transforming-growth-factor-alpha and Papilloma

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production of TGF alpha (Ellem et al. 1988, Pittelkow et al. 1989, Choi et al. 1991, Imamoto et al. 1991, J. DiGiovanni unpublished studies).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

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Disease Progression; ErbB Receptors; Mice; Models, Biological; Papilloma; Phorbol Esters; Precancerous Conditions; Skin Neoplasms; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF alpha) is one of a group of structurally-related growth factors (the epidermal growth factor family of ligands) that interact with one or other members of the epidermal growth factor family of protein tyrosine kinase receptors (EGF-R's). A number of excellent reviews detailing our knowledge of this area have been recently published (Carpenter and Wahl, 1991; Derynck, 1992; Prigent and Lemoine, 1992). Rather than add to their number, this review focuses on new insights into the importance of TGF alpha and signaling through the EGF receptor considered in the context of the laboratory mouse. The new information has emerged from analysis of mutant mice generated either by classical gene targeting in embryonic stem (ES) cells or by accidents of nature. In addition to their intrinsic interest, these mice are proving invaluable in determining the importance of EGF receptor signaling in wound healing and as a contributing factor in the conversion of a normal cell into its tumorigenic counterpart.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; Cocarcinogenesis; Epidermal Growth Factor; ErbB Receptors; Gene Targeting; Hair; Mice; Mice, Knockout; Mice, Mutant Strains; Multigene Family; Mutagenesis, Insertional; Papilloma; Recombination, Genetic; Signal Transduction; Skin Neoplasms; Transforming Growth Factor alpha; Vibrissae; Wound Healing

To measure HPV status, epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha) expression and Ki-67 index in exophytic papilloma (EP), inverted papilloma (IP) with dysplasia, IP with carcinoma and invasive squamous cell carcinoma (SCC).. Forty-four patients with sinonasal papilloma and invasive SCC were selected. The nasal tissues were stained with monoclonal antibodies to EGFR, TGF-alpha and Ki-67. The results were analysed using quantitative immunohistochemical analysis. In situ hybridization studies for HPV DNA for 6/11, 16/18 and 31/33 were also performed on the tissue.. Significant increase of EGFR and TGF-alpha was observed in IP with severe dysplasia, IP with carcinoma and invasive SCC compared to IP with mild dysplasia and control nasal mucosa. And a serial upreguration in terms of Ki-67 index in IP with dysplasia was observed. Among IP, HPV 6/11-positive was present in 42% tumour and HPV 16/18-positive was present in 31% of tumours. Among HPV 6/11 and 16/18-positive IP, significant increase of EGFR and Ki-67 index were observed.. Pre-cancerous lesions of IP exhibited elevated levels of EGFR and TGF-alpha and these expression may be associated with early events in IP carcinogenesis. HPV infection may be an early event in a multistep process of malignant formation of IP.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; ErbB Receptors; Female; Humans; In Situ Hybridization; Ki-67 Antigen; Male; Middle Aged; Nasal Mucosa; Neoplasm Invasiveness; Nose Neoplasms; Papilloma; Papilloma, Inverted; Papillomaviridae; Paranasal Sinus Neoplasms; Precancerous Conditions; Transforming Growth Factor alpha

