RESUMEN

Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARß/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARß/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARß/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparß/δ-null cells. Higher PPARß/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARß/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

Asunto(s)

Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , PPAR delta/metabolismo , PPAR-beta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas ras/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Western Blotting , Carcinogénesis/genética , Carcinogénesis/metabolismo , Células Cultivadas , Senescencia Celular/genética , Femenino , Células HEK293 , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Células 3T3 NIH , PPAR delta/genética , PPAR-beta/genética , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteínas ras/genética

RESUMEN

Chemical induction of squamous tumors in the mouse skin induces multiple benign papillomas: high-frequency terminally benign low-risk papillomas and low-frequency high-risk papillomas, the putative precursor lesions to squamous cell carcinoma (SCC). We have compared the gene expression profile of twenty different early low- and high-risk papillomas with normal skin and SCC. Unsupervised clustering of 514 differentially expressed genes (P<0.001) showed that 9/10 high-risk papillomas clustered with SCC, while 1/10 clustered with low-risk papillomas, and this correlated with keratin markers of tumor progression. Prediction analysis for microarrays (PAM) identified 87 genes that distinguished the two papilloma classes, and a majority of these had a similar expression pattern in both high-risk papillomas and SCC. Additional classifier algorithms generated a gene list that correctly classified unknown benign tumors as low- or high-risk concordant with promotion protocol and keratin profiling. Reduced expression of immune function genes characterized the high-risk papillomas and SCC. Immunohistochemistry confirmed reduced T-cell number in high-risk papillomas, suggesting that reduced adaptive immunity defines papillomas that progress to SCC. These results demonstrate that murine premalignant lesions can be segregated into subgroups by gene expression patterns that correlate with risk for malignant conversion, and suggest a paradigm for generating diagnostic biomarkers for human premalignant lesions with unknown individual risk for malignant conversion.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Transformación Celular Neoplásica/patología , Perfilación de la Expresión Génica , Papiloma/metabolismo , Neoplasias Cutáneas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Biomarcadores de Tumor/genética , Carcinógenos/toxicidad , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Femenino , Inmunidad Celular , Inmunofenotipificación , Ratones , Ratones Endogámicos SENCAR , Análisis de Secuencia por Matrices de Oligonucleótidos , Papiloma/inducido químicamente , Papiloma/patología , Valor Predictivo de las Pruebas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/toxicidad

RESUMEN

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-beta. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Inflamación/fisiopatología , Transactivadores/fisiología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/fisiopatología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Adhesión Celular , División Celular , Células Cultivadas , Quimiotaxis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Inflamación/genética , Queratinocitos/citología , Queratinocitos/fisiología , Masculino , Ratones , Ratones Noqueados , Monocitos/citología , Monocitos/fisiología , Transducción de Señal , Piel/lesiones , Proteína smad3 , Transactivadores/deficiencia , Transactivadores/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiología , Cicatrización de Heridas/genética , Heridas y Lesiones/genética , Heridas y Lesiones/patología

RESUMEN

Cyclin D1 is part of a cell cycle control node consistently deregulated in most human cancers. However, studies with cyclin D1-null mice indicate that it is dispensable for normal mouse development as well as cell growth in culture. Here, we provide evidence that ras-mediated tumorigenesis depends on signaling pathways that act preferentially through cyclin D1. Cyclin D1 expression and the activity of its associated kinase are up-regulated in keratinocytes in response to oncogenic ras. Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumors generated through either grafting of retroviral ras-transduced keratinocytes, phorbol ester treatment of ras transgenic mice, or two-stage carcinogenesis.

Asunto(s)

Ciclina D1/fisiología , Genes ras/fisiología , Proteínas Proto-Oncogénicas , Neoplasias Cutáneas/fisiopatología , Animales , Transformación Celular Neoplásica , Ciclina D1/deficiencia , Ciclina D1/genética , Ciclina E/genética , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Retroviridae/genética , Neoplasias Cutáneas/genética

