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Background: RNA-Seq is currently the most widely used tool to analyze whole-transcriptome profiles. There are numerous commercial kits available to facilitate preparing RNA-Seq libraries; however, it is still not clear how some of these kits perform in terms of: 1) ribosomal RNA removal; 2) read coverage or recovery of exonic vs. intronic sequences; 3) identification of differentially expressed genes (DEGs); and 4) detection of long non-coding RNA (lncRNA). In RNA-Seq analysis, understanding the strengths and limitations of commonly used RNA-Seq library preparation protocols is important, as this technology remains costly and time-consuming., Results: In this study, we present a comprehensive evaluation of four RNA-Seq kits. We used three standard input protocols: Illumina TruSeq Stranded Total RNA and mRNA kits, a modified NuGEN Ovation v2 kit, and the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5' and 3' end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes., Conclusions: At the manufacturers' recommended input RNA levels, all the RNA-Seq library preparation protocols evaluated were suitable for distinguishing between experimental groups, and the TruSeq Stranded mRNA kit was universally applicable to studies focusing on protein-coding gene profiles. The TruSeq protocols tended to capture genes with higher expression and GC content, whereas the modified NuGEN protocol tended to capture longer genes. The SMARTer Ultra Low RNA Kit may be a good choice at the low RNA input level, although it was inferior to the TruSeq mRNA kit at standard input level in terms of rRNA removal, exonic mapping rates and recovered DEGs. Therefore, the choice of RNA-Seq library preparation kit can profoundly affect data outcomes. Consequently, it is a pivotal parameter to consider when designing an RNA-Seq experiment.

The Slug transcription factor plays an important role in ultraviolet radiation (UVR)-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT) occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2) pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.

To determine whether the EP4 receptor for prostaglandin E2 (PGE2) contributes to the tumor promoting activity of PGs in murine skin, EP4 over-expressing mice (BK5.EP4) were generated and subjected carcinogenesis protocols. An initiation/promotion protocol resulted in 25-fold more squamous cell carcinomas (SCCs) in the BK5.EP4 mice than wild type (WT) mice. An increase in SCCs also occurred following treatment with initiator alone or UV irradiation. The initiator dimethylbenz[a]anthracene caused cytotoxicity in BK5.EP4, but not WT mice, characterized by sloughing of the interfollicular epidermis, regeneration and subsequent SCC development. A comparison of transcriptomes between BK5.EP4 and WT mice treated with PGE2 showed a significant upregulation of a number of genes known to be associated with tumor development, supporting a pro-tumorigenic role for the EP4 receptor., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The ultraviolet B (UVB) component of sunlight, which causes DNA damage and inflammation, is the major cause of nonmelanoma skin cancer (NMSC), the most prevalent of all cancers. Nonsteroidal anti-inflammatory drugs (NSAID) and coxibs have been shown to be effective chemoprevention agents in multiple preclinical trials, including NMSC, colon, and urinary bladder cancer. NSAIDs, however, cause gastrointestinal irritation, which led to the recent development of nitric oxide (NO) derivatives that may partially ameliorate this toxicity. This study compared the efficacy of several NSAIDs and NO-NSAIDs on UV-induced NMSC in SKH-1 hairless mice and determined whether various short-term biomarkers were predictive of long-term tumor outcome with these agents. Naproxen at 100 (P = 0.05) and 400 ppm (P < 0.01) in the diet reduced tumor multiplicity by 26% and 63%, respectively. The NO-naproxen at slightly lower molar doses shows similar activities. Aspirin at 60 or 750 ppm in the diet reduced tumor multiplicity by 19% and 50%, whereas the equivalent doses (108 and 1,350 ppm) were slightly less effective. Sulindac at 25 and 150 ppm in the diet, doses far below the human equivalent dose was the most potent NSAID with reductions of 50% and 94%, respectively. In testing short-term biomarkers, we found that agents that reduce UV-induced prostaglandin E2 synthesis and/or inhibit UV-induced keratinocyte proliferation yielded long-term tumor efficacy., (©2013 AACR.)