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was originally considered to have activity against malignant disease. However, recent studies suggest TNF-alpha may also act as an endogenous tumor promoter. In the present work, mice deficient in TNF-alpha either genetically (TNF-alpha(-/-)) or after blockade with a neutralizing antibody (cV1q) were used to investigate the role of TNF-alpha in skin tumor development. Papillomas were induced in wild-type (wt) mice after treatment of skin with the initiating agent 9,10-dimethyl-1,2-benzanthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 15 weeks. TNF-alpha(-/-) mice were resistant to papilloma development when compared with wt mice on C57Bl/6J, 129/SvEv, and BALB/c genetic backgrounds. Primary murine keratinocytes (newborn keratinocytes) and skin homogenates were used to characterize TPA-stimulated TNF-alpha expression. TPA induced TNF-alpha protein in newborn keratinocytes in vitro and epidermis in vivo. Neutralization of TNF-alpha protein with cV1q in vivo for 0-15 weeks of promotion significantly decreased skin tumor development after 9,10-dimethyl-1,2-benzanthracene/TPA treatment. cV1q treatment during the early stages of tumor promotion (0-6 weeks) was equally effective. These data suggest that early induction of TNF-alpha is critical for skin tumor promotion. cV1q also reduced TPA-stimulated expression of matrix metalloproteinase 9 and granulocyte macrophage colony-stimulating factor, proteins that are differentially regulated in wt and TNF-alpha(-/-) epidermis. Treatment of the 410.4 transplantable breast carcinoma with cV1q reduced tumor growth in vivo, illustrating that inhibition of tumor growth through neutralization of TNF-alpha is not limited to skin carcinogenesis. These results provide further evidence for procancer actions of TNF-alpha and give some rationale for use of TNF-alpha antagonists in cancer prevention and treatment.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antibodies, Monoclonal; Carcinogens; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immunity, Innate; Keratinocytes; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Staging; Papilloma; Rats; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

The enhanced proliferation of epithelial cells is a typical feature of respiratory papilloma. The mechanism or mechanisms leading to abnormal epithelial proliferation remain unclear. Overexpression of growth factors and their receptors and inactivation of tumor-suppressor proteins are known to cause cell transformation and proliferation. The objectives of this study were to evaluate the expression of these factors in juvenile respiratory papillomas with correlation to cellular proliferation activity, and to determine whether such expression is associated with the clinical course of the disease. The expression of transforming growth factor-alpha, epidermal growth factor receptor, p53 protein, retinoblastoma proteins and Ki-67 was quantified by immunohistochemistry in paraffin-embedded biopsy specimens taken at the initial surgical excision from children in whom respiratory papillomatosis was diagnosed. Clinical information regarding the number of disease sites, tracheobronchial spread, malignant transformation, and frequency of recurrences was reviewed. Thirty-five specimens were suitable for immunohistochemical evaluation. Ki-67 expression was significantly higher in patients with multiple sites of disease and frequent recurrences. High p53 expression was significantly associated with malignant transformation. We concluded that Ki-67 and p53 expression may be predictive of the clinical course in children with respiratory papillomatosis.

Topics: Adolescent; Biomarkers, Tumor; Cell Division; Cell Transformation, Neoplastic; Child; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Male; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Papilloma; Prognosis; Respiratory Mucosa; Respiratory Tract Neoplasms; Retinoblastoma Protein; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Previously, transgenic mice were generated that overexpressed v-Ha-ras or human transforming growth factor alpha (TGFalpha) exclusively in the epidermis, by means of a targeting vector based on the human keratin 1 gene (HK1). Both transgenics exhibited a similar neonatal phenotype of epidermal hyperplasia/hyperkeratosis and, in adults, spontaneous and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papilloma formation. To assess the synergism in vivo between Ha-ras and TGFalpha, mating experiments were performed. All ras/TGFalpha double genotype progeny (HK1 less than, with dotras/alpha) exhibited an increased epidermal hyperplasia/hyperkeratosis in neonates and accelerated spontaneous papillomatogenesis, compared with single transgenic siblings. HK1 less than, with dotras/alpha mice from the mild lines of HK1 less than, with dotrasxHK1 less than, with dotTGFalpha developed spontaneous papillomas that were not shown in either their parental mice or single transgenic littermates. Unlika in parental or single-genotype siblings, in which TPA promotion-elicited papillomas remained benign, TPA promotion elicited autonomous papillomas in HK1 less than, with dotras/alpha mice and exhibited a novel susceptibility to malignant conversion. Sequence analysis of the endogenous c-Ha-ras from spontaneous and TPA-induced HK1 less than, with dotras/alpha papillomas revealed wild-type sequence. However, carcinomas exhibited c-Ha-ras mutations at codon 61. All tumors analyzed to date expressed wild-type p53. These data provide in vivo evidence that Ha-ras and TGFalpha cooperate in the induction of epidermal hyperplasia and spontaneous tumor formation and predispose to malignant conversion via endogenous c-Ha-ras activation.

