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.

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.

Relationship between expression of tumor-specific transplantation antigens and neoplastic transformation in an ultraviolet radiation-induced murine skin cancer.

Ultraviolet radiation-induced murine skin cancers often express highly immunogenic tumor-specific transplantation antigens (TSTA). The relationship between expression of TSTA and neoplastic transformation is not clear. I have used DNA transfection techniques to determine whether expression of TSTA and the transformed phenotype are associated at the genetic level. C3H mouse embryo fibroblast 10T1/2 clone 8 cells were transfected with high-molecular-weight genomic DNA from a highly antigenic ultraviolet radiation-induced 2240 tumor cell line. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up foreign DNA. Morphologically transformed, G418-resistant colonies were isolated and tested for expression of 2240 tumor-specific antigens by means of a cytotoxic T-lymphocyte assay. None of the 12 morphologically transformed colonies tested expressed 2240 tumor-specific antigens on their cell surface as revealed by their inability to be killed by 2240 tumor-specific cytotoxic T-lymphocytes. In addition, the morphologically transformed cells did not inhibit the killing of 51Cr-labeled 2240 cells by 2240 tumor-specific cytotoxic T-lymphocytes in a cold-target inhibition assay. Cell surface expression of Class I major histocompatibility antigens was not significantly altered in 2240 DNA transformants. These results demonstrate that, in ultraviolet radiation-induced murine skin tumors, there is not coordinate expression of TSTA and the transformed phenotype, even though most ultraviolet radiation-induced skin tumors exhibit both characteristics. This finding suggests that the two phenotypes are controlled by separate genes.

Photoreactivation of ultraviolet radiation-induced pyrimidine dimers in neonatal BALB/c mouse skin.

The numbers of ultraviolet light (UV)-induced pyrimidine dimers in the DNA of neonatal BALB/c mouse skin were measured by assessing the sensitivity of the DNA to Micrococcus luteus UV endonuclease. Irradiation of neonatal BALB/c mice with FS40 sunlamps caused a dose-dependent induction of endonuclease-sensitive sites (pyrimidine dimers) in DNA extracted from back skin. Exposure of these UV-irradiated neonatal mice to photoreactivating (PR) light ("cool white" fluorescent lamp and incandescent lamp) caused a reduction in the number of pyrimidine dimers in the DNA, as revealed by a shift in low-molecular-weight DNA to high-molecular-weight DNA. In contrast, DNA profiles of the skin of either UV-irradiated mice or UV-irradiated mice kept in the dark for the same duration as those exposed to PR light did not show a loss of UV-induced endonuclease-sensitive sites. Furthermore, reversing the order of treatment, i.e., administering PR light first and then UV, did not produce a reduction in pyrimidine dimers. These results demonstrate that PR or UV-induced pyrimidine dimers occurs in neonatal BALB/c mouse skin. The optimal wavelength range for in vivo PR appears to be in the visible region of the spectrum (greater than 400 nm). Although dimer formation could be detected in both dermis and epidermis, PR occurred only in the dermis. Furthermore, the PR phenomenon could not be detected in the skin of adult mice from the same inbred strain.

In vitro transformation of primary cultures of neonatal BALB/c mouse epidermal cells with ultraviolet-B radiation.

Primary epidermal cultures from neonatal BALB/c mice were used to study the carcinogenic effects of ultraviolet radiation in vitro. These cultures were irradiated once through a Falcon plastic dish cover with an FS40 sunlamp [ultraviolet B, lambda approximately 290 to 400 nm] for various lengths of time and maintained for 8 to 12 weeks without subculturing. During this period, most of the cells in the untreated control showed signs of morphological differentiation and eventually died. The cultures irradiated with ultraviolet B radiation also behaved in the same manner except that, in some dishes, small populations of surviving cells began to proliferate and developed into morphologically distinct foci. Seven long-term cell lines were derived from these ultraviolet-irradiated primary epidermal cell cultures. Six of these cell lines produced tumors when injected s.c. into normal and/or immunosuppressed syngeneic recipients. These tumorigenic cell lines lacked definitive characteristics of differentiated epidermal cells, but the cells possessed intermediate junctions, suggesting that they were of epithelial origin. Some of these in vitro-transformed cell lines appeared to be highly antigenic inasmuch as they grew preferentially in immunosuppressed BALB/c mice as compared to their growth in normal syngeneic recipients.

Immune response to somatic cell hybrids between ultraviolet radiation-induced regressor and spontaneous progressor C3H mouse tumor cells.

