Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.

Prostaglandin E 2 (PGE 2 ) has been shown to promote the development of murine skin tumors. EP1 is 1 of the 4 PGE 2 G‐protein‐coupled membrane receptors expressed by murine keratinocytes. EP1 mRNA levels were increased ∼2‐fold after topical treatment with 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA) or exposure to ultraviolet (UV) light, as well as increased ∼3‐ to 12‐fold in tumors induced by 7,12‐dimethyl‐benz[ a ]anthracene (DMBA) initiation/TPA promotion or by UV exposure. To determine the effect of EP1 levels on tumor development, we generated BK5.EP1 transgenic mice that overexpress EP1 in the basal layer of the epidermis. Skins of these mice were histologically indistinguishable from wild type (WT) mice and had similar levels of proliferation after TPA treatment. Using a DMBA/TPA carcinogenesis protocol, BK5.EP1 mice had a reduced tumor multiplicity compared to WT mice, likely due to the observed down‐regulation of protein kinase C (PKC). However, the BK5.EP1 mice had an ∼8‐fold higher papilloma to carcinoma conversion rate. When DMBA/anthralin was used, BK5.EP1 mice produced more tumors than WT mice, as well as a ninefold increase in carcinomas, indicating that the tumor response is dependent on the type of tumor promoter agent used. Additionally, although almost undetectable in WT mice, cyclooxygenase‐2 (COX‐2) was expressed in the untreated epidermis of BK5.EP1 mice. While TPA highly induced COX‐2 in WT mice, COX‐2 expression in the BK5.EP1 mice did not change after TPA treatment; PGE 2 levels were likewise affected. These data indicate that EP1 is more important in tumor progression than in tumor promotion and that it indirectly regulates COX‐2 expression. © 2011 Wiley‐Liss, Inc.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5866 Chronically inflamed tissues are fertile environments for tumorigenesis. A major metabolite of cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2) is associated with changes in proliferation, angiogenesis, apoptosis, and immune function in inflammation-driven cancers. The BK5.COX-2 mouse is a model in which PGE2 overexpression leads to chronic pancreatitis and ultimately, pancreatic adenocarcinoma. Mice show signs of pancreatitis as early as 6 weeks of age, often becoming moribund by 6 months. We determined by Q-RT-PCR that COX-2 transgene-driven PGE2 production stimulates paracrine expression of endogenous COX-2, establishing a strongly inflammatory environment. Sequelae include acinar-ductal transdifferentiation, proliferation of ductal complexes, expansion of fibroinflammatory stroma, and influx of inflammatory cells. Levels of PGE2 as measured by ELISA are 15 fold higher in pretumor transgenic (TG) mice vs. wild-type (WT) mice (mean = 3623.25 (TG) vs 229 (WT) pg/mg protein); tumors in older transgenics show even greater increases (~120 fold vs. WT; mean = 27,466.2 pg/mg protein). Feeding BK5.COX-2 mice a diet containing the COX-2 selective inhibitor celecoxib lead to complete prevention of pancreatic disease, and verified that pathogenesis is prostaglandin driven. Other inflammatory mediators upregulated by PGE2 overexpression were identified using a TaqMan®Mouse Immune Panel. In young transgenics, elevated mRNAs were observed for IL-1β (TG 7X > WT), TNFα (TG 5X > WT), Csf-1 (TG 2.5X > WT) and a number of other markers, demonstrating early onset of immune dysregulation. These alterations persist, increasing with age and lesion progression. Inflammatory cells identified through histological and immunohistochemical means include granulocytes, macrophages, and mast cells, which appear early and persist as lesions progress; infiltration of B and T cells occurs in more advanced lesions/tumors. We explored the contribution of lymphoid cells to BK5.COX-2 tumor development by crossing transgenics with Rag1 null (KO) mice, lacking mature B and T cells. BK5.COX-2/Rag1KO mice develop more rapidly progressing pancreatic lesions, living an average of 13.9 weeks vs. 28.6 weeks for BK5.COX-2/Rag1WT. Although BK5.COX-2/Rag1KO tumors are not infiltrated by B and T lymphocytes, they have persistently high levels of myeloid lineage cells. These data show that the presence of lymphoid cells in BK5.COX-2/Rag1WT lesions is not required for progression, and suggests that these cells are perhaps limiting the inflammatory response established and promoted by myeloid cells. Future experiments will continue to explore interactions between immunocytes and chronically inflamed pancreatic epithelium, with the goal of assessing the impact on tumor development. Supported by T32 ES07247 from NIEHS, R25CA57730 from NCI, and CA100140 from NIH.