Topics: Animals; Base Sequence; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Epidermal Cells; Gene Expression Regulation; Genes, p53; Genes, ras; Mice; Mice, Transgenic; Mutation; Papilloma; Skin Neoplasms; Transforming Growth Factor alpha

To investigate the role of loss of the p53 tumor suppressor gene in skin carcinogenesis, p53 knockout (p53(-/-)) mice were mated with transgenic mice coexpressing v-Ha-ras, v-fos, or human transforming growth factor alpha (TGFalpha) exclusively in the epidermis by using human keratin 1 (HK1)-based vectors (HK1.ras/fos, HK1.ras/alpha, and HK1.fos/alpha). HK1.ras/fos and HK1.ras/alpha mice displayed epidermal hyperplasia and autonomous benign papillomas to an identical degree between p53(+/+) and p53(+/-) genotypes. However, HK1.ras/fos mice with the p53(-/-) genotype were born with papillomatous skin and died soon after birth. HK1.ras/alpha-p53(-/-) mice also exhibited an increased epidermal hyperplasia, and, similar to HK1.ras/alpha mice with p53(+/+) and p53(+/-) genotypes, these mice rapidly developed spontaneous and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomas. These results are in contrast to our previous observation that, HK1.ras, HK1.fos, and HK1.TGFalpha transgenic mice with the p53(-/-) genotype display an unexpected delay in both spontaneous and TPA-promoted papilloma formation compared with mice with p53(+/+) and p53(+/-) genotypes. Taken collectively, our mating experiments between HK1 oncogenic transgenic mice and p53 knockout mice may identify a backup system that effectively compensates for p53 loss. Activation of multiple oncogenes not only partly overcomes such compensation but also synergizes with p53 loss. However, HK1.fos/alpha-p53(-/-) mice failed to exhibit either an increased newborn epidermal hyperplasia or an accelerated spontaneous or TPA-induced papillomas, suggesting that certain combinations of oncogenes, such as with activated Ha-ras, are required for this process. Because neither spontaneous nor TPA-elicited papillomas in p53(-/-) mice progressed to malignancy, additional genetic insults appear to be required for malignant progression.

Topics: Animals; Carcinogens; Epidermis; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, p53; Genes, ras; Keratins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Transgenic; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

To study oncoprotein cooperation in vivo, transgenic mice were established that coexpressed human transforming growth factor-alpha (TGFalpha) and v-fos exclusively in the epidermis by means of a human keratin 1 (HK1)-based vector. HK1.fos/alpha mice exhibited aberrant epidermal proliferation and differentiation and formed spontaneous papillomas that achieved tumor autonomy but did not convert to malignancy. To determine the sensitivity to a chemical promotion stimulus, HK1.fos/alpha mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Previously, after 7 mo TPA promotion of HK1.TGFalpha mice that express moderate levels of TGFalpha elicited papillomas that remained regression-prone and benign for up to 2 yr. In HK1.fos mice, 6 mo TPA elicited papillomas that required spontaneous c-Ha-ras activation and converted to malignancy after 14-16 mo. We now show that in HK1.fos/alpha transgenic genotypes, TPA promotion accelerated papillomatogenesis, with the earliest papilloma appearance at 2 mo after initiation of TPA promotion. These papillomas started to convert to malignancy by 10 mo. Analysis of HK1.fos/alpha papillomas and carcinomas revealed that the endogenous c-Ha-ras gene possessed mutations at codons 12, 13, and 61 at the papilloma stage, but no mutations of the p53 tumor suppressor gene were detected. These data indicate that coexpression of fos and TGFalpha increased epidermal sensitivity to TPA promotion, which accelerated malignant conversion. However, in this transgenic model conversion always required additional genetic events, e.g., activation of the endogenous c-Ha-ras gene.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, ras; Humans; Mice; Mice, Transgenic; Oncogene Proteins v-fos; Papilloma; Proto-Oncogene Proteins p21(ras); Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Amphiregulin; Animals; Betacellulin; Carcinogens; Carcinoma, Squamous Cell; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ligands; Mice; Mice, Inbred SENCAR; Papilloma; RNA, Messenger; Skin; Skin Neoplasms; Transforming Growth Factor alpha; Up-Regulation

