The multistage evolution of squamous skin tumors induced by chemical or viral carcinogens on mice from different genetic backgrounds has been a valuable model to define low penetrance loci that determine cancer susceptibility or resistance. Susceptibility determinants are multigenic, stage specific, dependent on the carcinogenesis protocol, and in the case of initiating events, intrinsic properties of keratinocytes. In this study we examined the malignant conversion frequency of keratinocytes derived from FVB/N, inbred SENCARA/Pt, BALB/c, or C57BL/6 mouse strains that differ substantially in the frequency of progression from papilloma to carcinoma. Keratinocytes were cultured from newborn mice and tested in an in vitro malignant conversion assay induced by a chemical carcinogen or immortalized by infection with replication defective HPV-16 E6/E7 retroviruses and tested for malignancy by grafting immortalized cell lines to nude mice. In vitro, FVB/N keratinocytes were 10-fold more sensitive to chemically induced malignant conversion than keratinocytes from other strains, consistent with the known sensitivity of this strain to premalignant progression in vivo. The E6/E7 genes induced immortalization of keratinocytes from FVB/N, SENCARA/Pt, or C57BL/6 mice more efficiently than BALB/c, and HPV-16-immortalized FVB/N keratinocytes formed tumors more frequently (64%) than SENCARA/Pt (31%) BALB/c (1.9%) or C57BL/6 (2.5%). Furthermore, 78% of the tumors formed by FVB/N keratinocytes progressed to squamous carcinomas compared to 46% for SENCARA/Pt derived cells and less than 3% for the others. In F1 offspring of crosses from SENCARA/Pt and FVB/N mice, both the papilloma incidence and frequency of malignant conversion reflected the SENCARA/Pt parent indicating that predisposition to premalignant progression is not a dominant characteristic. This predisposition is an intrinsic property of the target keratinocytes and as such should be amenable to further study in isolated cells.mtCLIC/CLIC4 is a cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. In keratinocytes, CLIC4 is upregulated by p53 and TNF alpha, coinciding with the onset of apoptosis. Reporter assays indicate that the human CLIC4 promoter has a functional p53 binding sites. Overexpression of CLIC4 in keratinocytes induces apoptosis, and is associated with loss of mitochondrial membrane potential, cytochrome c release and caspase activation. CLIC4 mRNA expression is largely reduced in multiple cancer types in cDNA arrays comparing normal and matched tumor tissues. The reduction is particularly common in renal, ovarian and breast cancers. In contrast, immunohistochemical analyses of human tumor tissue arrays show variability in mtCLIC expression, indicating that suppression of CLIC4 may be tissue or cell-type dependent and post translationally regulated. Support for this idea is provided by carcinogen-induced murine skin tumors where mtCLIC protein is down-regulated as tumors progress from papilloma to carcinoma. Further, there is a shift from predominantly nuclear staining in benign lesions to more cytoplasmic staining in murine skin cancers that express mtCLIC. Thus, expression and subcellular localization of mtCLIC may be related to the differentiation status of a particular tumor. Together these results indicate that mtCLIC may be functionally important in carcinogenesis of certain epithelial tissues and may have diagnostic value for predicting the differentiation status of epithelial cancers. DNA damage, TNF alpha or elevation of p53 in several cell types is associated with translocation of cytoplasmic CLIC4 to the nucleus, suggesting that the apoptotic response may involve CLIC4 in more than one intracellular compartment. Biochemical and imaging analyses indicate that CLIC4 nuclear translocation is an early event in the response to apoptotic or stress signals. CLIC4 associates with the Ran, NTF2 and importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress inducing agents. Deletion or mutation of the nuclear localization signal in the C-terminus of CLIC4 eliminates nuclear localization, while N-terminus deletion enhances nuclear localization. We constructed adenoviruses that target CLIC4 to specific organelles. Overexpression of the nuclear-targeted CLIC4 causes influx of chloride ion to the nucleus, alters the nuclear pH, and accelerates apoptosis. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. CLIC4 is elevated during TNF alpha induced apoptosis in human osteosarcoma cell lines. In contrast, inhibition of NFkappaB results in an increase in TNFalpha-mediated apoptosis with a decrease in CLIC4 protein levels. Cell lines expressing an inducible CLIC4 antisense construct that also reduces the expression of several other CLIC family proteins were established in the human osteosarcoma lines SaOS and U2OS cells and a malignant derivative of the mouse squamous papilloma line SP1. Reduction of CLIC family proteins by antisense expression caused apoptosis in these cells. Moreover, CLIC4 antisense induction increased TNF alpha-mediated apoptosis in both the SaOS and U2OS derivative cell lines without altering TNF alpha-induced NFkappaB activity. Reducing CLIC proteins in tumor grafts of SP1 cells expressing a tetracycline-regulated CLIC4 antisense substantially inhibited tumor growth and induced tumor apoptosis. Administration of TNF alpha intraperitoneally modestly enhanced the antitumor effect of CLIC reduction in vivo. These results suggest that CLIC proteins could serve as drug targets for cancer therapy and reduction of CLIC proteins could enhance the activity of other anti-cancer drugs. Studies on the role of AP-1 in epidermal differentiation and function have revealed a critical function for AP-1 family members in maintaining the integrity of the epidermis as mice age. To study this in greater detail, we used mice in which a powerful AP-1 dominant-negative construct (A-FOS) is targeted to the epidermis with a keratin 5 promoter and regulated by a tetracycline sensitive transactivator. These mice develop ulcerative lesions and alopecia in a time dependant manner consistent with the level of AP-1 inhibition. This phenotype presents itself at 6-9 months in the A-FOS dominant-negative mice and after 1 year in c-fos null and heterozygous mice. Studies on keratin expression have not revealed abnormal expression patterns in vivo, however, examination of primary keratinocytes in culture has revealed expression of differentiation-associated keratins 1 and 10 in culture conditions favoring a basal cell phenotype. These results are supported by micro-array analysis of RNA from the skin of A-FOS expressing mice that reveals mis-expression of numerous differentiation specific genes.Genetic ablation of individual members of the AP-1 transcription factor family has revealed their participation in tumor formation in several target tissues. To assure that such results are not related to compensatory activities of other AP-1 family members, transgenic mice were generated that conditionally express a powerful inhibitor (A-FOS) of all AP-1 activity in the skin using the keratin 5 (K5) promoter. Here we show that when A-FOS was expressed during chemical-induced skin carcinogenesis, mice did not develop characteristic benign or malignant squamous lesions, but instead developed benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity following tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, sebaceous tumors transdifferentiate into squamous tumors when AP-1 is activated after tumor formation.
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