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.

The role of the EP receptors for prostaglandin E2 in skin and skin cancer.

One of the most common features of exposure of skin to ultraviolet (UV) light is the induction of inflammation, a contributor to tumorigenesis, which is characterized by the synthesis of cytokines, growth factors and arachidonic acid metabolites, including the prostaglandins (PGs). Studies on the role of the PGs in non-melanoma skin cancer (NMSC) have shown that the cyclooxygenase-2 (COX-2) isoform of the cyclooxygenases is responsible for the majority of the pathological effects of PGE(2). In mouse skin models, COX-2 deficiency significantly protects against chemical carcinogen- or UV-induced NMSC while overexpression confers endogenous tumor promoting activity. Current studies are focused on identifying which of the G protein-coupled EP receptors mediate the tumor promotion/progression activities of PGE(2) and the signaling pathways involved. As reviewed here, the EP1, EP2, and EP4 receptors, but not the EP3 receptor, contribute to NMSC development, albeit through different signaling pathways and with somewhat different outcomes. The signaling pathways activated by the specific EP receptors are context specific and likely depend on the level of PGE(2) synthesis, the differential levels of expression of the different EP receptors, as well as the levels of expression of other interacting receptors. Understanding the role and mechanisms of action of the EP receptors potentially offers new targets for the prevention or therapy of NMSCs.

Role of intracellular interleukin-1 receptor antagonist in skin carcinogenesis.

Interleukin 1 (IL-1) is a major mediator of inflammation and exerts pleiotropic effects on many systems. To elucidate the role of its endogenous inhibitor, intracellular IL-1 receptor antagonist (icIL-1Ra), in mouse skin, we produced an icIL-1Ra-overexpressing skin carcinoma cell line (icIL-1Ra-JWF2). Altered expression of icIL-1Ra did not change IL-1alpha mRNA levels in these transfected cells. In icIL-1Ra-JWF2 cells, however, cyclooxygenase-2 mRNA levels were dramatically reduced and shown to be transcriptionally regulated by icIL-1Ra. To determine the effect of icIL-1Ra on cell proliferation, cell counts were done 24 h after plating equal numbers of cells. Cells from three icIL-1Ra-JWF2 clones showed significantly reduced growth rates compared with parental JWF2 cells. We subcutaneously injected five independent clones of icIL-1Ra-JWF2 cells into nude mice and measured the tumor doubling time by weekly measurements of tumor volume. IcIL-1Ra appeared to significantly slow the growth of tumors in vivo. Collectively these observations suggest that IL-1Ra has antiproliferative effects in murine skin carcinoma cells.

8S-lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes.

To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor alpha (PPARalpha) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPARalpha, PPARgamma, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPARalpha.

Development and initial characterization of several new inbred strains of SENCAR mice for studies of multistage skin carcinogenesis.

The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12-dimethylbenz[a]anthracene and 0.25 microg 12-O-tetradecanoylphorbol-13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4-20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system.

Changes in protein expression during multistage mouse skin carcinogenesis.

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

Opposite effect of stable transfection of bioactive transforming growth factor-beta 1 (TGF beta 1) versus exogenous TGF beta 1 treatment on expression of 92-kDa type IV collagenase in mouse skin squamous cell carcinoma CH72 cells.