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Topics: Animals; Antibodies; Arsenic; Cell Division; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Papilloma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Diseases; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tissue Distribution; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGF alpha) can stimulate keratinocyte proliferation and function as an autocrine tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated TGF alpha-transgenic mouse skin. In this study, we examined the effect of ectopic TGF alpha transgene expression on skin tumor growth and progression after DMBA initiation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the multiplicity and size of skin tumors arising in TGF alpha-transgenic mice were significantly higher than those of the nontransgenic parental CD-1 strain. There were more dysplastic papillomas and squamous cell carcinomas (SCCs) in the transgenic animals as well. ProTGF alpha protein was expressed in transgenic papillomas, but mature TGF alpha was not detected. The epidermal growth factor receptor (EGFR) appeared to be downregulated and was associated with enhanced tyrosine phosphorylation of several substrates in TGF alpha-transgenic mouse tumors. Characteristic codon 61 mutations in the Ha-ras gene were found in most of the papillomas and SCCs induced by DMBA and TPA in transgenic as well as nontransgenic mice. However, no p53 gene mutations were found in any skin tumors from either transgenic or control animals. Analysis of cellular proliferation in both DMBA-TPA-induced papillomas and in skin 48 h after TPA treatment alone revealed significantly more DNA synthesis in TGF alpha-transgenic mice relative to controls. These results demonstrate that TGF alpha, through EGFR overstimulation, can act synergistically with TPA to induce the formation, growth, and development of DMBA-initiated skin tumors containing classic Ha-ras gene mutations but not p53 gene inactivation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cloning, Molecular; ErbB Receptors; Exons; Genes, p53; Humans; Mice; Mice, Transgenic; Papilloma; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Recombinant Proteins; Skin; Skin Neoplasms; Transforming Growth Factor alpha

To assess the synergistic effect of growth and transcription factor deregulation on carcinogenesis in vivo, mating experiments were performed between transgenic mice expressing human TGF alpha or v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos, HK1.TGF alpha and HK1.fos/alpha). While HK1.TGF alpha mice exhibited mild epidermal hyperplasia resulting in a wrinkled appearance, this hyperplasia was significantly increased in HK1.fos/alpha mice which also exhibited a novel opalescent and peeling skin phenotype. HK1.fos/alpha keratinocyte differentiation was considerably deregulated with cornified cells appearing in the granular layer, granular cells in the spinous layer and a sixfold increase in BrdU labeling over normal. In addition, hyperplastic HK1.fos/alpha epidermis exhibited aberrant loricrin, filaggrin and novel K13 expression associated with v-fos expression. Unlike adult HK1.TGF alpha controls, hyperplasia persisted in HK1.fos/alpha adults which also rapidly developed autonomous squamous cell papillomas. These results demonstrate that v-fos and TGF alpha over-expression can cooperate to reprogram keratinocyte differentiation and elicit the early stages of neoplasia. Moreover, TGF alpha over-expression appeared to play an early, initiating role in HK1.fos/alpha papilloma etiology, and a promotion role in the accelerated appearance of v-fos wound-associated preneoplastic phenotypes. However, the stable persistence of HK1.fos/alpha papillomas for up to 12 months, suggests that additional events are required for malignant conversion.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Filaggrin Proteins; Genetic Vectors; Keratins; Mice; Mice, Transgenic; Microscopy, Electron; Molecular Sequence Data; Oncogene Proteins v-fos; Papilloma; Skin; Skin Neoplasms; Transforming Growth Factor alpha

Autocrine epidermal growth factor receptor activation by transforming growth factor alpha (TGF alpha) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGF alpha expression, we tested whether TGF alpha is required for transformation by the v-rasHa oncogene. Introduction of v-rasHa into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGF alpha +/+, BALB/c) and TGF alpha-deficient (TGF alpha -/-, wa-1) keratinocytes. Moreover, v-rasHa elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGF alpha +/+ and TGF alpha -/- keratinocytes. v-rasHa markedly increased secreted (> 10-fold) and cell-associated (2-3-fold) TGF alpha levels in keratinocytes from TGF alpha +/+ and BALB/c mice, but not TGF alpha -/- or wa-1 mice. Based on Northern blot analysis, v-rasHa induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGF alpha transcript, wa-1 keratinocytes expressed two additional TGF alpha transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to v-rasHa, as well as exogenous TGF alpha or keratinocyte growth factor treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-rasHa keratinocytes from control as well as TGF alpha-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGF alpha status. These findings indicate that TGF alpha is not essential for epidermal neoplasia induced by the v-rasHa oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ras-transformed keratinocytes.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, ras; Keratinocytes; Ligands; Mice; Mice, Inbred BALB C; Mice, Nude; Papilloma; Phenotype; Skin Neoplasms; Transcription, Genetic; Transforming Growth Factor alpha