RESUMEN

Previous studies have demonstrated a role for the fos gene in promoting malignant conversion of mouse skin tumors. In the study reported here, differential display was performed to identify fos- and jun-regulated genes that are differentially expressed during premalignant progression. Total RNA isolated from variants of the papilloma cell line SP-1 transduced with retroviral vectors expressing v-jun and v-fos alone or in tandem was analyzed for the presence of differentially expressed transcripts by using 35 different primer combinations. Differentially expressed clones were rescreened by dot-blot analysis by using cDNA from chemically induced tumors with a high or low risk of malignant conversion. Three differentially displayed fragments were isolated in this analysis. Homology searches indicated that these fragments shared significant homology with the apoptosis inhibitor bcl-2, human alternative splicing factor/splicing factor 2 (ASF/SF2), and a novel gene not present in the GenBank or EMBL databases. In situ hybridization indicated that the expression levels of the bcl-2 homolog increased with malignant potential in chemically derived mouse skin tumors. A similar analysis indicated that expression of the ASF/SF2 homolog was greater in papillomas than in normal skin or in squamous cell carcinomas. Transcripts for this gene were most abundant in the granular layer. The expression pattern of the third differential display fragment was consistent with that of a tumor suppressor gene. This gene was expressed at very high levels in normal skin and benign papillomas but was essentially undetectable in squamous cell carcinomas. Through this approach, we identified known and novel genes that may contribute to malignant progression in epidermal tumors.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Genes fos , Genes jun , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Animales , Carcinoma/inducido químicamente , Carcinoma/genética , Carcinoma/metabolismo , Línea Celular , ADN de Neoplasias/metabolismo , ADN Viral/metabolismo , Femenino , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos SENCAR , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/metabolismo , Factores de Riesgo , Neoplasias Cutáneas/genética , Factor de Transcripción AP-1/metabolismo

RESUMEN

Alterations in expression of or responsiveness to transforming growth factor beta (TGF-beta) are frequently found in human and animal epithelial cancers and are though to be important for loss of growth control in the neoplastic cell. We show here that keratinocyte cell lines from mice with a targeted deletion of the TGF-beta 1 gene have significantly increased frequencies of gene amplification in response to the drug N-phosphonoacetyl-L-aspartate (PALA) compared to TGF-beta 1-expressing control keratinocyte cell lines. In contrast to the control lines, the PALA-mediated G1 arrest did not occur in the TGF-beta 1 null keratinocytes despite the presence of wild-type p53 in both genotypes. Exogenous TGF-beta 1 suppresses gene amplification in the null keratinocytes at concentrations that do not cause a G1 growth arrest and in human tumor cell lines that are insensitive to TGF-beta 1-mediated growth inhibition. The pathway of TGF-beta 1 suppression is independent of the p53 and Rb genes, but requires an intact TGF-beta type II receptor. These studies reveal a novel TGF-beta-mediated pathway regulating genomic stability and suggest that defects in TGF-beta signaling may have profound effects on tumor progression independent of cell proliferation.

Asunto(s)

Fase G1 , Genes de Retinoblastoma/fisiología , Genes p53/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Células Cultivadas , Fase G1/efectos de los fármacos , Amplificación de Genes , Ratones , Ácido Fosfonoacético/análogos & derivados , Ácido Fosfonoacético/farmacología , Factor de Crecimiento Transformador beta/genética

RESUMEN

The multistage evolution of squamous cell cancer on mouse skin has provided a model to dissect the biological and genetic changes that contribute to specific stages of carcinogenesis. Keratinocyte cell culture models have been developed that reproduce the genetic and epigenetic events in multistage skin carcinogenesis, and these provide insights into the biochemistry of the process. When the v-rasHa oncogene is transduced into normal mouse keratinocytes, the resultant papilloma phenotype is characterized by a high rate of cell proliferation, an aberrant program of differentiation marker expression, and resistance to terminal differentiation. These changes are attributed to differential effects on isoforms of protein kinase C coupled to activation of the epidermal growth factor receptor. Premalignant progression requires additional genetic changes in the v-rasHa-transduced cells. The acquisition of these changes is suppressed by transforming growth factor beta (TGF beta), and the absence of TGF beta in premalignant tumors indicates a high risk for malignant progression. Keratinocytes that are genetically null for the TGF beta 1 gene rapidly progress to squamous cell carcinomas when transduced with the v-rasHa oncogene. In culture, TGF beta 1 null keratinocytes exhibit a high rate of gene amplification that can be suppressed by concentrations of exogenous TGF beta well below those required to inhibit proliferation. This model is well-suited to identifying critical genetic changes that contribute to premalignant progression.