We have previously shown that transforming growth factor-beta 1 (TGF beta 1) mRNA is consistently overexpressed in squamous cell carcinomas relative to normal mouse skin. Here we show that 92-kDa type IV collagenase (matrix metalloproteinase) (MMP-9) mRNA was likewise progressively overexpressed during mouse skin carcinogenesis. To determine if overexpression of MMP-9 and TGF beta 1 are linked, we stably transfected a bioactive TGF beta 1 into a mouse skin squamous cell carcinoma cell line (CH72), which resulted in about twofold to three-fold higher levels of secreted active TGF beta 1. Active TGF beta 1-transfected cells grew only slightly, but not significantly, more slowly in vitro and in vivo than vector-only transfectants. Two clones overexpressing active TGF beta 1 secreted much reduced levels of MMP-9 activity, as determined by zymogram analyses. However, treatment of these clones with 40 pM exogenous TGF beta 1 for 48 h enhanced secretion of MMP-9 activity. Constitutive mRNA expression of MMP-9 was reduced twofold to 70-fold in five untreated active TGF beta 1-transfected clones relative to the other transfectants. In contrast, treatment with 40 pM exogenous TGF beta 1 induced MMP-9 mRNA expression in a time-dependent fashion, from twofold to fourfold after 4 h to a maximum of 12- to 19-fold after 24-48 h. Induction of MMP-9 mRNA was dose dependent at TGF beta 1 concentrations of 4-400 pM. Thus, stable transfection of bioactive TGF beta 1 downregulated whereas exogenous TGF beta 1 treatment upregulated MMP-9 activity and expression. Treatment of transfectants with a neutralizing TGF beta 1 antibody slightly downregulated constitutive MMP-9 mRNA (20-30%) but completely blocked induction by exogenous TGF beta 1. Thus, the effect of TGF beta 1 transfection was not due to secreted TGF beta 1 but may have been a secondary effect.

Uncoordinated regulation of mRNA expression of the three isoforms of transforming growth factor-beta in the mouse skin carcinogenesis model.

The mRNA expression and autoregulation of expression of the three isoforms of transforming growth factor-beta (TGFbeta) were examined in the mouse skin carcinogenesis model by northern analyses. We found that TGFbeta3 mRNA levels followed a pattern similar to those of TGFbeta1 during carcinogenesis: the levels were somewhat low in normal skin but became highly overexpressed in late-stage papillomas and squamous cell carcinomas (15- to 20-fold higher than in normal skin). On the other hand, the TGFbeta2 mRNA levels remained relatively low in all benign and malignant tumors, even though the levels were higher than the nearly undetectable levels in normal skin. In a squamous cell carcinoma cell line (CH72), stable transfection and expression of a mutated simian TGFbeta1 cDNA producing bioactive TGFbeta1 significantly downregulated (mean greater than ten-fold) TGFbeta2 mRNA levels and modestly downregulated (about twofold) murine TGFbeta1 expression but had no effect on TGFbeta3 mRNA. In contrast, treatment of all CH72 clones with exogenous TGFbeta1, TGFbeta2, or TGFbeta3 either had no effect or slightly downregulated TGFbeta1 mRNA, upregulated TGFbeta2 mRNA expression an average of twofold to threefold, and strongly upregulated (mean 13- to 27-fold) TGFbeta3 mRNA levels. TGFbeta treatment of primary cultures of mouse skin keratinocytes upregulated all three TGFbeta mRNA levels slightly to moderately (1.3- to 5-fold). Thus, although TGFbeta1 and TGFbeta3 mRNA expressions were apparently coordinately upregulated during mouse skin carcinogenesis, the three TGFbeta mRNAs were differentially regulated by stable transfection of active TGFbeta1 versus exogenous TGFbeta treatment in CH72 cells and by TGFbeta treatments of normal keratinocytes versus carcinoma CH72 cells.

Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-beta s.

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-beta (TGF beta) and to be growth inhibited by exogenous TGF beta. The purpose of the present studies was to determine whether the endogenous TGF beta(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGF beta 1 and TGF beta 2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8- and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGF beta 2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGF beta s were acting locally. Anti-TGF beta 1 treatment of cultures at various stages of growth resulted in 1.2-1.7-fold increases in DNA synthesis above controls, whereas anti-TGF beta 2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGF beta s inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4-70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGF beta 1 and TGF beta 2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGF beta s.

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-beta 1 with time in culture.

