine malignant melanoma cells varied greatly in their ability to produce metastatic colonies in the lungs upon intravenous inoculation into syngeneic mice. This suggests that the parent tumor is heterogeneous and that highly metastatic tumor cell variants preexist in the parental population.

Antigenic similarity between cells transformed by ultraviolet radiation in vitro and in vivo.

Cells of the 10T 1/2 mouse fibroblast line transformed in vitro by ultraviolet radiation are antigenically similar to those from skin cancers produced in mice by repeated exposure to ultraviolet radiation. Both types of tumor cells grew preferentially in ultraviolet-irradiated syngeneic mice relative to untreated animals, and both were recognized by ultraviolet radiation-induced tumor-specific suppressor lymphocytes. These properties were not shared by 10T 1/2 cells transformed in vitro by x-rays or 3-methylcholanthrene.

Fas ligand: a sensor for DNA damage critical in skin cancer etiology.

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.

Immunosuppressive effects of ultraviolet (280-320 nm) radiation and psoralen plus ultraviolet (320-400 nm) radiation in mice.

Contact hypersensitivity (CHS), a cell-mediated immunologic reaction, can be induced in mice by application of a contact-sensitizing chemical to the shaved skin. Exposing the animals to UV radiation from FS40 sunlamps inhibits this immune response. This inhibition is systemic, since the sensitizer need not be applied to the irradiated site of the animal. The mechanism whereby UV radiation prevents CHS appears to involve the production of suppressor T-lymphocytes. Recent evidence suggests that UV exposure of mice alters the way in which certain antigens are processed, and this altered processing or presentation of antigen results in the activation of the suppressor cell pathway, rather than leading to immunization. Treatment of mice with a photosensitizer, psoralen, plus UV (320-400 nm) radiation also suppresses CHS systemically, but whether the cellular mechanisms are the same as those underlying the suppression from the shorter UV wavelengths remains to be determined. The possible role of these immunosuppressive events in photocarcinogenesis is discussed.

Immunoregulation of carcinogenesis: past, present, and future.

Twenty years ago the concept that immunological mechanisms might regulate carcinogenesis was highly controversial. Today this concept survives in modified form and continues to evolve as our knowledge of the immune system, the etiology of cancer, and tumor antigens expands. This review illustrates how our ideas about immune surveillance have changed in recent years and considers the significance of these changes for the future directions of cancer immunobiology.

Ultraviolet carcinogenesis in T-cell-depleted mice.

The induction of skin tumors by chronic UV irradiation was compared in normal C3H/HeN(MTV-) mice, T-cell-depleted mice, and T-cell-depleted mice reconstituted with thymus grafts. The T-cell-depleted mice were thymectomized as young adults, lethally X-irradiated, and repopulated with neonatal liver cells. Skin tumors appeared earlier in the immunosuppressed T-cell-depleted mice than in the normal mice. However, tumors developed most rapidly in the group that was T-cell depleted and immunologically restored with thymus grafts. In the latter group, most of the tumors were squamous cell carcinomas, whereas in the normal mice, fibrosarcomas predominated. These experiments illustrated the limitations of this approach to studying the role of the immune response in carcinogenesis.

Influences of age and anatomical site on ultraviolet carcinogenesis in BALB/c mice.

Young adult BALB/c mice were mor susceptible to the induction of skin tumors from FS40 sunlamps than were 18-month-old animals. The relative contributions of tissue and host factors to this difference in susceptibility to carcinogenesis were analyzed by reciprocal grafting of skin between young and old animals, followed by repeated exposure of the grafts to UV radiation. More tumors developed in ear skin grafted to the middorsum of young recipients than in that of old recipients, regardless of the age of the skin donor. These ear skin grafts were more susceptible to tumor induction than were comparable grafts of back skin. When large areas of dorsal skin (16 cm2) were grafted to young adult mice, very old skin (greater than 2 yr) was more susceptible to tumor induction than skin that was 1 year old at the start of irradiation.

Immunologic parameters of ultraviolet carcinogenesis.

Skin tumors induced in mice by UV light are usually immunologically rejected by normal syngeneic recipents. We evaluated the immune status of primary hosts against these highly antigenic tumors immediately after surgical removal of the primary tumor. All primary hosts were susceptible to challenge with their autochthonous tumors, though most of these were rejected by untreated control mice. Primary hosts were also susceptible to challenge with isografts of antigenically dissimilar UV-induced neoplasms. The susceptibility of the primary hosts to tumor challenge was probably induced by chronic exposure to UV light, since UV-irradiated non-tumor-bearing mice were also susceptible to challenge with these tumors. Although UV-treated mice were unalbe to reject these syngeneic tumors, they could reject skin and tumor allografts. Further, UV irradiation did not interfere with the second-set rejection of syngeneic UV-induced tumors in mice that were specifically immunized before UV treatment.