We used somatic cell hybridization to determine whether the regressor phenotype exhibited by UV-induced murine tumors was dominant or recessive and whether this technique could confer immunogenic properties on nonimmunogenic syngeneic tumors. We transfected a highly antigenic UV-induced C3H mouse tumor cell line (UV-2240) with the plasmid pSV2-neo and selected G418-resistant clones. The resulting cell line was fused with a spontaneously transformed nonimmunogenic C3H progressor tumor cell line (SF-2T) that had been selected previously for resistance to 3.0 mM ouabain. These two cell lines were fused by a brief exposure to polyethylene glycol and heterokaryons isolated by growth in medium containing both G418 and ouabain. Hybrid cell lines established from individual colonies and from pools of colonies were tested for tumorigenicity in normal C3H and athymic nude mice. The results indicated that all the hybrid cell lines tested were highly antigenic in that they were completely rejected when transplanted into normal syngeneic mice but grew progressively in nude mice. Furthermore, immunization of C3H mice with the hybrid cell lines induced protective immunity against challenge with the immunizing tumor and generated cross-protective immunity against challenge with the regressor parental cell line but not against challenge with the progressor parental cell line. These results demonstrate that the regressor phenotype of the UV-2240 tumor is dominant in nature and that the immune response induced by somatic cell hybrids is uniquely directed against the dominant tumor-specific transplantation antigens expressed on the regressor tumor. This implies that introduction of tumor-specific transplantation antigens from an immunogenic tumor into a nonimmunogenic tumor, although sufficient to confer immunogenic properties to the hybrid, is insufficient to induce cross-protective transplantation immunity against the nonimmunogenic tumor.

N-ras mutation in ultraviolet radiation-induced murine skin cancers.

UV radiation is a potent DNA-damaging agent and a known inducer of skin cancer in experimental animals. To elucidate the role of oncogenes in UV carcinogenesis, we analyzed UV-induced murine skin tumors for mutations in codon 12, 13, or 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to UV-induced C3H mouse skin tumors, we also analyzed skin tumors induced in the same strain of mice by other carcinogenic agents such as 8-methoxypsoralen + UVA, angelicin + UVA, dimethylbenz-[a]anthracene + UV + croton oil, and 4-nitroquinoline-1-oxide. We found that 4 of 20 UV-induced skin tumors contained either C----A or A----G base substitutions at N-ras codon 61. In addition, 2 of 5 melanomas possessed a G----A transition in N-ras codon 13 and an A----T transversion in N-ras codon 61, respectively. Interestingly, none of the 8-methoxypsoralen + UVA- or angelicin + UVA-induced tumors we analyzed contained mutations in any of the ras genes. However, 1 of 4 4-nitroquinoline-1-oxide-induced tumors exhibited a G----T transversion at Ki-ras codon 12, a potential site for formation of a 4-nitroquinoline-1-oxide adduct with a guanine residue. We also found that 2 nonmelanoma tumors induced by dimethylbenz[a]anthracene + UV + croton oil contained an A----T transversion at Ha-ras codon 61 position 2, which is characteristic of most dimethylbenz[a]anthracene-induced tumors. These results suggest that UV-induced C3H mouse tumors display mutations preferentially in the N-ras oncogene. Since most N-ras mutations in UV-induced tumors occurred opposite dipyrimidine sequences (T-T or C-C), one can infer that these sites are the targets for UV-induced mutation and transformation.

Afferent and efferent specificity in the induction and elicitation of parental cross-protective immunity by an immunogenic murine tumor variant: associative recognition of a unique tumor-specific antigen on somatic cell hybrids.

Highly immunogenic (Imm+) murine tumor cell variants can engender a strong tumor-specific, cross-protective immune response against challenge with the weakly immunogenic parental tumor cell line. We examined the afferent induction and efferent specificity of the parental cross-protective immunity observed following immunization with the Imm+ variant of the murine fibrosarcoma MCA-F, designated MCA-FM1. Specificity of the afferent and efferent responses against the parental tumor in mice immunized with the MCA-FM1 variant were monitored by challenge with the tumor MCA-D, which expresses a tumor-specific antigen that is immunologically distinct from but biochemically related to the MCA-F antigen. We observed that mixture of MCA-D and MCA-FM1 cells at immunization failed to elicit a strong tumor rejection response against challenge with MCA-D. Challenge of MCA-FM1-immune mice with a mixture of MCA-FM1 and MCA-D cells resulted in a significant bystander effect at the site of Imm+ rejection, with reduced growth of the MCA-D tumor. To test the hypothesis that the induction of parental cross-protective immunity required the associative recognition of both the Imm+ neoantigen and the parental tumor antigen on the same cell, we constructed somatic cell hybrids of MCA-D with either MCA-F or MCA-FM1. Surprisingly, the hybrids did not express either parental tumor-specific antigen present on the fusion partners but displayed a unique antigenic specificity designated F/D. Expression of the F/D antigen by both the immunogenic and nonimmunogenic hybrid cell lines demonstrated that the tumor-specific F/D antigen was the focus of the cross-protective immunity. These results demonstrate that associative recognition of the tumor-specific parental antigen with the strongly immunogenic neoantigen coexpressed on the surface of the Imm+ variant is responsible for the afferent induction and efferent elicitation of anti-parental cross-protective immunity. Furthermore, this study is the first to report that the fusion of two syngeneic tumor cell lines reproducibly results in a new tumor antigen specificity at the expense of the original parental specificities.