Studies show that elevated insulin-like growth factor-1 (IGF-1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the pro-tumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA-induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE(2) levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE(2) showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE(2) /EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE(2) levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE(2) /EP3 signaling pathway is involved in IGF-1-stimulated mammary tumorigenesis and that COX-2-selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans. © 2011 Wiley Periodicals, Inc., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

High levels of prostaglandin E2 (PGE2) synthesis resulting from the up-regulation of cyclooxygenase (COX)-2 has been shown to be critical for the development of non-melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G-protein coupled membrane receptors, EP1-4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCCs) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity, and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX-2 levels in the epidermis and SCC could be completely prevented in DMBA-treated BK5.EP1 mice either by feeding the selective COX-2 inhibitor celecoxib in their diet or by crossing them onto a COX-2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX-2., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

Background: A number of studies have shown that hydroxyethyl starch (HES) solutions are able to down-regulate the expression of inflammatory mediators and inhibit neutrophil-mediated tissue injuries when they are used in patients with sepsis or other diseases with severe inflammatory responses. However, our knowledge about the underlying mechanisms is limited. Toll-like receptor 4 (TLR4) signaling has a pivotal role in inflammatory processes. In this study, we examined the possible involvement of TLR4 signaling in the antiinflammatory effects of HES., Methods: Male Sprague-Dawley rats were exposed to lipopolysaccharide (LPS) (10 mg/kg, IV) and received IV saline (30 mL/kg) or HES 130/0.4 (15 or 30 mL/kg). Six hours after LPS challenge, rats were killed and their lungs harvested. Lung injury was examined by hematoxylin and eosin staining. TLR4 mRNA expression, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 MAPK activation, and activator protein 1 (AP-1) activity in the lungs were detected with quantitative polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay, respectively., Results: Compared with saline, HES profoundly attenuated the histological changes induced by LPS in the lungs at both dose levels. Molecular analysis showed that both 15 and 30 mL/kg HES significantly decreased TLR4 mRNA levels and inhibited activation of p38 MAPK and AP-1 in rats challenged with LPS, whereas activation of extracellular signal-regulated kinases 1/2 MAPK was not affected by either dose of HES., Conclusions: These findings indicate that the beneficial effects of HES 130/0.4 on inflammation are mediated at least in part by inhibiting the TLR4/p38 MAPK/AP-1 pathway in lungs from rats challenged with LPS.

Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-a and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.

Estrogen receptor alpha (ER) and p53 are critical prognostic indicators in breast cancer. Loss of functional p53 is correlated with poor prognosis, ER negativity, and resistance to antiestrogen treatment. Previously, we found that p53 genotype was correlated with ER expression and response to tamoxifen in mammary tumors arising in mouse mammary tumor virus-Wnt-1 transgenic mice. These results lead us to hypothesize that p53 may regulate ER expression. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated a 5-fold increase in p53 expression. ER expression was also increased 4-fold over a 24-h time frame. In cells treated with small interfering RNA (siRNA) targeting p53, expression of both p53 and ER was significantly reduced (>60%) by 24 h. Induction of ER by DNA-damaging agents was p53 dependent as either ionizing radiation or doxorubicin failed to up-regulate ER after treatment with p53-targeting siRNA. To further investigate whether p53 directly regulates transcription of the ER gene promoter, MCF-7 cells were transiently transfected with a wild-type (WT) p53 expression vector along with a luciferase reporter containing the proximal promoter of ER. In cells transfected with WT p53, transcription from the ER promoter was increased 8-fold. Chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun, and Sp1 and that this multifactor complex was formed in a p53-dependent manner. These data show that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer.

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

Although prostaglandin E2 (PGE2) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study, we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly regulated kinase 1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-kappa B, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse keratinocytes. Similarly, in vivo, we found that WT mice treated with PGE2 and untreated cyclooxygenase-2-overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear factor-kappa B, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-2 overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE2 in mouse skin.