Asunto(s)

Neoplasias Cutáneas/etiología , Animales , Calcio/fisiología , Humanos , Ratones , Papiloma/etiología , Lesiones Precancerosas/etiología , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/genética

RESUMEN

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.

Asunto(s)

Empalme Alternativo , Carcinoma/genética , Transformación Celular Neoplásica/genética , Integrinas/genética , Neoplasias Cutáneas/genética , Animales , Western Blotting , Carcinoma/inducido químicamente , Carcinoma/etiología , Adhesión Celular , Técnica del Anticuerpo Fluorescente , Integrina alfa6 , Integrinas/biosíntesis , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Proteínas Oncogénicas v-fos/genética , Papiloma/genética , Reacción en Cadena de la Polimerasa , Lesiones Precancerosas , ARN Mensajero/genética , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/etiología

RESUMEN

Retinoids are powerful regulators of epidermal cell growth and differentiation and are widely used in the prevention and treatment of skin disorders and cancers in humans. Since many of the effects of retinoids on cell growth and differentiation are mediated by nuclear retinoid receptors (RARs and RXRs), we were interested in determining RAR and RXR gene expression during mouse skin tumor progression. The two-stage system of mouse skin carcinogenesis was used to generate papillomas and carcinomas, and the different stages of malignant progression (papillomas, differentiated squamous cell carcinomas, undifferentiated squamous cell carcinomas, and spindle cell carcinomas) were characterized in each tumor by specific keratin expression prior to receptor characterization. Using in situ hybridization analysis, we show that the two major RAR isoforms (alpha 1 and gamma 1), which account for most of RARs in the skin, were expressed in both the basal and suprabasal layers in mouse epidermis. In contrast, RXR alpha transcripts were compartmentalized to the basal cell layers and concentrated in hair follicles. During skin tumor progression, RAR (alpha 1 and gamma 1) transcripts were down-modulated in malignant tumor cells, whereas RXR (alpha and beta) transcript expression was expanded in papillomas and carcinomas as the number of undifferentiated cells also increased. RXR gamma was not detected in the skin or at any stage during skin tumor progression. Spindle cell tumors lacked markers of the keratinocyte phenotype and lost RAR expression, yet retained expression of RXR alpha and beta. The increased abundance of transcripts for RXRs and decreased presence of RARs in skin tumor progression may favor other nuclear signal transduction pathways requiring RXR for heterodimer formation and contribute to phenotypic progression of cancer cells.

Asunto(s)

Carcinoma/metabolismo , Papiloma/metabolismo , Receptores de Ácido Retinoico/metabolismo , Neoplasias Cutáneas/metabolismo , Factores de Transcripción/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Compartimento Celular , Epidermis/metabolismo , Hibridación in Situ , Queratinas/genética , Ratones , Receptores X Retinoide , Acetato de Tetradecanoilforbol

Asunto(s)

Transformación Celular Neoplásica , Técnicas de Cultivo/métodos , Células Epidérmicas , Queratinocitos/citología , Queratinocitos/metabolismo , Oncogenes , Transfección/métodos , Animales , Animales Recién Nacidos , Biomarcadores de Tumor/análisis , Diferenciación Celular , Células Cultivadas , Técnicas de Cultivo/instrumentación , Epidermis/metabolismo , Feto , Expresión Génica , Humanos , Indicadores y Reactivos , Recién Nacido , Masculino , Neoplasias/genética , Neoplasias Experimentales/genética , Plásmidos

RESUMEN

The introduction of the techniques of molecular biology as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer pathogenesis: initiation, exogenous promotion, premalignant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozygous activating mutations in c-rasHa are dominant, and affected keratinocytes hyperproliferate and are resistant to signals for terminal differentiation. An important pathway impacted by c-rasHa activation is the protein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKC alpha and suppression of PKC delta by tyrosine phosphorylation contribute to the phenotypic consequences of rasHa gene activation in keratinocytes. Tumor promoters disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differentiation and enhanced growth rate of initiated cells impart a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progression varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the alpha 6 beta 4 integrin and are deficient in transforming growth factor beta 1 and beta 2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The introduction of an oncogenic rasHa gene into epidermal cells derived from transgenic mice with a null mutation in the TGF beta 1 gene have an accelerated rate of malignant progression when examined in vivo. Thus members of the TGF beta gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasHa transduced keratinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant conversion by initiated keratinocytes from TG beta 1 and p53 null geneotypes suggest that a common, growth-related pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis model.