Immune response to progressor variants derived from transfection of an ultraviolet radiation-induced C3H mouse regressor tumor cell line with activated Harvey-ras oncogene.

Skin cancers induced in mice by UV radiation often exhibit a regressor phenotype. In order to determine how tumors escape the immune defenses of the normal immunocompetent host, we sought to isolate progressor variants from a UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select transfected cells. Injection of Ha-ras-transfected UV-2240 cells s.c. into immunocompetent C3H mice produced tumors in four of 36 animals. In contrast, UV-2240 cells transfected with pSV2-neo DNA alone or mock transfected with CaPO4 did not produce tumors in normal C3H mice. DNAs from cell lines established from Ha-ras-induced tumors contained unique Ha-ras sequences in addition to those sequences endogenous to UV-2240 cells. However, the Ha-ras-induced progressor variants did not overexpress the Mr 21,000 protein. The Ha-ra-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. In addition, all four Ha-ras-induced progressor variants produced significantly more experimental lung metastases in nude mice than did the parent UV-2240 cell line. However, both the parental UV-2240 cell line and the Ha-ras-induced progressor variants expressed similar levels of H-2Kk and H-2Dk antigens and were immunologically cross-reactive, as determined by in vitro cytotoxic T-lymphocyte and in vivo immunization-challenge assays. These results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of tumor-specific transplantation or Class I major histocompatibility complex antigens. This implies that some tumor cells can escape the immune defenses of the normal immunocompetent host by mechanisms other than loss of tumor-specific transplantation and Class I major histocompatibility antigens.

Detection and identification of activated oncogenes in human skin cancers occurring on sun-exposed body sites.

High-molecular-weight DNA isolated from eight fresh human skin cancers occurring on sun-exposed body sites were assayed for their ability to transform NIH 3T3 cells. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up the transfected DNA. About 2 weeks after transfection, G418-resistant colonies were pooled and injected s.c. into athymic nude mice. The NIH 3T3 cells transfected with DNA from six of the human skin cancers induced tumors in nude mice. DNAs from all six tumor cell lines contained human alu sequences. Southern blot hybridization with ras-specific probes revealed that DNAs from the four alu-rich tumors contained the human Ha-ras oncogene, in addition to that of the NIH 3T3 controls. In contrast, DNAs from the other two tumors did not contain any of the known oncogenes tested, except those endogenous to NIH 3T3 cells. DNAs from three of four first cycle tumorigenic transformants gave rise to morphologically transformed foci when assayed in a second cycle of transfection. DNAs from all three secondary transformants contained discrete human alu sequences, and in addition, contained Ha-ras sequences similar to those present in their respective primary transformants. Interestingly, DNA from both primary and secondary transformants of one particular human squamous cell carcinoma contained highly amplified copies of the Ha-ras oncogene. These results suggest that activation of the Ha-ras oncogene may be common in human skin cancers originating on sun-exposed body sites. Further characterization of the Ha-ras oncogenes present in these human skin cancers may provide information on the molecular mechanisms by which UV radiation of the sun induces human neoplasms on exposed body sites.

Hypersensitivity of skin fibroblasts from basal cell nevus syndrome patients to killing by ultraviolet B but not by ultraviolet C radiation.