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-beta 1 (TGF beta 1) when treated within 1-2 days after plating. The purpose of the present studies was to examine the effects of TGF beta 1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGF beta 1 decreased dramatically as the cultures aged. The IC50 for inhibition of colony forming efficiency was 0.18 pM when TGF beta 1 was added 24 h after cell plating. When TGF beta 1 treatment was begun on day 5 of culture, the IC50 was 3-4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGF beta 1 was begun on day 19, the IC50 was 65 pM or greater than 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGF beta 1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGF beta 1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGF beta 1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGF beta 1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGF beta 1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-alpha, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGF beta 1 by about twofold at both early and late times in culture. This indicates that the changes in TGF beta 1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events.

Transformed rat tracheal epithelial cells exhibit alterations in transforming growth factor-beta secretion and responsiveness.

The purpose of our studies was to define abnormalities in the transforming growth factor-beta (TGF-beta) system of transformed rat tracheal epithelial (RTE) cells that might cause their abnormal growth behavior. We found that many, but not all, of the transformed cell lines were hyporesponsive or unresponsive to the growth inhibitory effects of TGF-beta 1. Scatchard and receptor cross-linking analyses indicated that loss of TGF-beta 1 responsiveness of transformed cells was probably not due to changes in receptor number or affinity, or to changes in expression of the three TGF-beta-binding protein subtypes. Transformed cells were found to secrete far less TGF-beta-like activity (less than 1/10) than primary cells. Cultured normal and transformed RTE cells expressed three TGF-beta 1 transcripts of 2.5, 1.9, and 1.4 kb. In contrast, rat kidney tissue, a rat embryo fibroblast cell line, and a rat liver cell line expressed only the typical 2.5-kb mRNA transcript commonly reported in the literature. In spite of the marked differences in TGF-beta secretion between normal and transformed cells, their levels of TGF-beta 1 mRNA expression were similar. This suggests a change in the posttranscriptional regulation of TGF-beta 1 expression. TGF-beta 2 message was not detected in either normal or transformed RTE cells in culture. These findings are consistent with the hypothesis that the abnormal growth behavior of transformed RTE cells is at least in part due to disturbances of the TGF-beta system.

Abnormalities of growth factor systems in transformed airway epithelial cells.

The role of the peptide growth factors transforming growth factor alpha (TGF alpha) and transforming growth factor beta (TGF beta) in the regulation of proliferation of normal and transformed airway epithelial cells was studied. Normal as well as transformed rat tracheal epithelial (RTE) cell cultures secrete similar amounts of TGF alpha during logarithmic growth. Once normal RTE cell cultures reach the plateau phase of growth, they down-regulate TGF alpha expression at the RNA and protein level; in contrast, transformed cells do not down-regulate TGF alpha. Using neutralizing TGF alpha antiserum and a tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor, we show that the secreted TGF alpha is utilized by both normal and transformed cells as an autocrine mitogenic factor. Normal RTE cells are highly sensitive to the growth inhibitory effects of TGF beta, particularly during early phases of logarithmic growth. At late logarithmic and plateau phases of proliferation, cultures of normal RTE cells secrete large amounts of TGF beta. That the endogenous TGF beta is exerting growth inhibitory effects can be demonstrated by adding TGF beta antisera to the cultures which causes a burst of proliferation. Many transformed RTE cell lines exhibit a markedly reduced sensitivity to the growth inhibitory effects of TGF beta. However, the cells remain responsive to regulation of ECM genes by TGF beta. The transformed cell lines examined secrete less than one tenth the amount of TGF beta of normal cells. Our studies show that the autocrine TGF beta growth restraining mechanism is inoperative in many of the RTE cell transformants. We conclude, therefore, that alterations in TGF alpha and TGF beta regulation of cell proliferation are important factors contributing to the abnormal growth behavior of transformed RTE cells.

Development of in vitro systems for chemoprevention research.