Asunto(s)

Genes Supresores de Tumor , Neoplasias Experimentales/genética , Oncogenes , Animales , Transformación Celular Neoplásica , Genes ras , Sustancias de Crecimiento/fisiología , Humanos , Estadificación de Neoplasias , Neoplasias Experimentales/patología , Proteína p53 Supresora de Tumor/genética

RESUMEN

To study the contribution of autocrine and paracrine TGF-beta 1 to tumor progression in a well-defined system of multistage carcinogenesis, keratinocytes with a targeted deletion of the TGF-beta 1 gene were initiated in vitro with the v-rasHa oncogene and their in vivo tumorigenic properties were determined by skin grafting initiated cells onto athymic mice in combination with either wild-type or null dermal fibroblasts. Grafts of v-rasHa-initiated null keratinocytes progressed rapidly to multifocal squamous cell carcinomas within dysplastic papillomas irrespective of the fibroblast genotype, whereas the initiated control genotypes formed well-differentiated papillomas. Malignant progression was not associated with mutations in the c-rasHa gene, alterations in p53 protein, or loss of responsiveness to TGF-beta 1. The tumor cell labeling index was elevated in grafts of initiated null keratinocytes with wild-type fibroblasts compared to tumors of other genotypes. However, labeling index in all tumors was reduced when TGF-beta 1 null fibroblasts formed the stroma. The null tumor cells could not accumulate TGF-beta 1 from the host, but grafts of uninitiated null keratinocytes, which formed a normal epidermis, became TGF-beta 1 positive even though they did not express TGF-beta 1 mRNA. These results demonstrate that autocrine TGF-beta 1 suppresses the frequency and rate of malignant progression, and that autocrine and paracrine TGF-beta 1 can have opposing effects on tumor cell proliferation. The lack of paracrine inhibition of tumor cell progression appears to result from the inability of tumor cells to localize host-derived TGF-beta 1 by a mechanism that operates in normal cells.

Asunto(s)

Carcinoma de Células Escamosas/etiología , Eliminación de Gen , Neoplasias Cutáneas/etiología , Factor de Crecimiento Transformador beta/genética , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , División Celular , Cocarcinogénesis , Cartilla de ADN/genética , ADN de Neoplasias/genética , Expresión Génica , Genes p53 , Genes ras , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Trasplante de Piel

RESUMEN

The widespread use of FVB/N mice for the establishment of transgenic lines containing active oncogenes suggested the importance of testing the parent FVB/N mice for sensitivity to experimental carcinogenesis. After initiation of mouse skin by a single treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), the skin tumor incidence was compared in FVB/N mice, TPA-sensitive (SENCAR and CD-1) and TPA-resistant mice (BALB/c and C57BL/6). Initiation by 25 micrograms DMBA followed by promotion with a low dose of TPA (2 micrograms/week) induced one or more papillomas in only 25% of FVB/N mice, compared with 100% in SENCAR, 53% in CD-1, 17% in BALB/c and 0% in C57BL/6 mice. At a more effective dose of TPA (5 micrograms/week), FVB/N mice initiated by 5, 25 or 100 micrograms DMBA developed 3.4, 6.9 and 11.8 papillomas per mouse. In contrast, the incidence of squamous cell carcinomas (SCCs) (17-18/30 mice) did not increase with DMBA dose. TPA promotion of non-initiated mice induced only six papillomas, but three progressed to SCCs, a high rate of malignant conversion. Skin tumor induction by 20 weekly treatments with 10 micrograms DMBA produced few papillomas, but 50.0% of the papillomas progressed to carcinomas in FVB/N mice, compared with 9.15% in SENCAR, 37.5% in CD-1, 23.1% in BALB/c and 15.0% in C57CL/6 mice. The first carcinomas appeared after 14 weeks in FVB/N, 24 weeks in SENCAR, 26 weeks in CD-1 and C57BL/6 and 34 weeks in BALB/c mice. Thus, FVB/N mice develop an unusually high incidence of SCCs after treatment with repeated DMBA, DMBA initiation-TPA promotion and even TPA alone.