Effect of sunscreens on UV radiation-induced enhancement of melanoma growth in mice.

BACKGROUND: Epidemiologic evidence suggests that exposure to UV radiation plays a significant role in the development of melanoma skin cancers. As early surgical removal of the melanoma is the only effective therapy, current strategies for reducing mortality from melanoma focus on prevention of the disease. Chemical sunscreens protect mice from development of skin cancers that resemble sunlight-induced human squamous cell cancers, but there appears to be a complex relationship between UV radiation exposure and development of melanoma. PURPOSE: We asked whether common sunscreens would protect mice against UV radiation-induced enhancement of melanoma incidence. METHODS: C3H mice were exposed to 4.8 kJ/m2 UVB from FS40 sunlamps twice a week for 3 weeks. Sunscreens containing 7.5% 2-ethylhexyl-p-methoxycinnamate, 8% octyl-N-dimethyl-p-aminobenzoate, 6% benzophenone-3, or the oil-in-water vehicle alone were applied to the ears and tails of the mice 20 minutes before irradiation. At various times during and after exposure, we determined UV radiation-induced inflammation by measuring ear swelling. We also examined the ears histologically for UV radiation-induced alterations. One day after the final irradiation, 2.5 x 10(4) syngeneic K1735 melanoma cells were injected into the external ears. Mice were examined weekly for tumor growth for 5-8 weeks after tumor cell injection. Control mice were treated in the identical way except for exposure to UV radiation. RESULTS: The incidence of melanomas was significantly higher in the UV-irradiated mice. All three sunscreens protected against UV radiation-induced ear swelling and clearly diminished histopathologic alterations, including sunburn cell formation, epidermal hyperplasia, and mononuclear cell infiltrate in the dermis. However, the sunscreens failed to protect against UV radiation-induced increase in melanoma incidence. The sunscreens or vehicle alone did not significantly alter tumor growth. CONCLUSIONS: Protection against sunburn does not necessarily imply protection against other possible UV radiation effects, such as enhanced melanoma growth. IMPLICATIONS: Sunscreen protection against UV radiation-induced inflammation may encourage prolonged exposure to UV radiation and thus may actually increase the risk of melanoma development. These findings suggest that further research on the ability of sunscreens to prevent melanoma is urgently needed.

Enhancement of two-stage skin carcinogenesis by exposure of distant skin to UV radiation.

Exposure of C3H/HeN mice to UV 280-320 nm (UVB) radiation induces a systemic, immunologic alteration that interferes with the rejection of highly antigenic UVB radiation-induced skin cancers. The effect of this systemic alteration, induced by ventral UVB irradiation of mice, was tested on the induction of dorsal skin tumors resulting from initiation with UVB radiation and promotion with 12-O-tetradecanoylphorbol 13-acetate (TPA). The systemic effect of UVB radiation markedly potentiated carcinogenesis at the distant site. More important, mice treated with TPA alone on the dorsal skin developed a significant number of dorsal tumors if the mice also had been exposed ventrally to UVB radiation. Treatment of dorsal skin with UVB radiation alone did not result in the development of cancers, regardless of whether the mice received ventral irradiation. These results suggest that the systemic effect of UVB radiation is exerted during the promotion phase of two-stage carcinogenesis. Furthermore, they imply that a systemic effect of UVB radiation interferes with a natural host control mechanism that ordinarily holds skin cancers in check.

Promotion of syngeneic intraocular tumor growth in mice by anterior chamber-associated immune deviation.

Regressor tumors (P91 and UV-5C25), known to express potent tumor-specific transplantation antigens, were briskly rejected when transplanted either sc or ip into syngeneic mice (DBA/2 and BALB/c, respectively). These tumors demonstrated markedly different behavior following transplantation into the anterior chamber of syngeneic mice. Tumors grew to significant masses and survived for prolonged periods within the anterior chamber of the eye. The extended growth and survival of regressor tumors in the anterior chamber (i.e. immune privilege) were abrogated by splenectomy and thus resemble anterior chamber-associated immune deviation. However, this immune privilege proved to be temporary and was superseded by complete tumor resolution. Studies of mice immunosuppressed with UV light or X-ray irradiation demonstrated that spontaneous intraocular resolution of regressor tumors was due to specific systemic immunity that not only led to intraocular tumor resolution but also prevented the spread of the primary intraocular tumor to distant organs and rendered the hosts highly resistant to secondary challenge with sc tumor inocula. The present findings were relevant to understanding human retinoblastoma, an intraocular neoplasm demonstrating a high incidence of spontaneous resolution, and will hopefully form a foundation for designing, immunotherapeutic strategies for treating human intraocular neoplasms.