The C3H/10T1/2 mouse fibroblast cell line has been developed as a predictive model for cancer chemopreventive agents. The retinoids, which are currently being evaluated as chemopreventive agents in the clinic, are potent inhibitors or chemically induced neoplastic transformation in this cell line. Mechanistic studies suggest that retinoids stabilize chemically initiated cells and prevent their transformation by enhancing gap junctional communication between these cells and adjacent growth-inhibited normal 10T1/2 cells. Carotenoids also prevent chemically and physically induced neoplastic transformation of 10T1/2 cells. beta-Carotene is active without evidence of bioconversion to retinoids, implying that this dietary constituent has intrinsic chemopreventive activity. This cell culture system mimics many aspects of carcinogenesis in animals and man and appears well suited to mechanistic studies at the cellular and molecular level.

Beta-carotene and canthaxanthin inhibit chemically- and physically-induced neoplastic transformation in 10T1/2 cells.

We have studied the effects of beta-carotene (beta-C), a vitamin A precursor of plant origin, and canthaxanthin (CTX), a non-provitamin A carotenoid, on the neoplastic transformation of C3H/10T1/2 murine fibroblast cells. Chemical transformation in this well-characterized cell system has previously been shown to be reversibly inhibited by retinoids, compounds with vitamin A-like activity. Here we show that both beta-C and CTX inhibit 3-methylcholanthrene (MCA)-induced transformation with ED50s of 9 x 10(-7) M and 2 x 10(-7) M, respectively. Both carotenoids failed to inhibit X-ray-induced transformation when the cells were treated prior to and during irradiation. However, when the drugs were added 1 week after X-irradiation and maintained in the medium thereafter, as in the chemical transformation protocol, both carotenoids inhibited subsequent development of transformed foci in a dose-dependent manner. Again, CTX was more effective than beta-C, such that 3 x 10(-6) M completely inhibited radiogenically-induced foci. Similar to the previously described action of retinoids, the inhibition of MCA-induced transformation was reversible; upon removal of the drug, transformed foci developed within 2 weeks, indicating that the carotenoids were not specifically toxic to initiated cells. Although both carotenoids caused a small dose-dependent decrease in the growth rate of both parental and initiated 10T1/2 cells, they did not markedly affect colony size or number when the cells were treated as in the transformation assays, nor did they influence the expression of neoplasia of two transformed cell lines. Although the actions of beta-C and CTX are similar to those of retinoids in the 10T1/2 system, we suggest that the carotenoids act via a different mechanism, since CTX cannot be converted to active retinoids in mammalian cells, and there is no evidence that 10T1/2 cells can convert beta-C to vitamin A. We suggest that the carotenoids' lipid anti-oxidant properties may be responsible for their inhibitory actions on transformation.

Uptake and metabolism of beta-carotene and retinal by C3H/10T1/2 cells.

Both beta-carotene (beta-C), a vitamin A precursor, and vitamin A itself have been shown to reversibly inhibit neoplastic transformation in 10T1/2 cells during the progression phase of carcinogenesis. In order to determine whether the activity of beta-C in these cells may be attributed to conversion to vitamin A or is intrinsic to the carotenoid molecule, the uptake and metabolism of beta-C, and of retinal, the immediate product of dioxygenase-cleavage of beta-C, was studied in 10T1/2 cells. Cellular uptake of 2.6 nmol/10(6) cells occurred 24 h after treatment with 10(-5) M beta-C. Thereafter, cell levels remained relatively stable between 1 and 2 nmol/10(6) cells over the 1-week treatment period. Upon removal of beta-C from the medium, cellular levels decreased by approximately 80% in 2 weeks, then stabilized. Retinal was rapidly and quantitatively converted to retinol by 10T1/2 cells, suggesting that the inhibitory action of retinal on neoplastic transformation in these cells is due to its conversion to retinol, and that any enzymatic conversion of beta-C to retinal by these cells would be expected to result in retinol as the end product. Using [14C]beta-C, we found no evidence for formation of [14C]retinol, [14C]retinal or [14C]retinoic acid using sensitive HPLC. We therefore conclude that beta-C has intrinsic chemopreventive activity in 10T1/2 cells, perhaps due to its anti-oxidant properties.