MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor.

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.

Fluorescence spectroscopy of epithelial tissue throughout the dysplasia-carcinoma sequence in an animal model: spectroscopic changes precede morphologic changes.

BACKGROUND AND OBJECTIVE: The hamster cheek pouch carcinogenesis model, using chronic treatments of dimethylbenz[alpha]anthracene (DMBA) was used as a model system to investigate changes in epithelial tissue autofluorescence throughout the dysplasia-carcinoma sequence. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence emission spectra were measured weekly from 42 DMBA-treated animals and 20 control animals at 337, 380, and 460 nm excitation. A subset of data in which histopathology was available was used to develop diagnostic algorithms to separate neoplastic and non-neoplastic tissue. The change in fluorescence intensity over time was examined in all samples at excitation-emission wavelength pairs identified as diagnostically useful. RESULTS: Algorithms based on autofluorescence can separate neoplastic and non-neoplastic tissue with 95% sensitivity and 93% specificity. Greatest contributions to diagnostic algorithms are obtained at 380 nm excitation, and 430, 470, and 600 nm emission. Changes in fluorescence intensity are apparent as early as 3 weeks after initial treatment with DMBA, whereas morphologic changes associated with dysplasia occur on average at 7.5-12.5 weeks after initial treatment. CONCLUSIONS: Fluorescence spectroscopy provides a potential tool to identify biochemical changes associated with dysplasia and hyperplasia, which precede morphologic changes observed in histologically stained sections.

Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.

Genetic analysis of neoplasia induced by N-nitroso-N-methylurea in Xiphophorus hybrid fish.

Interspecific crosses within the genus Xiphophorus have historically been used to study the genetic aspects of melanoma formation. Melanomas typically occur as a result of deregulation of polymorphic, naturally occurring macromelanophore pigment patterns. Hybrid crosses also have been used to study the inducibility of melanoma by physical sources (such as UV light) and chemicals (such as N-methyl-N-nitrosourea, MNU). We previously defined a genomic region that is implicated in fish melanomagenesis and identified a candidate tumor suppressor gene (CDKN2X) within this genomic area. Highly significant associations between BC(1)-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC(1) hybrids are exposed to MNU as the tumor induction agent, a significant association between inheritance of CDKN2X alleles and tumor development is not observed. These data suggest there is mechanistic and genetic heterogeneity in melanomas derived from different etiologies within BC(1) hybrid fish.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Activators of peroxisome proliferator-activated receptor-alpha partially inhibit mouse skin tumor promotion.

Several recent reports have suggested that peroxisome proliferator-activated receptors (PPARs) may be involved in the development of neoplasias in different tissue types. The present study was undertaken to determine whether PPARs play a role in skin physiology and tumorigenesis. In an initiation-promotion study, SENCAR mice treated topically with the PPARalpha ligands conjugated linoleic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14643) exhibited an approximately 30% lower skin tumor yield compared with mice treated with vehicle. The PPARgamma and PPARdelta activators troglitazone and bezafibrate, respectively, exerted little, if any, inhibitory activity. PPARalpha was detected in normal and hyperplastic skin and in papillomas and carcinomas by immunohistochemistry. In addition, PPARalpha, PPARdelta/PPARbeta, and PPARgamma protein levels were analyzed by immunoblotting in normal epidermis and papillomas. Surprisingly, the levels of all three isoforms were increased significantly in tumors as opposed to normal epidermis. In primary keratinocyte cultures, protein levels of PPARalpha and, to a lesser extent, PPARgamma were markedly increased when the cells were induced to differentiate with high-calcium (0.12 mM) conditions. In addition, we observed that Wy-14643 enhanced transcriptional activity of a peroxisome proliferator-response element-driven promoter in a mouse keratinocyte cell line. These results demonstrate that keratinocytes express functional PPARalpha, that PPARalpha may play a role in differentiation, and that ligands for PPARalpha are moderately protective against skin tumor promotion. We conclude that selective PPARalpha ligands may exert their protective role against skin tumor promotion by ligand activation of 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.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: results from an animal model.

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

E2F1 has both oncogenic and tumor-suppressive properties in a transgenic model.

