Mitochondrial respiratory uncoupling promotes keratinocyte differentiation and blocks skin carcinogenesis.

Decreased mitochondrial oxidative metabolism is a hallmark bioenergetic characteristic of malignancy that may have an adaptive role in carcinogenesis. By stimulating proton leak, mitochondrial uncoupling proteins (UCP1-3) increase mitochondrial respiration and may thereby oppose cancer development. To test this idea, we generated a mouse model that expresses an epidermal-targeted keratin-5-UCP3 (K5-UCP3) transgene and exhibits significantly increased cutaneous mitochondrial respiration compared with wild type (FVB/N). Remarkably, we observed that mitochondrial uncoupling drove keratinocyte/epidermal differentiation both in vitro and in vivo. This increase in epidermal differentiation corresponded to the loss of markers of the quiescent bulge stem cell population, and an increase in epidermal turnover measured using a bromodeoxyuridine (BrdU)-based transit assay. Interestingly, these changes in K5-UCP3 skin were associated with a nearly complete resistance to chemically-mediated multistage skin carcinogenesis. These data suggest that targeting mitochondrial respiration is a promising novel avenue for cancer prevention and treatment.

Alteration of Egr-1 mRNA during multistage carcinogenesis in mouse skin.

Immediate early genes, including fos, jun, and early growth response-1 (Egr-1), are induced during cellular response to changes in extracellular environment. These immediate early genes are believed to mediate processes of cell growth and differentiation. In particular, Egr-1 is induced during mitogenic stimulation of a variety of cell types, including fibroblasts, B cells, and epithelial cells. In the present study, we examined Egr-1 gene expression during multistage carcinogenesis in mouse skin. After a single topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse skin, Egr-1 mRNA was induced, and maximal induction was observed at 2 h in both epidermis and dermis. Induction of Egr-1 mRNA by TPA was inhibited by fluocinolone acetonide, a potent inhibitor of tumor promotion by TPA. Egr-1 mRNA was present in primary keratinocytes derived from adult SENCAR mice. The keratinocyte cultures were maintained in low Ca(2+) medium, and Egr-1 mRNA levels became significantly elevated after the cultures were switched to high Ca(2+) medium. Additionally, a large proportion of primary papillomas and carcinomas generated from SENCAR mice by standard initiation-promotion regimens exhibited elevated Egr-1 mRNA compared with normal epidermis. Taken together, these data suggest a possible role of Egr-1 during multistage carcinogenesis in mouse skin.

Altered expression of insulin-like growth factor I and its receptor during multistage carcinogenesis in mouse skin.

We examined the possible role of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-Ir) during multistage carcinogenesis in mouse skin. For this purpose, the expression of both IGF-I and IGF-Ir was investigated in mouse skin during tumor promoter treatment and in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation-promotion regimens. IGF-I transcripts were not detectable or only weakly detectable in normal SENCAR mouse epidermis by northern or reverse transcription (RT)-polymerase chain reaction (PCR) analysis, respectively, whereas IGF-I transcripts (primarily a 7.0-kb transcript) were readily detected in RNA preparations from the dermis by both northern blot analysis and RT-PCR analysis. In contrast, IGF-Ir transcripts were observed in RNA samples from both epidermis and dermis of control SENCAR mice. Single and multiple topical treatments with 3.4 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on dermal or epidermal IGF-I and IGF-Ir mRNA levels. In contrast, the levels of IGF-I transcripts were elevated (2.5- to 15-fold) in a significant number of mouse skin tumors (71% of all tumors examined). Transcripts of 7.0, 2.5, and 1.3 kb were more consistently overexpressed in skin tumors compared with epidermis, whereas the two smaller transcripts were most consistently overexpressed compared with the dermis. The levels of an 11.0-kb IGF-Ir transcript were also elevated (2.5- to 8-fold) in some papillomas (20%) and SCCs (55%), but the percentage of tumors exhibiting this property (32% of all tumors examined) was lower than the percentage overexpressing IGF-I. These data suggest that altered expression of IGF-I and IGF-Ir may play a role in multistage carcinogenesis in the mouse skin model. The inability of TPA to induce elevated IGF-I or IGF-Ir expression suggests that these changes in skin tumors are coincident with tumor formation and not a direct result of altered epidermal proliferation per se. Altered expression of IGF-I in a high percentage of papillomas may indicate that IGF-I has an important role in the development of autonomous growth in these tumors. The higher percentage of SCCs with altered levels of IGF-Ir mRNA may indicate a role for these changes in the later stages (i.e., tumor progression) of carcinogenesis in this model system.

Elevation of transforming growth factor-alpha mRNA and protein expression by diverse tumor promoters in SENCAR mouse epidermis.

The study presented here was designed to further investigate the role of transforming growth factor-alpha (TGF alpha) in skin tumor promotion by examining the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) and several non-phorbol ester promoters to alter TGF alpha mRNA and protein levels in mouse epidermis. Total RNA was isolated from SENCAR mouse epidermis at various times after single topical treatments with TPA (3.4 nmol), chrysarobin (220 nmol), okadaic acid (2.5 nmol), and thapsigargin (8.5 nmol). Northern analyses of these isolated RNA samples revealed that all four tumor promoters transiently elevated TGF alpha mRNA levels. Whereas TPA, okadaic acid, and thapsigarin elevated TGF alpha mRNA levels over similar time courses (peak at 4-8 h), chrysarobin elevated TGF alpha mRNA levels with a markedly delayed time course (peak at 24-48 h). More detailed studies with TPA also revealed that multiple treatments (four over a 2-wk period) transiently elevated TGF alpha mRNA in both the epidermis and the dermis. The time courses for changes in TGF alpha mRNA after multiple TPA treatments were similar for both tissues. To facilitate studies of altered TGF alpha mRNA expression in mouse epidermis and possibly other mouse tissues, a semiquantitative reverse transcriptase-polymerase chain reaction method was developed. This method faithfully revealed changes in TGF alpha mRNA levels with all four tumor-promoting agents similar to those determined by northern blot analyses. Immunofluorescence analysis of frozen sections from promoter-treated skin revealed elevated TGF alpha protein levels in both epidermis and dermis, although staining was most intense in the epidermal layer. Immunofluorescence analysis of epidermal hyperplasia adjacent to a full-thickness wound also demonstrated significant epidermal TGF alpha staining. Collectively, these results indicate that mechanistically diverse tumor promoter stimuli elevate TGF alpha mRNA and protein in SENCAR mouse epidermis. Elevated levels of TGF alpha may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli.