Besides induction of DNA damage and p53 mutations, chronic exposure to UV irradiation leads to the constitutive up-regulation of cyclo-oxygenase-2 (COX-2) expression and to increased production of its primary product in skin, prostaglandin E2 (PGE2). COX-2 has also been shown to be constitutively overexpressed in mouse, as well as human, UV-induced skin cancers and premalignant lesions. UV exposure results in ligand-independent activation of the epidermal growth factor receptor and subsequent activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways leading to transcriptional activation of the COX-2 gene. Use of COX-2-specific inhibitors and genetic manipulation of COX-2 expression have demonstrated that UV induction of COX-2 in the skin contributes to the induction of epidermal hyperplasia, edema, inflammation, and counters the induction of apoptosis after UV exposure. Likewise, inhibition of COX-2 activity or reduced expression in COX-2 knockout mice resulted in significantly reduced UV-induced tumorigenesis, while overexpression of COX-2 in transgenic mice enhanced UV-induced tumor development. A combination of signaling from the PGE2 EP1, EP2 and/or EP4 receptors mediates the effects of COX-2 overexpression. These studies demonstrate the crucial role of COX-2 in the development of UV-related nonmelanoma skin cancers.

The up-regulation of the inducible form of cyclooxygenase (COX-2), a central enzyme in the prostaglandin (PG) biosynthetic pathway, occurs in many epithelial tumors and has been associated with tumor cell proliferation and angiogenesis. To better understand the role of COX-2 in skin tumor development, we generated transgenic mice that overexpress COX-2 under the control of the keratin 14 promoter. We previously reported (Cancer Res. 62: 2516, 2002) that these mice, referred to as keratin 14 (K14).COX2 mice, were unexpectedly very resistant to 12-O-tetradecanoylphorbol 13-acetate (TPA) tumor promotion. The current studies were undertaken to determine the mechanism of this resistance and determine if it was restricted to TPA promotion. Transgenic and wild-type mice were subjected to a complete carcinogenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) only, as well as a two-stage protocol using DMBA plus an unrelated tumor promoter, anthralin. In addition, the responses of transgenic and wild-type mice to TPA in terms of induction of proliferation and various down-stream mediators were examined. The TPA resistance phenotype correlated with a reduced ability to induce ornithine decarboxylase, interleukin-1alpha, and tumor necrosis factor-alpha and a reduced proliferation response. This resistance phenotype appears to be restricted to phorbol ester promotion because K14.COX2 mice developed six times more tumors than wild-type mice when anthralin was used as the tumor promoter. Additionally, K14.COX2 mice treated only with DMBA developed approximately 3.5 times more tumors than wild-type mice, suggesting that PGs have intrinsic tumor promoting activity. We conclude that the role of PGs in skin tumorigenesis is context dependent., (Copyright 2007 Wiley-Liss, Inc.)

Nonmelanoma skin cancer is the most prevalent cancer in the United States and its incidence is on the rise. These cancers generally arise on sun-exposed areas of the body and the ultraviolet (UV) B spectrum of sunlight has been clearly identified as the major carcinogen responsible for skin cancer development. Besides inducing DNA damage directly, UV exposure of the skin induces the expression of the enzyme cyclooxygenase-2 (COX-2), which catalyzes the first step in the conversion of arachidonic acid to prostaglandins, the primary product in skin being prostaglandin E(2) (PGE(2)). COX-2 has been shown to be overexpressed in premalignant lesions as well as in nonmelanoma skin cancers in both humans and mice chronically exposed to UV. Through the use of COX-2-selective inhibitors and COX-2 knockout mice, it has been shown that UV-induced COX-2 expression plays a major role in UV-induced PGE(2) production, inflammation, edema, keratinocyte proliferation, epidermal hyperplasia, and generation of a pro-oxidant state leading to oxidative DNA damage. Chronic exposure to UV leads to chronic up-regulation of COX-2 expression and chronic inflammation along with the accumulation of DNA damage and mutations, all of which combine to induce malignant changes in epidermal keratinocytes and skin cancers. Both inhibition of COX-2 activity and reduction in COX-2 expression by genetic manipulations significantly reduce, while overexpression of COX-2 in transgenic mice significantly increases UV-induced skin carcinogenesis. Together these studies demonstrate that COX-2 expression/activity is critical to the development of UV-related nonmelanoma skin cancers.