Using a transgenic mouse model expressing the E2F1 gene under the control of a keratin 5 (K5) promoter, we previously demonstrated that increased E2F1 activity can promote tumorigenesis by cooperating with either a v-Ha-ras transgene to induce benign skin papillomas or p53 deficiency to induce spontaneous skin carcinomas. We now report that as K5 E2F1 transgenic mice age, they are predisposed to develop spontaneous tumors in a variety of K5-expressing tissues, including the skin, vagina, forestomach, and odontogenic epithelium. On the other hand, K5 E2F1 transgenic mice are found to be resistant to skin tumor development following a two-stage carcinogenesis protocol. Additional experiments suggest that this tumor-suppressive effect of E2F1 occurs at the promotion stage and may involve the induction of apoptosis. These findings demonstrate that increased E2F1 activity can either promote or inhibit tumorigenesis, dependent upon the experimental context.

Deregulated expression of cell-cycle proteins during premalignant progression in SENCAR mouse skin.

It is now evident that several genes encoding regulatory activities that control the mammalian cell cycle, particularly some that control the progression of quiescent cells through G1 and into S phase, are targets for alterations that underlie the development of neoplasms. Here, we made a sequential study of alterations in cell cycle protein expression and complex formation among cyclin, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs) during premalignant progression in SENCAR mouse skin tumors. Changes in the level of expression were observed in positive (cyclin D1, D2, and E2F family members) and negative regulators (p16Ink4a, p57Kip2) of the cell cycle. Also, we observed the formation of cyclin/CDK/CKI complexes. The amounts of these proteins and complexes increased substantially at specific times during promotion but not during malignant conversion to carcinomas. These data show that deregulation of growth control occurs in benign tumors and that subsequent mutations not involved cell-cycle regulation are probably necessary to induce invasive behavior.

Suppression or elevation of cytosolic phospholipase A2 alters keratinocyte prostaglandin synthesis, growth, and apoptosis.

Prostaglandins and other arachidonic acid (AA) metabolites are synthesized by keratinocytes in response to tumor promoters and are produced at very high levels in tumors. After phorbol ester treatment, AA is hydrolyzed from keratinocytes primarily by the cytosolic form of phospholipase A2 (cPLA2), which exhibited a strong substrate preference for phosphatidylcholine over phosphatidylethanolamine and AA over other fatty acids. Phorbol esters increase cPLA2 activity but not the level of expression. To dissociate increased cPLA2 activity from other phorbol ester effects and thus determine the effects of altered AA release on cell growth, the murine keratinocyte cell line, HEL-30, was stably transfected with the sense or antisense cDNA for cPLA2. The resulting cell lines displayed corresponding over- or underexpression and up to 23-fold differences in cPLA2 activity between them. Phorbol ester caused a 15-fold difference in AA release between sense and antisense transfectants. Prostaglandin E2 levels correlated with AA release levels. The sense transfectants showed an enhanced proliferative capacity, based on increased cell number over time and [3H]thymidine incorporation. The antisense transfectants had significantly (>60%) reduced growth rates, compared with both parental cells and sense transfectants. The extent of apoptosis was determined in tumors from cell lines grown in graft chambers in vivo. The number of apoptotic cells was significantly greater in tumors from the sense transfectants, based on terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, compared with the parental or antisense lines. These data are in agreement with a recent study (M. C. Stern et al., Mol. Carcinog., 20: 137-142, 1997) showing a correlation between increased apoptosis and tumor progression in this model system. These results suggest that the elevated eicosanoid synthesis that is observed in skin carcinomas contributes to the growth and progression of these tumors.

Alterations of p53, cyclin D1, Rb, and H-ras in human oral carcinomas related to tobacco use.

BACKGROUND: Epidemiologic studies have indicated that environmental and personal habits, particularly tobacco use and alcohol abuse, are major etiologic factors in the induction and progression of head and neck squamous cell carcinomas (HNSCC). Molecular studies have focused on HNSCC related to smoking but not those associated with smokeless tobacco. METHODS: The authors studied immunohistochemical evidence of alterations of p53, cyclin D1, and Rb in 34 human oral carcinomas related to tobacco use. They also examined p53 and H-ras using single strand conformation polymorphism (SSCP) and sequencing analysis. RESULTS: Overexpression of cyclin D1 was found in 41% of cases, and accumulation of p53 was found in 59%. Only 9% of the samples did not show Rb staining. In SSCP and sequencing analysis, 17 cases showed mutations in the conserved region of the p53 gene. No mutations were detected in codons 12, 13, or 61 of the H-ras gene. CONCLUSIONS: Overexpression of cyclin D1 and p53 mutations are common alterations in HNSCC. In contrast, the loss of Rb function seems to occur infrequently, and mutations in the H-ras gene apparently do not play a role in this cancer. HNSCC associated with smokeless tobacco contained the same alterations as those related to smoking.

Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53.

The p16(INK4a)-cyclin D-retinoblastoma tumor suppressor pathway is disrupted in most human cancers, and it has been suggested that the subsequent release of E2F transcription factors from inhibitory complexes may be a key event in tumor development. We described recently the generation of transgenic mice with E2F1 gene expression targeted to squamous epithelial tissues by a keratin 5 (K5) promoter. In the present study, K5 E2F1 transgenic mice were crossed with p53 null mice to examine functional interactions between E2F1 and p53 in vivo. We find that E2F1-induced apoptosis of epidermal keratinocytes is reduced in K5 E2F1 transgenic mice lacking p53, whereas E2F1-induced hyperproliferation is unaffected by p53 status. We also find that K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin carcinomas, which normally are rare in p53-deficient mice. The timing of tumor development correlates with the level of E2F1 transgene expression and the status of p53. In primary transgenic keratinocytes, the major change in E2F1 DNA-binding activity is the generation of a complex also containing the retinoblastoma tumor suppressor protein. Nevertheless, the expression and associated kinase activity of cyclin E, a known target for E2F transcriptional activity, is elevated significantly in K5 E2F1 transgenic keratinocytes. These findings firmly establish that increased E2F1 expression can contribute to tumor development and suggest that p53 plays an important role in eliminating cells with deregulated E2F1 activity.

Comparison of the skin tumor-promoting potential of different organic peroxides in SENCAR mice.

The skin tumor-promoting activities of three organic peroxides were evaluated and compared to the activity of benzoyl peroxide, a well-characterized tumor promoter. Two of the compounds (di-t-butyl peroxide and dicumyl peroxide) were dialkyl peroxides and the other (di-m-chlorobenzoyl peroxide) was a diacyl peroxide. These compounds were selected based on a previous study in which we evaluated their capacity to induce epidermal hyperplasia, ornithine decarboxylase activity, and dark basal keratinocytes, which have been reliable short-term markers of tumor promotion. Dicumyl peroxide was a weak tumor promoter despite its high activity in inducing hyperplasia. Like benzoyl peroxide, di-m-chlorobenzoyl peroxide generally had intermediate activity as an inducer of short-term markers of tumor promotion and was a moderately effective tumor promoter. However, compared to benzoyl peroxide, di-m-chlorobenzoyl peroxide was more toxic to the skin, which may have limited its tumor-promoting activity. The final compound, di-t-butyl peroxide, which was essentially inactive in short-term assays, was also totally inactive in promoting papillomas or carcinomas in initiated skin. Tumor-promoting efficacy generally showed an inverse association with thermal stability for the compounds tested, suggesting that the rate of formation of free radicals is a key factor contributing to tumor promotion by organic peroxides. However, a number of other factors can potentially affect the activity of different organic peroxides as tumor promoters. Each compound evaluated had a different spectrum of activities, and these compounds should be useful for studying mechanisms of organic peroxide-induced tumor promotion.

Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter.

Transgenic mice were developed to explore the role of the erbB2 during epithelial homeostasis and tumorigenesis, through targeted expression of the neu oncogene (neu*). Expression of a neu* cDNA was targeted to the basal layer of skin epidermis as well as other epithelial tissues of transgenic mice via the bovine keratin 5 promoter. Two transgenic founders were obtained that were morphologically distinguishable from non-transgenic littermates by their visibly thickened skin and patchy hair growth by day 3 after birth. The presence of the transgene was confirmed by polymerase chain reaction analysis of tail DNA and immunofluorescence analysis of neu* protein in skin sections. Histological evaluation revealed significant hyperplasia of the follicular and interfollicular epidermis, the abnormal presence of horny material in the dermis and hypodermis, and a dramatic increase in epidermal proliferation. Many areas of the dermis involving this abnormal epithelial proliferation exhibited a squamous cell carcinoma-like appearance. In addition, there was unusual proliferation of the sebaceous glands. One founder died at day 14 and the other at day 20. The latter founder had two papillomas at the time of death. Additional phenotypic changes resulting from the expression of neu* in other tissues included hyperkeratosis in the forestomach and esophagus. In addition, there was a lack of distinction of the cortical-medullary boundaries and an increased rate of cell death in lymphocytes in the thymus. The phenotypic changes in these other tissues correlated with transgene expression. The data suggest that erbB2 signaling has an important role in epidermal proliferation. In addition, the data provide strong support for a role for erbB2 signaling during epidermal carcinogenesis in mouse skin.