Altered expression of the epidermal growth factor receptor and transforming growth factor-alpha during multistage skin carcinogenesis in SENCAR mice.

In the study presented here, we examined the possible role of the transforming growth factor-alpha (TGF alpha)/epidermal growth factor receptor (EGFR) system during multistage carcinogenesis in mouse skin. In this regard, the expression (mRNA and protein) of both TGF alpha and EGFR was examined in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation-promotion regimens and compared with the levels of expression in normal epidermis. The level of a 4.8-kb TGF alpha transcript was elevated in 100% of the skin tumors examined (both papillomas and SCCs), including papillomas obtained 13 wk after the start of promotion, compared with normal epidermis. Immunohistochemical analyses detected elevated levels of TGF alpha protein in these skin tumors and in papillomas as early as 10 wk after the start of promotion. The levels of EGFR transcripts were also significantly elevated in most (90%) of the skin tumors examined, including again those harvested after 13 wk of promotion. Interestingly, multiple EGFR transcripts (10.5, 5.8, 2.8, and 1.8 kb) were detected in both papillomas and SCCs. The two smaller transcripts appeared to encode truncated versions of the EGFR, and the 1.8-kb transcript appeared to be unique to RNA samples isolated from skin tumors, based on comparative analyses of several normal tissues. As with TGF alpha, immunohistochemical analyses detected elevated levels of EGFR protein in these skin tumors (both papillomas and SCCs), including papillomas harvested as early as 10 wk after the start of promotion. Southern analyses of genomic DNAs for TGF alpha and EGFR failed to detect any cases of gene rearrangements or amplification as a possible explanation for the elevated levels of the transcripts of these two genes. These results support the hypothesis that a key step in the development of autonomous growth in mouse skin papillomas generated in SENCAR mice by an initiation-promotion regimen may involve alterations in the synthesis of TGF alpha and its cognate receptor.

Role of the epidermal growth factor receptor and transforming growth factor alpha in mouse skin carcinogenesis.

The mouse skin model of multistage carcinogenesis continues to serve as a major in vivo model for studying the sequential and stepwise evolution of the cancer process by chemical and physical carcinogens. The initiation stage of mouse skin carcinogenesis involves genetic damage in the form of DNA adducts or initiator-induced DNA base changes. These changes ultimately lead to mutations in critical target genes of epidermal stem cells. The rasHa gene, and to a limited extent the N-ras gene, have been identified as target genes for certain tumor initiators in this model system (reviewed in DiGiovanni 1992). The promotion stage of mouse skin carcinogenesis involves the production and maintenance of a chronic state of hyperplasia and cell proliferation and ultimately the selective clonal expansion of initiated cells. The hallmark of all tumor promoters that have been adequately tested is their ability to induce a potentiated hyperplasia after several treatments that is greater than that observed after a single application. Tumor promoters produce many effects when applied topically to mouse skin. Many of the effects that occur after a single application of phorbol esters such as TPA appear to be mediated by its interaction with PKC (Nishizuka 1989). An important question is whether the activation of PKC per se is responsible for tumor promotion by TPA. Because repetitive treatments with TPA lead to a sustained loss of PKC, it is possible that other effects not mediated by PKC but produced by phorbol esters and related compounds may play an important role in the production and maintenance of chronic hyperplasia and cell proliferation in the skin and for skin tumor promotion. More attention should be placed on studying the promoting actions of other compounds outside of the most commonly studied phorbol esters. Investigations of some of these compounds already have and will continue to provide important clues regarding possible common pathways shared by diverse promoting agents. One such pathway may involve the EGFr and its ligand TGF alpha. As discussed in this review, it is now evident that many different types of promoting agents increase production of TGF alpha (Ellem et al. 1988, Pittelkow et al. 1989, Choi et al. 1991, Imamoto et al. 1991, J. DiGiovanni unpublished studies). Although many tumor promoters initially decrease the binding of 125I-EGF to EGFr in specific cell types, including mouse epidermal cells, the long-term effects of tumor promoters, especially after repetitive treatments, may be considerably different.+4

The proximal end of mouse chromosome 17: new molecular markers identify a deletion associated with quakingviable.

Five randomly identified cosmids have been mapped proximal to the Leh66D locus on mouse chromosome 17. Two of these cosmids, Au10 and Au119, map near the neurological mutation quaking. Au119 is deleted in qkviable/qkviable DNA, whereas Au10 is not. Au76 maps to a gene-rich region near the Time locus. The Au76 locus encodes a member of a low copy gene family expressed in embryos, the adult central nervous system and testis. A second member of this family has been mapped to chromosome 15 near c-sis (PDGF-B). At the centromeric end of chromosome 17, Au116 maps near the Tu1 locus, and along with Au217rs identifies a region of unusually high recombinational activity between t-haplotypes and wild-type chromosomes. Au217I and II map to the large inverted repeats found at the proximal end of the wild-type chromosome. In addition, the Au217I and/or II loci encode testis transcripts not expressed from t-haplotypes.