RESUMEN

Squamous differentiation is controlled by key transcription factors such as Sp1 and E2F. We have previously shown that E2F1 can suppress transcription of the differentiation-specific gene, transglutaminase type 1 (TG1), by an indirect mechanism mediated by Sp1. Transient transfection of E2F1-E2F6 indicated that E2F-mediated reduction of Sp1 transcription was not responsible for E2F-mediated suppression of squamous differentiation. However, we found that E2F4 and E2F7, but not E2Fs 1, 2, 3, 5, or 6, could suppress the activation of the Sp1 promoter in differentiated keratinocytes (KCs). E2F4-mediated suppression could not be antagonized by E2Fs 1, 2, 3, 5, or 6 and was localized to a region of the human Sp1 promoter spanning -139 to + 35 bp. Chromatin immunoprecipitation analysis, as well as transient overexpression and short hairpin RNA knockdown experiments indicate that E2F7 binds to a unique binding site located between -139 and -119 bp of the Sp1 promoter, and knockdown of E2F7 in proliferating KCs leads to a derepression of Sp1 expression and the induction of TG1. In contrast, E2F4 knockdown in proliferating KCs did not alter Sp1 expression. These data indicate that loss of E2F7 during the initiation of differentiation leads to the derepression of Sp1 and subsequent transcription of differentiation-specific genes such as TG1.

Asunto(s)

Diferenciación Celular/genética , Factor de Transcripción E2F7/metabolismo , Regulación Enzimológica de la Expresión Génica , Queratinocitos/citología , Factor de Transcripción Sp1/metabolismo , Transglutaminasas/genética , Células Cultivadas , Factor de Transcripción E2F7/genética , Humanos , Queratinocitos/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética

RESUMEN

Tumor initiation (TI) in xenotransplantation models of head and neck squamous cell carcinoma (HNSCC) is an inefficient process. Poor TI could be due to (1) posttransplant cell loss, (2) a rare sub-population of cancer stem cells or (3) a requirement for specific cellular interactions, which rely on cell number. By tracking GFP-expressing HNSCC cells, we conclude that the posttransplant loss of cancer cells is minimal in the xenotransplant model. Furthermore, an examination of putative cancer stem cell markers (such as CD133, CD44, SP and label retention) in HNSCC cell lines revealed no correlation between marker expression and tumorigenicity. In addition, single-cell clones randomly isolated from HNSCC cell lines and then transplanted into mice were all capable of initiating tumors with efficiencies varying almost 34-fold. As the observed variation in the clones was both more and less tumorigenic than the parental cells, a combination of two clones, at suboptimal cell numbers for TI, was implanted into mice and was found to modulate the tumor-initiating activity, thus indicating that TI is dependent on a 'critical' number of cells and, for the first time, that interactions between clonal variants within tumors can modulate the overall tumor-initiating activity. Put in context with previous literature on tumorigenic activity, we believe that interactions between clonal variants within a tumor as well as (1) stromal interactions, (2) angiogenic activity, (3) immunocompetence and (4) cancer stem cells may all contribute to tumorigenic potential and the propensity for tumor growth and recurrence.

Asunto(s)

Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Antígeno AC133 , Animales , Antígenos CD/análisis , Carcinoma de Células Escamosas/etiología , Moléculas de Adhesión Celular/análisis , Línea Celular Tumoral , Proteínas Ligadas a GPI , Glicoproteínas/análisis , Neoplasias de Cabeza y Cuello/etiología , Humanos , Receptores de Hialuranos/análisis , Ratones , Ratones SCID , Péptidos/análisis

RESUMEN

The E2F family of transcription factors plays a crucial role in the regulation of genes involved in cell proliferation, differentiation, and apoptosis. In keratinocytes, the inhibition of E2F is a key step in the control and initiation of squamous differentiation. Because the product of the recently identified E2F7a/E2F7b gene has been shown to repress E2F-regulated promoters, and to be abundant in skin, we examined its role in the epidermis. Our results indicate that E2F7b mRNA expression is selectively associated with proliferation-competent keratinocytes. Moreover, E2F7 was able to antagonize E2F1-induced proliferation and apoptosis. In contrast, although E2F7 was able to inhibit proliferation and initiate differentiation, it was unable to antagonize the differentiation suppression induced by E2F1. These data indicate that E2F7-mediated suppression of proliferation and apoptosis acts through E2F1-dependent pathways, whereas E2F7-induced differentiation acts through an E2F1-independent pathway. These data also suggest that proliferation, differentiation, and survival of primary human keratinocytes can be controlled by the relative ratio of E2F1 to E2F7. Because deregulated proliferation, differentiation, and apoptosis are hallmarks of cancer, we examined the expression levels of E2F1 and E2F7 in cutaneous squamous cell carcinomas (CSCC). We found that both genes were overexpressed in CSCCs compared with normal epidermis. Furthermore, inhibition of E2F7 in a SCC cell line sensitized the cells to UV-induced apoptosis and doxorubicin-induced apoptosis. Combined, these data suggest that the selected disruption of E2F1 and E2F7 in keratinocytes is likely to contribute to CSCC formation and may prove to be a viable therapeutic target.