Basal cell nevus syndrome (BCNS) is an autosomal dominant genetic disorder in which the afflicted individuals are extremely susceptible to sunlight-induced skin cancers, particularly basal cell carcinomas. However, the cellular and molecular basis for BCNS is unknown. To ascertain whether there is any relationship between genetic predisposition to skin cancer and increased sensitivity of somatic cells from BCNS patients to killing by UV radiation, we exposed skin fibroblasts established from unexposed skin biopsies of several BCNS and age- and sex-matched normal individuals to either UV-B (280-320 nm) or UV-C (254 nm) radiation and determined their survival. The results indicated that skin fibroblasts from BCNS patients were hypersensitive to killing by UV-B but not UV-C radiation as compared to skin fibroblasts from normal individuals. DNA repair studies indicated that the increased sensitivity of BCNS skin fibroblasts to killing by UV-B radiation was not due to a defect in the excision repair of pyrimidine dimers. These results indicate that there is an association between hypersensitivity of somatic cells to killing by UV-B radiation and the genetic predisposition to skin cancer in BCNS patients. In addition, these results suggest that DNA lesions (and repair processes) other than the pyrimidine dimer are also involved in the pathogenesis of sunlight-induced skin cancers in BCNS patients. More important, the UV-B sensitivity assay described here may be used as a diagnostic tool to identify presymptomatic individuals with BCNS.

High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor heterogeneity.

Exposure to UV radiation has long been associated with the development of skin cancers. To identify the molecular targets in UV carcinogenesis, we analyzed 11 UV-induced murine skin cancers for mutations in the p53 tumor suppressor gene and found a 100% incidence rate. Such a high frequency of p53 mutations is unprecedented and suggests that this gene plays an important role in the development of UV-induced skin cancers. The mutations were predominantly "UV-signature" transitions (C-->T and CC-->TT) at pyrimidine-rich sequences located on the nontranscribed strand of the gene. In addition, seven tumors harbored multiple mutant alleles of p53, providing strong evidence for tumor heterogeneity at the molecular level.

Generation of antigenic variants from a nonantigenic murine tumor cell line by transfection with a gene encoding a novel tumor-specific transplantation antigen.

Tumors induced in mice by UV radiation often express highly immunogenic tumor-specific transplantation antigens (TSTA). The 216 gene, which encodes a TSTA of the C3H tumor UV-1591, has been cloned and characterized as a novel major histocompatibility complex Class I antigen. The purpose of this study was to determine whether the 216 gene-encoded TSTA can function as a tumor rejection antigen when expressed on unrelated, nonantigenic murine tumor cells or whether its function is restricted to UV-induced tumors. A cell line (10T-1) derived from a spontaneous transformant of C3H 10T1/2 cells was cotransfected with DNA from p216 and pSV2-neo plasmids. About 2 wk after transfection, G418-resistant colonies were isolated randomly and tested for cell surface expression of the 216 gene product using a monoclonal antibody specific for 216 gene-encoded TSTA. Of 20 clones tested, four expressed high levels of 216 gene-encoded TSTA. These four clones were highly antigenic in that they were completely rejected in normal mice but grew progressively in nude mice. Furthermore, the 216-positive clones were immunologically cross-reactive with UV-1591, as determined by in vitro cytotoxic T-lymphocyte and in vivo immunization and challenge assays. Surprisingly, 216-positive 10T-1 transfectants also cross-reacted with 10T-1 cells, both in vitro and in vivo. These results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.

Direct correlation between nitric oxide synthase II inducibility and metastatic ability of UV-2237 murine fibrosarcoma cells carrying mutant p53.

The relationship between nitric oxide synthase II (NOS II) inducibility and the metastatic ability of UV-2237 murine fibrosarcoma cells was determined. Highly metastatic cells survived to produce numerous lung metastases after i.v. injection in syngeneic C3H/HeN mice, whereas poorly metastatic cells did not. Highly metastatic clones exhibited higher levels of NOS II than did poorly metastatic clones in response to interleukin 1alpha and IFN-gamma stimulation. Furthermore, both poorly and highly metastatic clones contained an identical p53 mutation. Overexpression of NOS II in a highly metastatic clone by transfection with NOS II gene retarded tumor growth and completely suppressed metastasis. Our data indicate that a low to moderate level of NOS II expression directly correlates with metastatic ability of UV-2237 fibrosarcoma cells carrying mutant p53 and that a high level of nitric oxide production suppresses tumor growth and metastasis.

Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras.

We sought to determine whether the transfection of tumorigenic but not metastatic cells with the activated c-Ha-ras oncogene was invariably associated with acquisition of the metastatic phenotype. Three clonally derived lines of the K-1735 murine melanoma, characterized as nonmetastatic or poorly metastatic, were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, that confers resistance to neomycin (pSV2neoEJ). Cells transfected with pSV2neo, a plasmid containing the neo gene, served as controls for the procedure of Polybrene-mediated transfection. All cell lines were injected into syngeneic C3H/HeN and into athymic mice, and the results were compared with those produced by highly metastatic K-1735 M-2 cells. Although the pSV2neoEJ-transfected cells produced more rapidly growing s.c. tumors than the control cell lines did, the incidence of spontaneous metastasis was not increased. Following i.v. inoculation, the c-Ha-ras transfectants were retained in lung vasculature in greater proportions than pSV2neo counterpart transfectants were. The c-Ha-ras transfectants also produced significantly more lung tumor colonies, which grew faster than the few lung tumor colonies in mice given injections of control melanoma cells. We concluded that transfection of the activated c-Ha-ras oncogene into nonmetastatic K-1735 melanoma cells leads to accelerated tumor growth in vivo and can confer the ability to form lung colonies after i.v. injection but not the ability to metastasize from a primary s.c. tumor.