While it has been established that both the constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2, respectively) play important roles in chemical initiation-promotion protocols with phorbol ester tumor promoters, the contribution of these two enzymes to ultraviolet (UV) light-induced skin tumors has not been fully assessed. To better understand the contribution of COX-1 and COX-2 to UV carcinogenesis, we transferred the null allele for each isoform onto the SKH-1 hairless strain of mouse. Due to low viability on this background with complete knockout of COX-2, heterozygous mice were used in UV carcinogenesis experiments. While the lack of one allele of COX-1 had no effect on tumor outcome, the lack of one allele of COX-2 resulted in a 50-65% reduction in tumor multiplicity and a marked decrease in tumor size. Additionally, transgenic SKH-1 mice that overexpress COX-2 under the control of a keratin 14 promoter developed 70% more tumors than wild-type SKH-1 mice. The lack of one allele of either COX-1 or COX-2 reduced prostaglandin (PG) E2 levels in response to a single UV treatment. The proliferative response to UV was significantly reduced in COX-2, but not COX-1, heterozygous mice. UV-induced apoptosis, however, was greater in COX-2 heterozygous mice. Collectively, these results clearly establish the requirement for COX-2 in the development of skin tumors., (Copyright (c) 2007 Wiley-Liss, Inc.)

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.

The levels of 8S-lipoxygenase (8S-LOX) expression and of its arachidonic acid metabolite, 8-hydroxyeicosatetraenoic acid (8-HETE), are highly elevated in the early stages of mouse skin carcinogenesis. On the other hand, several reports showing that 8-HETE is also closely associated with keratinocyte differentiation raise a question concerning the role of 8S-LOX/8-HETE in skin carcinogenesis. To address that question, here we conducted a series of gain-of-function studies. Skin targeted loricrin 8S-LOX/C57BL/6J transgenic mice showed a more differentiated epidermal phenotype as well as a 64% reduced papilloma development in a two-stage skin carcinogenesis protocol. Forced expression of 8S-LOX in MT1/2 cells, a murine papilloma cell line, also caused a more differentiated appearance as well as keratin 1 expression. Overexpression of 8S-LOX in CH72 cells, a murine carcinoma cell line, inhibited cell proliferation by 30% in vitro and by 86% in in vivo xenografts. Exogenous addition of 5 muM 8-HETE to CH72 cells caused cell cycle arrest at the G1 phase. Finally, immunohistochemical analyses showed 8S-LOX protein expression was strictly confined to the differentiated compartment of mouse skin and throughout tumorigenesis. Collectively, these data suggest that 8S-LOX plays a role as a prodifferentiating, antitumorigenic, and tumor suppressing gene in mouse skin carcinogenesis.

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

Interleukin-1 receptor antagonist (IL-1Ra) is involved in many processes, including epidermal inflammation and hyperplasia after irritation or injury. However, the mechanism by which intracellular IL-1Ra (icIL-1Ra) expression is regulated in mouse keratinocytes has not been reported. We found that the CH72 mouse carcinoma cell line constitutively expresses the icIL-1Ra mRNA. To study the transcriptional factors responsible for the constitutive expression of icIL-1Ra, we functionally characterized 4.5 kb of the 5' flanking region of the human icIL-1Ra gene in these cells. We first demonstrated that icIL-1Ra expression in these cells was regulated at the level of transcription. Deletion analysis of the promoter showed that regulatory elements for constitutive expression were located -158 to -49 bp upstream of the transcription start site for icIL-1Ra. We investigated the cis- and trans-acting factors required for icIL-1Ra expression. An activating protein-1 (AP-1) site was identified as the positive regulatory element necessary for the constitutive expression of the icIL-1Ra promoter in CH72 cells. Moreover, electrophoretic mobility shift assay and cotransfection experiments showed that c-jun and c-fos proteins bound to the AP-1 site and functionally transactivated the icIL-1Ra promoter in mouse carcinoma CH72 cells., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.