Asunto(s)

Apoptosis , Carcinoma de Células Escamosas/etiología , Factor de Transcripción E2F7/fisiología , Queratinocitos/citología , Neoplasias Cutáneas/etiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F7/análisis , Humanos

RESUMEN

The role of thymic versus peripheral epithelium in the regulation of the antigen-specific CD8 T-cell repertoire is still largely unresolved. We generated TCR-beta chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H-2D(b) MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T-cell development in the thymus was observed in these double-transgenic mice. In contrast, peripheral CD8 T-cell responses in the single-transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T-cell responses and antibody production are unchanged in these mice. Peripheral non-responsiveness among CD8 T cells was mediated largely by CD4(+)CD25(+) T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down-regulate CD8 T-cell responses in the periphery. This may have important consequences for the treatment of cervical pre-cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Proteínas Oncogénicas Virales/inmunología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Epitelio/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Queratina-14/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Linfocitos T Reguladores/metabolismo

RESUMEN

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.

Asunto(s)

Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp1/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transglutaminasas/biosíntesis , Biomarcadores de Tumor/análisis , Regulación hacia Abajo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Ensayo de Cambio de Movilidad Electroforética , Humanos , Queratinocitos , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

The AP-2 transcription factor family is presumed to play an important role in the regulation of the keratinocyte squamous differentiation program; however, limited functional data are available to support this. In the present study, the activity and regulation of AP-2 were examined in differentiating human epidermal keratinocytes. We report that (1) AP-2 transcriptional activity decreases in differentiated keratinocytes but remains unchanged in differentiation-insensitive squamous cell carcinoma cell lines, (2) diminished AP-2 transcriptional activity is associated with a loss of specific DNA-bound AP-2 complexes, and (3) there is an increase in the ability of cytoplasmic extracts, derived from differentiated keratinocytes, to phosphorylate AP-2 alpha and AP-2 beta when cells differentiate. In contrast, extracts from differentiation-insensitive squamous cell carcinoma cells are unable to phosphorylate AP-2 proteins. Finally, the phosphorylation of recombinant AP-2 alpha by cytosolic extracts from differentiated keratinocytes is associated with decreased AP-2 DNA-binding activity. Combined, these data indicate that AP-2 trans-activation and DNA-binding activity decrease as keratinocytes differentiate, and that this decreased activity is associated with an enhanced ability to phosphorylate AP-2 alpha and beta.

Asunto(s)

Proteínas de Unión al ADN/genética , Queratinocitos/metabolismo , Factores de Transcripción/genética , Western Blotting , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunohistoquímica , Queratinocitos/citología , ARN Mensajero/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo

RESUMEN

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation.

Asunto(s)

Antígenos de Diferenciación/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Queratinocitos/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Biomarcadores , Proteína Quinasa CDC2/genética , División Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Reporteros/genética , Humanos , Queratina-14 , Queratinocitos/citología , Queratinas/genética , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína smad7 , Transactivadores/genética , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética

RESUMEN

BACKGROUND: Normal keratinocytes (KC) and neoplastic cells derived from a head and neck lesion (SCC-25) were grown as organotypic raft cultures to mimic in vivo architecture in the absence of contaminating cell types. Alterations in gene expression between normal keratinocytes and a head and neck squamous cell carcinoma (HNSSC) cell line (SCC-25) were analysed using gene arrays. MATERIALS AND METHODS: RNA from the organotypic raft cultures were used to probe four gene arrays. Gene expression alterations between the normal and neoplastic cells were identified and analysed using both fold differences and 2-tailed t-test. Four genes from different functional groups were used for immunohistochemical staining of patient tumours to confirm the gene array data. RESULTS: Statistical analysis of the array data revealed 124 significantly altered genes between normal and neoplastic HNSCC cells. These gene expression alterations are associated with a variety of different functional groups and indicate the complexity of gene de-regulation associated with HNSCC. CONCLUSION: This study identified many novel gene alterations associated with HNSCC. The significantly altered gene alterations belong in a variety functional groups including: growth control, apoptosis and detoxication and present new targets for investigating the molecular basis of HNSCC formation.

RESUMEN

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.

Asunto(s)

Proteínas de Ciclo Celular , Diferenciación Celular , Proteínas de Unión al ADN , Queratinocitos/citología , Factores de Transcripción/farmacología , Sitios de Unión , Biomarcadores/análisis , Carcinoma de Células Escamosas/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , División Celular , ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Epidermis , Mutación del Sistema de Lectura , Expresión Génica , Humanos , Mutación Puntual , Isoformas de Proteínas/genética , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Transfección , Células Tumorales Cultivadas

RESUMEN

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLO-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P

Asunto(s)

Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Lesiones Precancerosas/genética , Neoplasias Cutáneas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Queratinocitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas

RESUMEN

In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2. These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2).

Asunto(s)

Carcinoma Basocelular , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas de la Membrana/genética , Neoplasias Cutáneas , Biomarcadores de Tumor , Pruebas de Carcinogenicidad , Regulación Neoplásica de la Expresión Génica , Humanos , Riñón/citología , Receptores Patched , Receptor Patched-1 , Receptor Patched-2 , ARN Mensajero/análisis , Receptores de Superficie Celular , Células Tumorales Cultivadas