1504 Background: Pancreatic cancer is the 4th leading cause of cancer death in both men and women in the U.S. Increased obesity has emerged as a risk factor for pancreatic cancer. Calorie restriction (CR), a dietary strategy that prevents or reverses obesity, has significant anti-cancer effects in a variety of tumor types and in both spontaneous and chemically-induced tumors. We have found in the K5.COX-2 transgenic mouse model of pancreatitis-driven pancreatic cancer that CR significantly protects from spontaneous pancreatic lesion formation as compared to the overweight and obese groups. Also, CR mice had significantly reduced serum IGF-1 levels and pro-inflammatory cytokine levels as compared to ad libitum fed (AL) and high fat (HF) fed mice. We hypothesized that decreased serum IGF-1 levels were responsible for the decreased tumor burden. Methods: Orthotopic injections were performed on 6–8 week old Liver-specific IGF-1- Deficient (LID) and control mice (FVB/N and floxed IGF-1) with pancreatic tumor cells (JC101) derived from a transgenic K5.COX-2 animal. Tumors and pancreata were harvested and weighed 28 days after tumor injection. Results: LID mice exhibited significantly reduced tumor burden (0.23±0.04) than either the floxed IGF-1 control (0.71±0.10) or FVB/N wild-type animals (0.71±0.04) following 28 days of growth. In addition to a reduction in serum IGF-1 in the LID mice, we also found a substantial decrease in serum levels of a panel of pro-inflammatory cytokines such as IFN-g, IL-1b, IL-4, IL-5, IL-10, IL-12, and TNF-a as compared to sera of control animals. Furthermore, we saw reduced proliferation and microvessel density in the tumor tissue of LID animals compared to controls by immunohistochemical staining. Conclusions: Using a model of pancreatitis-induced or orthotopically injected pancreatic tumorigenesis, our findings indicate that either transgenic manipulation or diet modulation of serum IGF-1 can alter the development of pancreatic cancer. Moreover, our findings suggest a strong link between expression of IGF-1 and pro-inflammatory cytokines in response to CR and the prevention of tumor growth. This could have direct implications for the prevention and control of pancreatic tumors due to chronic inflammation and/or obesity. No significant financial relationships to disclose.

Cyclooxygenase-2 (COX-2) overexpression is an established factor linking chronic inflammation with metaplastic and neoplastic change in various tissues. We generated transgenic mice (BK5.COX-2) in which elevation of COX-2 and its effectors trigger a metaplasia-dysplasia sequence in exocrine pancreas. Histologic evaluation revealed a chronic pancreatitis-like state characterized by acinar-to-ductal metaplasia and a well-vascularized fibroinflammatory stroma that develops by 3 months. By 6 to 8 months, strongly dysplastic features suggestive of pancreatic ductal adenocarcinoma emerge in the metaplastic ducts. Increased proliferation, cellular atypia, and loss of normal cell/tissue organization are typical features in transgenic pancreata. Alterations in biomarkers associated with human inflammatory and neoplastic pancreatic disease were detected using immunohistochemistry. The abnormal pancreatic phenotype can be completely prevented by maintaining mice on a diet containing celecoxib, a well-characterized COX-2 inhibitor. Despite the high degree of atypia, only limited evidence of invasion to adjacent tissues was observed, with no evidence of distant metastases. However, cell lines derived from spontaneous lesions are aggressively tumorigenic when injected into syngeneic or nude mice. The progressive nature of the metaplastic/dysplastic changes observed in this model make it a valuable tool for examining the transition from chronic inflammation to neoplasia.