Analysis of two inbred strains of mice derived from the SENCAR stock with different susceptibility to skin tumor progression.

The SENCAR stock of mice has proved to be a useful model in dissecting out the multistage nature as well as the critical mechanisms involved in skin tumorigenesis. This outbred stock was selectively bred to be susceptible to initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to obtain mice more suitable for genetic analyses of tumor susceptibility and tissue transplantation studies, several inbred lines of mice were derived from the SENCAR stock. One of these lines, the SSIN mice, has a higher susceptibility to tumor promotion compared to the SENCAR stock but is very resistant to tumor progression. On the other hand, the SENCAR B/Pt mice, derived also from the outbred stock, not only have a tumor promotion susceptibility almost identical to the SSIN mice, but they also have a high susceptibility to tumor progression. In order to understand the nature of the phenotypic differences between these two inbred lines we have characterized them using several parameters and markers that are associated with the progression of papillomas to squamous cell carcinoma (SCC). In this sense we analysed the tumor multiplicity and SCC incidence, and the expression of markers of progression and cell cycle related proteins in papillomas derived from both strains. Our results showed that while both strains have a similar papilloma multiplicity and incidence the SENCAR B/Pt mice have 67% incidence of SCC, compared to 0% in the SSIN. SENCAR B/Pt papillomas at 30 weeks of promotion have a higher and aberrant expression of K13, and loss of connexin 26. TGF-beta1 was found to be over-expressed in the suprabasal and superficial cells in the SENCAR B/Pt papillomas, while it was only expressed in the superficial cell layer in those derived from SSIN. The SENCAR B/Pt papillomas also showed an enlarged proliferative compartment with overexpression of cyclin D1 and PCNA as seen by immunohistochemistry and Western blot.

Angiogenesis is an early event in the development of chemically induced skin tumors.

In this study we have analyzed the vascular response induced in the two-stage carcinogenesis model in SENCAR mice. The role of angiogenesis has not been explored in this model, which is the paradigm of multistage carcinogenesis and a model for neoplastic lesions derived from exophytic premalignant lesions (e.g. colon carcinoma, bladder papilloma). We investigated if angiogenesis is involved in the formation of papillomas and in the progression from papilloma to carcinoma. To this end we analyzed the vasculature of normal and hyperplastic skin, focal epidermal hyperplasias that are precursors of papillomas, papillomas at different stages and squamous cell carcinomas. We also analyzed the vascularization of papillomas induced in two strains of mice that differ in their susceptibility to malignant progression. We show here that angiogenesis is turned on in the earliest stages of papilloma formation. In late stages, regardless of state of progression, the predominant response is an increase in the size of blood vessels. Thus, in the SENCAR mouse model, representative of exophytic tumors, the angiogenesis switch is a very early event, probably mechanistically related to the development of the primarily exophytic lesions. Therefore, the density of blood vessels cannot be used as a predictor of malignant progression in this model.

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.

Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice.

Transgenic animals were developed to assess the role of insulin-like growth factor 1 (IGF-1) in skin growth, differentiation and organization, as well as its importance in tumor formation. Expression of a human IGF-1 cDNA was targeted to the interfollicular epidermis of transgenic mice using a human keratin 1 promoter construct (HK1). Transgenic animals (HK1.IGF-1 mice) could be identified at birth by early ear unfolding and excessive ear and skin growth compared to non-transgenic littermates. Further examination of the skin from these mice showed epidermal hyperplasia and hyperkeratosis, marked thickening of the dermis and hypodermis, and early hair follicle generation in newborns. The severity of this phenotype correlated with transgene expression both of which subsided with age. Adult HK1.IGF-1 mice developed spontaneous tumors following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and exhibited an exaggerated epidermal proliferative response following treatment with the tumor promoter compared to non transgenic littermates. Additionally, HK1.IGF-1 transgenic mice developed papillomas faster and in markedly greater numbers compared to non-transgenic littermates in standard initiation-promotion experiments. The data presented suggest an important role for IGF-1 in the process of multistage carcinogenesis in mouse skin.