Asunto(s)

9,10-Dimetil-1,2-benzantraceno/toxicidad , Carcinoma de Células Escamosas/inducido químicamente , Neoplasias Cutáneas/inducido químicamente , Acetato de Tetradecanoilforbol/toxicidad , Acetona/toxicidad , Animales , Carcinoma de Células Escamosas/patología , Resistencia a Medicamentos/genética , Femenino , Predisposición Genética a la Enfermedad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Papiloma/inducido químicamente , Papiloma/patología , Neoplasias Cutáneas/patología , Especificidad de la Especie

RESUMEN

Enhanced expression of the alpha 6 beta 4 integrin complex has been linked to malignant progression in mouse skin carcinogenesis. To determine if alpha 6 beta 4 expression can predict risk for malignant conversion among populations of benign skin tumors, we analyzed the distribution of alpha 6 beta 4 and other markers of progression in papillomas at high and low risk for malignant progression. After initiation with 7,12-dimethylbenz[a]anthracene, mice were promoted with 12-O-tetradecanoylphorbol-13-acetate to induce predominantly low risk tumors or promoted with mezerein to produce predominantly high risk tumors. When tumors first appeared at 8 weeks after promotion, high risk papillomas demonstrated basal and suprabasal alpha 6 beta 4 expression, loss of keratin 1, and aberrant expression of keratin 13. In these tumors alpha 6 beta 4 expression coincided with an expansion of the proliferating compartment as indicated by suprabasal bromodeoxyuridine labeling. In contrast, alpha 6 beta 4 immunostaining was confined to basal cells in low risk tumors, keratin 1 was abundant, and keratin 13 was absent in the majority of this group, while proliferating cells were largely in the basal compartment. By 33 weeks, alpha 6 beta 4 suprabasal expression continued to distinguish groups at higher risk for malignant conversion, but keratin 13 was expressed in all groups. At this time, high risk tumors displayed focal expression of keratin 8 and gamma-glutamyltranspeptidase, markers also found in chemically induced carcinomas. Keratin 8 and gamma-glutamyltranspeptidase were expressed only in alpha 6 beta 4 positive cells. These results indicate that expression of alpha 6 beta 4 integrin in suprabasal strata serves as an early predictive marker to identify benign squamous tumors at high risk for malignant progression.

Asunto(s)

Antígenos de Superficie/análisis , Biomarcadores de Tumor/análisis , Diterpenos , Papiloma/patología , Neoplasias Cutáneas/patología , Piel/patología , 9,10-Dimetil-1,2-benzantraceno , Animales , Antígenos de Superficie/biosíntesis , Femenino , Inmunohistoquímica , Integrina alfa6beta4 , Cinética , Ratones , Ratones Endogámicos , Papiloma/inducido químicamente , Papiloma/metabolismo , Factores de Riesgo , Piel/efectos de los fármacos , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Terpenos , Acetato de Tetradecanoilforbol , Factores de Tiempo

RESUMEN

Mouse skin carcinomas arise from a small subpopulation of benign papillomas with an increased risk of malignant conversion. These papillomas arise with limited stimulation by tumor promoters, appear rapidly, and do not regress, suggesting that they differ in growth properties from the majority of benign tumors. The transforming growth factor beta (TGF-beta) proteins are expressed in the epidermis and are growth inhibitors for mouse keratinocytes in vitro; altered TGF-beta expression could influence the growth properties of high-risk papillomas. Normal epidermis, tumor promoter-treated epidermis, and skin papillomas at low risk for malignant conversion express TGF-beta 1 in the basal cell compartment and TGF-beta 2 in the suprabasal strata. In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high-risk papillomas are devoid of both TGF-beta 1 and TGF-beta 2 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers. Squamous cell carcinomas are also devoid of TGF-beta, suggesting that they arise from the TGF-beta-deficient high-risk papillomas. In some high-risk papillomas, TGF-beta 1 loss can occur first and correlates with basal cell hyperproliferation, while TGF-beta 2 loss correlates with suprabasal hyperproliferation. Similarly, TGF-beta 1-null transgenic mice, which express wild-type levels of TGF-beta 2 in epidermis but no TGF-beta 1 in the basal layer, have a hyperproliferative basal cell layer without suprabasal proliferation. In tumors, loss of TGF-beta is controlled at the posttranscriptional level and is associated with expression of keratin 13, a documented marker of malignant progression. These results show that TGF-beta expression and function are compartmentalized in epidermis and epidermal tumors and that loss of TGF-beta is an early, biologically relevant risk factor for malignant progression.