Abstract Pancreatic cancer is the fourth leading cause of cancer death in both men and women in the United States. Obesity, which has increased dramatically over the past 40 years, has emerged as an important risk factor for pancreatic cancer, although the mechanisms underlying the obesity-pancreatic cancer association have not been identified. Calorie restriction (CR), a dietary strategy for inducing negative energy balance and preventing or reversing obesity, has significant anti-cancer effects in several species, for a variety of tumor types, and for both spontaneous and chemically-induced tumors. However, the effects of dietary energy balance modulation on pancreatic tumor formation have not been well studied. We hypothesized that spontaneous tumor development in the K5.COX-2 transgenic mouse model of pancreatitis-driven pancreatic cancer would be reduced in lean mice, relative to overweight or diet-induced obese (DIO) mice. In order to test this hypothesis, we placed 6-8 week old K5.COX-2 transgenic mice on one of three diets for 14 weeks (n=12/group): lean (30% CR), overweight (AIN-76A diet fed ad libitum), or a high calorie/high fat DIO regimen. K5.COX-2 transgenic mice develop spontaneous pancreatic lesions as early as 4 weeks with 100% becoming moribund within 6-8 months. Based on histologic analysis of the percentage of pancreas that is comprised of enlarged and dilated ductal lesions, the CR animals were significantly protected from spontaneous pancreatic lesion formation (7.5%±1.44) as compared to the overweight (45%±10.5) and DIO (57.5%±9.46) groups. Furthermore, CR mice had smaller lesions at time of harvest with none exhibiting signs of moribundity. Conversely, the DIO group generally had the largest lesions, although there was no statistical difference between the overweight and DIO groups. In addition, several mice in the overweight and DIO groups had to be sacrificed early due to extensive abdominal distention, morbidity from enlarged cystic lesions, and other signs of distress. Further investigation is needed to discern how CR exerts its effects. Preliminary analyses suggest that IGF-1 may be playing a significant role. Moreover, pro-inflammatory cytokines were shown to be significantly reduced in sera of CR-animals as compared to the other two groups. In conclusion, using a model of pancreatitis-induced pancreatic tumorigenesis, our findings indicate that dietary energy balance modulation can alter the development of pancreatic cancer. This could have direct implications for the prevention and control of pancreatic tumors due to chronic inflammation and/or obesity.

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‐1α, and tumor necrosis factor‐α 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. © 2007 Wiley‐Liss, Inc.

5664 Cyclooxygenase (COX) enzymes catalyze the rate-limiting step in the conversion of arachidonic acid to prostaglandins. While COX-1 is constitutively expressed in most tissues and is involved in tissue homeostasis, COX-2 is highly inducible, is frequently overexpressed in cancers, and plays roles in inflammation, immune function, angiogenesis, and tumor invasiveness. Both general COX inhibitors, as well as COX-2-specific inhibitors, have been shown to inhibit carcinogenesis in a number of animal models. However, COX inhibitors have been shown to have COX-independent effects. So we have used both COX-2 transgenic and COX-2 knockout mice to determine the role of COX-2 in carcinogenesis using the mouse skin model. FVB mice overexpressing COX-2 in the skin via the keratin 14 (K14) promoter were initially shown to be resistant to skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. However, using DMBA only or using the nonphorbol ester tumor promoter, anthralin, with DMBA, K14.COX2 transgenic mice developed many more tumors (3.7 and 5.5 fold, respectively) than wild type FVB mice. Thus, overexpression of COX-2 appears to promote skin tumorigenesis and resistance to tumorigenesis is limited specifically to TPA tumor promotion, which correlated to a reduced TPA induction of proliferation, ornithine decarboxylase, interleukin-1α, and tumor necrosis factor-α expression in transgenic versus wild type mice. Similarly, when SKH-1 hairless mice overexpressing COX-2 in the skin via the keratin 5 (K5) promoter were subjected to ultraviolet (UV) light-induced tumorigenesis, transgenic mice developed 2.5 fold more tumors/mouse than wild type SKH-1 mice. To complement these studies with transgenic overexpressing mice, COX-1 and COX-2 knockout mice were backcrossed onto the SKH-1 background for UV carcinogenesis studies (others have already shown that COX-1 and COX-2 knockout mice are resistant to DMBA/TPA skin tumorigenesis; Cancer Res., 62:3395, 2002). However, since homozygous COX-2 -/- mice do not survive beyond ~6 weeks of age on the SKH-1 background, heterozygous COX-1 +/- and COX-2 +/- mice were used. While COX-1 +/- mice developed UV-induced tumors similarly to wild type SKH-1 mice, COX-2 +/- mice developed 3 fold fewer tumors/mouse with a 10-week increase in latency compared to wild type mice. Thus, loss of only one allele of COX-2 was enough to decrease tumorigenesis in this system. Overall, mouse skin COX-2 expression levels were directly correlated to susceptibility to tumorigenesis. Supported by NIH grants CA100140, ES07784.

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) E 2 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. © 2007 Wiley‐Liss, Inc.

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.

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.

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. © 2002 Wiley‐Liss, Inc.