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

Topics: Animals; Carcinoma, Squamous Cell; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Inbred SENCAR; Neoplasm Proteins; Papilloma; RNA, Neoplasm; Skin Neoplasms; Transforming Growth Factor alpha

To assess the requirements for papilloma formation in transgenic mice that overexpress transforming growth factor-alpha (TGF-alpha) in the epidermis (HK1.TGF alpha), we tested the sensitivity of HK1.TGF alpha mice to tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) and analyzed the resultant papillomas for synergic c-Ha-ras activation and overexpression. We observed that HK1.TGF alpha mice were highly sensitive to TPA promotion, exhibiting multiple papillomas as early as the third week of treatment. After 60 wk of promotion, malignant conversion was not observed and tumors regressed upon removal of the TPA promotion stimulus. Most of the TPA-induced papillomas did not have detectable c-Ha-ras mutations at codons 12, 13, or 61, but three papillomas arising after long-term TPA promotion (5-7 mo) exhibited c-Ha-ras activation at codon 61 (A-->T and A-->G). Conversely, spontaneous papillomas arising without TPA promotion, including persisting autonomous papillomas, were all negative for activating c-Ha-ras mutations. Both spontaneous and TPA-induced HK1.TGF alpha papillomas expressed c-Ha-ras message levels similar to those in normal, nontransgenic epidermis or HK1.TGF alpha hyperplastic epidermis. These data demonstrate that TGF-alpha overexpression can be an initiating event for TPA promotion, that papillomatogenesis in HK1.TGF alpha mice proceeds frequently via a pathway independent of Ha-ras activation or overexpression, and, thus, that other events are required for autonomous growth and malignant conversion.

Topics: Animals; Base Sequence; DNA Primers; Gene Expression; Genes, ras; Mice; Mice, Transgenic; Molecular Sequence Data; Papilloma; Proto-Oncogene Proteins p21(ras); RNA, Messenger; RNA, Neoplasm; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

To assess the effects of transforming growth factor alpha (TGF-alpha) on mammalian skin in vivo, we have targeted its expression to the epidermis of transgenic mice using a vector based on the human K1 (HK1) gene. Neonatal mice expressing the HK1.TGF-alpha transgene were often smaller than normal littermates and had precocious eyelid opening and wrinkled, scaly skin with diffuse alopecia. Juvenile transgenic mouse epidermis was uniformly hyperkeratotic, but this pattern was generally less pronounced in adult transgenic mice unless they expressed high levels of the HK1.TGF-alpha transgene. Spontaneous, squamous papillomas occurred at sites of wounding in adult mice expressing high levels of HK1.TGF-alpha; however, most were prone to regression. Immunoreactive TGF-alpha was 2-6 times higher in the epidermis of these HK1.TGF-alpha lines. Immunoreactive epidermal growth factor receptor had a normal pattern of expression in nonphenotypic adult epidermis, but a marked reduction in the receptor population was detected in hyperplastic newborn epidermis and phenotypic adult epidermis. Autoradiographic localization of 125I-epidermal growth factor showed a similar pattern of distribution, suggesting that the sites of increased TGF-alpha expression induced epidermal growth factor receptor down-regulation. These data demonstrate the in vivo effect of deregulated TGF-alpha expression on epidermal proliferation and differentiation and suggest a potential role for TGF-alpha in carcinogenesis and other hyperproliferative epidermal disorders.

Topics: Animals; Base Sequence; Cell Division; Epidermal Growth Factor; Epidermis; ErbB Receptors; Hyperplasia; Keratosis; Mice; Mice, Transgenic; Molecular Sequence Data; Papilloma; Skin Neoplasms; Transforming Growth Factor alpha

The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.