Asunto(s)

Transformación Celular Neoplásica , Epidermis/química , Neoplasias Cutáneas/química , Factor de Crecimiento Transformador beta/análisis , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patología , División Celular , Células Epidérmicas , Femenino , Queratinas/análisis , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Papiloma/química , Papiloma/etiología , Papiloma/patología , Riesgo , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología

RESUMEN

Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillomas already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated rasHa gene produced cells with a carcinoma phenotype, i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the rasHa gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The rasHa/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated rasHa oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the rasHa-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.

Asunto(s)

Transformación Celular Neoplásica , Oncogenes , Neoplasias Cutáneas/patología , Animales , Carcinógenos , Línea Celular , Regulación de la Expresión Génica , Genes fos , Genes ras , Queratinocitos/patología , Ratones , Mutagénesis , Papiloma/inducido químicamente , Papiloma/patología , Neoplasias Cutáneas/inducido químicamente

RESUMEN

Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-beta 1, revealed increased extracellular and decreased intracellular expression of transforming growth factor-beta 1 in retinoic acid-treated, compared to vehicle-treated, skin. Transforming growth factor-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF-beta 2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming growth factor-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-beta 1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-beta 1 protein, without any detectable change in transforming growth factor-beta 2. These data demonstrate disassociation of modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin.

Asunto(s)

Mucinas/análisis , Piel/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Factor de Crecimiento Transformador beta/análisis , Tretinoina/farmacología , Adulto , Células Cultivadas , Humanos , Queratinocitos/química , Queratinocitos/efectos de los fármacos , Persona de Mediana Edad , Mucinas/metabolismo , ARN Mensajero/análisis , Piel/química , Factor de Crecimiento Transformador beta/genética

RESUMEN

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.

Asunto(s)

Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Tretinoina/farmacología , Deficiencia de Vitamina A/metabolismo , Animales , Colon/metabolismo , Epidermis/metabolismo , Femenino , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Pulmón/metabolismo , Ratas , Ratas Endogámicas , Factor de Crecimiento Transformador beta/análisis , Vagina/metabolismo

RESUMEN

Genomic DNA extending over 10 kb 5' of the transforming growth factor-beta 2 (TGF-beta 2) coding region was isolated from a human lung fibroblast lambda phage library. A 5.6 kb Hind III fragment containing the 5'-untranslated region and flanking sequences was subcloned and sequenced. S1 nuclease protection analysis identified a transcriptional initiation site 1357 nucleotides 5' of the methionine initiation codon (ATG). A "TATA box" consensus sequence was identified 30 bp from this transcriptional start site; however, consensus "CAT box" sequences were not observed. Approximately 50 nucleotides of homopurine-pyrimidine [d(GA.CT)50] sequence were identified in the 5'-untranslated region, as well as two short open reading frames of 5 and 45 amino acids. Several AP-1, AP-2, CRE and SP1-like DNA consensus sequence elements were also identified surrounding the transcription initiation site. 5'-deletion mutants of the promoter region were fused to the chloramphenicol acetyl transferase (CAT) gene and promoter activity of the isolated genomic DNA was demonstrated in several cell lines. DNA constructs containing nucleotides between -508 to +63 demonstrated high levels of promoter activity. However, sequences between -778 and -508 nucleotides modulated this promoter activity in a manner which was dependent upon the cell line utilized, suggesting that regulation of TGF-beta 2 gene transcription may be dependent upon the cellular background. The TGF-beta 2 promoter is markedly different from the promoters that have been recently characterized for TGF-beta 1 and TGF-beta 3.

Asunto(s)

ADN/genética , Regiones Promotoras Genéticas/genética , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Regulación de la Expresión Génica , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Mapeo Restrictivo , Transcripción Genética , Factor de Crecimiento Transformador beta/aislamiento & purificación

RESUMEN

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.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Genes ras , Queratinocitos/metabolismo , Papiloma/genética , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador beta/genética , Animales , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes ras/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Papiloma/patología , Células Tumorales Cultivadas