Transformation of NIH3T3 cells by transfection with UV-irradiated human c-Ha-ras-1 proto-oncogene DNA.

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain G----T mutations at the second position of Ha-ras codon 12. In this study, we investigated whether the c-Ha-ras-1 proto-oncogene could be activated by in vitro UV-irradiation of pEC plasmid DNA, which contains the 6.6 kb BamHI fragment of the human c-Ha-ras-1 proto-oncogene. Focus formation and nude mouse tumorigenicity assays showed that UV-irradiated pEC DNA induced morphologic and tumorigenic transformation of NIH3T3 cells in multiple cycles of transfection, whereas unirradiated pEC DNA did not. DNAs from secondary cycle foci and tertiary cycle tumors were analyzed for mutations in Ha-ras codons 12 and 61 using the polymerase chain reaction and synthetic oligonucleotide probes. Eleven of 11 secondary cycle foci analyzed possessed a G----T mutation at the second position of Ha-ras codon 12. However, the nude mouse tumors exhibited a G----A mutation at position 1 of the Ha-ras codon 12. These results suggest that in vitro UV irradiation of the c-Ha-ras-1 proto-oncogene DNA can induce mutations that are similar to those found in human skin cancers that originated on sun-exposed body sites.

Differential sensitivity of normal and Ha-ras-transformed C3H mouse embryo fibroblasts to tumor necrosis factor: induction of bcl-2, c-myc, and manganese superoxide dismutase in resistant cells.

In this study, we investigated the role of activated Ha-ras oncogene on the growth-regulatory properties of tumor necrosis factor (TNF) in C3H mouse embryo fibroblasts. TNF-resistant 10T1/2 cells transfected with an activated Ha-ras oncogene not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. TNF-induced cell death was mediated through apoptosis. The differential sensitivity of normal and Ha-ras transformed cells to TNF was not due to differences in the number of TNF receptors on their cell surface. However, TNF-resistant cells, but not sensitive cells, overexpressed bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. In addition, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. These results suggest that TNF-induced cytotoxicity involves apoptosis and that TNF-induced over-expression of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts.

p53 protects against skin cancer induction by UV-B radiation.

To assess the role of the p53 tumor suppressor gene in skin carcinogenesis by UV radiation, mice constitutively lacking one or both copies of the functional p53 gene were compared to wild-type mice for their susceptibility to UV carcinogenesis. Heterozygous mice showed greatly increased susceptibility to skin cancer induction, and homozygous p53 knockout mice were even more susceptible. Accelerated tumor development in the heterozygotes was not associated with loss of the remaining wild-type allele of p53, as reported for tumors induced by other carcinogens, but in many cases was associated with UV-induced mutations in p53. Tumors arose on the ears and dorsal skin of mice of all three genotypes, and homozygous knockout mice also developed ocular tumors, mainly melanomas. Skin tumors in the p53 knockout mice were predominately squamous cell carcinomas and were associated with premalignant lesions resembling actinic keratoses, whereas those in the heterozygous and wild-type mice were mainly sarcomas. These results demonstrate the importance of p53 in protecting against UV-induced cancers, particularly in the eye and epidermis.

Differential regulation of endogenous endonuclease activation in isolated murine fibroblast nuclei by ras and bcl-2.

Transfection of a murine fibroblast cell line with an activated form of the Harvey ras oncogene conferred sensitivity to apoptosis induced by various agents. This intrinsic sensitivity to apoptosis correlated with the expression of endogenous endonuclease activity in isolated nuclei that was undetectable in the untransfected parental cell line. Subsequent transfection with the human bcl-2 oncogene prevented the morphological and biochemical features of apoptosis in whole cells, although it failed to confer complete protection against cell death. Furthermore, transfection of the bcl-2 oncogene also inhibited the enhanced endonuclease activity in isolated nuclei. Our results indicate that some of the effects of Ha-ras and bcl-2 and potentially other oncogenes, are exerted on the biochemical machinery of apoptosis at the level of the nucleus.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.