Ultraviolet radiation of mouse skin leads to epidermal hyperplasia, inflammation, and subsequent tumor development. In this study we determined to what extent the cell cycle machinery is altered during epidermal proliferation after ultraviolet B radiation. A minimal erythema dose, 90 mJ per cm(2), increased the protein expression of the G1 phase cyclins, cyclin DI and E, by 12 h. The majority of epidermal cells entered S phase between 18 and 24 h as determined by 5'-bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen, and cyclin A immunohistochemistry. An increase in cyclin-dependent kinase 2 (cdk-2) protein expression occurred after 12 h, but no changes in cdk-4 or cdk-6 protein levels were observed. The increase in cyclin DI, E, and A protein expression was associated with an increase in cyclin D1-cdk-4, cyclin E-cdk-2, and cyclin A-cdk-2 complex formation. p53 protein expression was elevated through 48 h, and the cdk inhibitor protein p21(Cip1/WAF1) was elevated 6-fold to 7.5-fold between 12 and 24 h. The elevated p21(Cip1/WAF1) protein contributed to an enhanced association with cdk-2 and cdk-4 at 3-24 h and 6-24 h post-ultraviolet B irradiation, respectively. These data indicate that 90 mJ per cm 2 of ultraviolet B irradiation induces a DNA damage response, by increasing p53 and p21(Cip1/WAF1) protein expression, but also induces a rapid and sustained increase in S phase by 18 h.

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 car-: cinogenesis. 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 threefold 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 rime-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. (C) 1997 Wiley-Liss, Inc.

Ear/footpad swelling following sensitization and challenge with 2,4-dinitrofluorobenzene (DNFB)/allogenic splenocytes (AS) were used to monitor the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on contact hypersensitivity (CHS) and delayed hypersensitivity (DHS) reactions, respectively. Topical treatment of dorsal or ventral SENCAR mouse skin 4x with 2 micrograms of TPA prior to sensitization of dorsal skin with DNFB suppressed attempts to induce CHS by subsequent challenge with DNFB. The adoptive transfer of splenocytes isolated from mice pretreated on the dorsum with TPA prior to dorsal sensitization with DNFB inhibited the development of CHS to DNFB in recipient mice. Conversely, topical treatment with TPA prior to s.c. sensitization with AS neither suppressed subsequent attempts to induce DHS, nor resulted in the generation of a splenocyte population capable of suppressing DHS reactions in adoptive transfer studies. Thus, promoting doses of topically applied TPA has differential effects on CHS and DHS reactions.

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c‐ jun and c‐ fos , and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD‐1 mice and from discarded adult human skin were grown in primary culture in a high‐calcium serum‐free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKCα, PKCβ, and PKCδ; no PKCγ, PKCϵ, or PKCζ were detected. In fresh MEKs, PKCα, PKCβ, PKCΔ, and PKCζ were observed, but not PKCγ or PKCζ. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKCγ was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA‐induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c‐ jun and c‐ fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c‐ jun , c‐ fos , and ODC mRNA in HEKs. However, c‐ jun and c‐ fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.

12-O-tetradecanoylphorbol-13-acetate (TPA) and its analogs were surveyed for their abilities to modify contact hypersensitivity (CHS) responses in SENCAR mice. Sensitization of dorsal skin with 2,4-dinitrofluorobenzene (DNFB) and subsequent challenge of the ear 5 d later resulted within 24 h in ear swelling and increased vascular permeability (as measured by the extravasation of Evans Blue dye). Treatment of dorsal or ventral skin with TPA 4 times (application made every 3 or 4 d) prior to sensitization on the dorsum inhibited subsequent induction of CHS by DNFB challenge. Maximum suppression of CHS required sensitization at the site of TPA treatment. Suppression occurred over a narrow dose range of TPA (0.1-1.0-mu-g), and qualitatively correlated with the tumor incidences scored in an initiation-promotion multistage skin carcinogenesis experiment. Multiple applications (4 x) of the promoters phorbol-12,13-dibenzoate (10-mu-g) and mezerein (2-mu-g) also suppressed CHS, whereas the non-promoter phorbol (20-mu-g) and the first stage tumor promoter 4-O-methyl TPA (20-mu-g) had no effect. Adoptive transfer of splenocytes isolated from mice pre-treated with TPA prior to DNFB sensitization inhibited the development of CHS in recipient mice that were sensitized and challenged with DNFB, but not oxazolone. Splenocyte preparations depleted of T lymphocytes prior to transfer could not suppress CHS in recipient mice. Conversely, suppressive activity was concentrated in splenocyte preparations depleted of adherent cells/monocytes. Collectively, these studies demonstrate that TPA treatment of murine epidermis prior to sensitization with hapten can inhibit subsequent hapten-dependent elicitation of CHS. This suppression is mediated in part by antigen-specific suppressor T cells. Furthermore, there is a qualitative correlation between the complete and second stage in vivo tumor-promoting activities of TPA and its analogs, and their abilities to inhibit CHS.