Topics: Animals; Base Sequence; Disease Models, Animal; DNA, Neoplasm; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, ras; Mice; Mice, Transgenic; Molecular Sequence Data; Mutation; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Wounds and Injuries

Transforming growth factor-alpha (TGF-alpha) is thought to be the major autocrine factor controlling growth in epidermal cells. To explore further the role of TGF-alpha in epidermal growth and differentiation, we used a human keratin K14 promoter to target expression of rat TGF-alpha cDNA to the stratified squamous epithelia of transgenic mice. Unexpectedly, the only regions of epidermis especially responsive to TGF-alpha overexpression were those that were normally thick and where hair follicle density was typically low. This included most, if not all, body skin from 2-day- to 2-week-old mice, and ear, footpad, tail, and scrotum skin in adult mice. In these regions, excess TGF-alpha resulted in thicker epidermis and more stunted hair growth. Epidermal thickening was attributed both to cell hypertrophy and to a proportional increase in the number of basal, spinous, granular, and stratum corneum cells. During both postnatal development and epidermal differentiation, responsiveness to elevated TGF-alpha seemed to correlate with existing epidermal growth factor (EGF) receptor levels, and we saw no evidence for TGF-alpha-mediated control of EGF receptor (EGFR) expression. In adults, no squamous cell carcinomas were detected, but benign papillomas were common, developing primarily in regions of mechanical irritation or wounding. In addition, adult transgenic skin that was still both sensitive to TGF-alpha and subject to mild irritation displayed localized regions of leukocytic infiltration and granular layer loss, characteristics frequently seen in psoriasis in humans. These unusual regional and developmental effects of TGF-alpha suggest a natural role for the growth factor in (1) controlling epidermal thickness during development and differentiation, (2) involvement in papilloma formation, presumably in conjunction with TGF-beta, and (3) involvement in psoriasis, in conjunction with some as yet unidentified secondary stimulus stemming from mild mechanical irritation/bacterial infection.

Topics: Animals; Animals, Newborn; Blotting, Northern; Cell Differentiation; Cell Division; Cells, Cultured; Epidermis; ErbB Receptors; Humans; Keratins; Mice; Mice, Transgenic; Papilloma; Phenotype; Psoriasis; Transforming Growth Factor alpha; Wounds and Injuries

The influence of an oncogenic v-Ha-ras gene on the expression of TGF-beta and TGF-alpha by mouse keratinocytes and derived tumors has been investigated. Normal mouse keratinocytes cultured as basal cells in 0.05 mM Ca2+ secreted low levels of TGF-beta 2 peptide, and this increased markedly following culture in 1.4 mM Ca2+, retinoic acid, or phorbol esters. In contrast, introduction of a v-Ha-ras gene into normal keratinocytes increased basal expression and secretion of TGF-beta 1 (rather than TGF-beta 2) in response to all three agents. The selective secretion of TGF-beta 1 in v-Ha-ras keratinocytes in response to 1.4 mM Ca2+ occurred even though the four TGF-beta 2 transcripts were induced and the TGF-beta 1 transcript decreased, suggesting that the activated v-Ha-ras gene product regulates expression of the TGF-beta isoforms at the posttranscriptional level. Immunohistochemical analysis of papillomas formed following skin grafting of v-Ha-ras keratinocytes onto nude mice indicated that TGF-beta 1 was abundant in the basal and spinous layers, while there was no expression of TGF-beta 1 in normal skin. In contrast, both normal and neoplastic tissues expressed TGF-beta 2 and TGF-beta 3 in the granular layers. Furthermore, TGF-alpha mRNA expression was also elevated fivefold in cultured v-Ha-ras keratinocytes, and TGF-alpha protein was overexpressed in the grafted papillomas, but there was no detectable expression in normal skin. Elevated expression of both TGF-beta 1 and TGF-alpha in the basal and spinous layers of benign tumors may be important for the high proliferation rate in these tumors as well as for increased proliferation in the suprabasal layer.

Topics: Animals; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, ras; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Papilloma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured