Alison L. Dahler

E2F7 Can Regulate Proliferation, Differentiation, and Apoptotic Responses in Human Keratinocytes: Implications for Cutaneous Squamous Cell Carcinoma Formation

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.

Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma

Background:We examine the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC).Methods:Head and neck squamous cell carcinoma cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were then determined in vitro and in vivo.Results:Phosphatidylinositol-3-phosphate kinase, AKT and dual PI3K-mTOR inhibitors caused marked in vitro enhancement of cytotoxicity induced by HDACIs in HNSCC cancer cells. This effect correlates with AKT inhibition and is attenuated by expression of constitutively active AKT. Histone deacetylase inhibitor and phosphatidylinositol-3-phosphate kinase inhibitors (PI3KIs) inhibited tumour growth in xenograft models of HNSCC. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining, respectively. However, we saw no evidence of improved efficacy with an HDACI/PI3KI combination.Interpretation:That PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against HNSCC in vivo.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Cytochrome P450, CYP26AI, is expressed at low levels in human epidermal keratinocytes and is not retinoic acid-inducible

Retinoids, and their synthetic analogues, are well‐established regulators of the squamous differentiation programme both in vivo and in vitro. Despite this, very few studies have focused on the mechanism by which retinoid action is terminated, e.g. metabolism. Recently, a new cytochrome P450 family member (CYP26AI) was cloned. CYP26AI was reported to have substrate specificity for retinoids and to be retinoid‐inducible. In this study, we have examined the expression and retinoic acid (RA) inducibility of CYP26AI in human epidermis and cultured keratinocytes. We found very low levels of CYP26AI mRNA expression in both epidermis and keratinocytes. Furthermore, we found no evidence for RA inducibility of CYP26 mRNA expression. This lack of RA inducibility was not due to inactivity of the retinoids, as we show that transglutaminase was still repressed by RA in the same cultures. Despite the low levels of CYP26AI expression in the keratinocytes, the keratinocytes were still capable of significant RA metabolism. In conclusion, our study reports, for the first time, that CYP26AI is unlikely to contribute to RA metabolism in keratinocytes. These studies also indicate that as yet unknown isoforms of cytochrome P450 may be involved in RA metabolism in keratinocytes.

E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase type 1, in a squamous cell carcinoma cell line

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.

Keratinocyte growth arrest is associated with activation of a transcriptional repressor element in the human cdk1 promoter

In this study we examined the regulation of cdk1 expression in normal human epidermal keratinocytes (HEKs) and neoplastic keratinocytes. Keratinocytes were growth‐arrested by allowing the cells to grow to confluence or by treating them with interferon‐gamma (IFNγ) or 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA). RT‐PCR and Western blot analysis demonstrated that cdk1 was profoundly reduced in growth‐arrested HEKs when compared with dividing HEKs. In contrast, a squamous carcinoma cell line, SCC25, did not growth‐arrest in response to growth inhibitors and did not downregulate cdk1 expression. Transfection of HEKs with a reporter gene driven off a 2.5‐kb fragment of the human cdk1 promoter indicated that the downregulation of cdk1 upon growth arrest was transcriptional. Deletion mapping of the cdk1 promoter indicated that a repressor region was located between −949–−722 bp. This repressor region was not operative in the SCC25 cells. Examination of DNA:protein binding complexes by gel‐shift analysis indicated that nuclear factors from both proliferative and growth‐arrested cells bound to the DNA fragment spanning −949–−722 bp. Further analysis revealed that this binding could be resolved into a constitutive and growth arrest‐specific complex that bound in a similar fashion to regions spanning −892–−831 bp and −831–−774 bp, respectively. The putative growth arrest‐specific complex was not found in contact‐inhibited fibroblasts and was found at very low levels in SCC25 cells, indicating that the putative repressor binding was growth arrest‐specific and possibly keratinocyte‐specific. The binding complexes bound to these two fragments were localized, by competition analysis, to regions −874–−853 bp and −830–−800 bp. This is the first report of a transcriptional repressor being operative during keratinocyte growth arrest. J. Cell. Physiol. 177:474–482, 1998.

Histone deacetylase inhibitors: novel anticancer agents.

Previous studies have established that the regulation of gene expression is dependent upon the nucleosomal integrity of nuclear DNA. To a large extent, this integrity is dictated by the acetylation status of the core histone particles. The acetylation of histones is, in turn, controlled by the combined activity of specific acetylases and deacetylases. Moreover, disruption of histone acetylases and deacetylases has been linked to a wide variety of human cancers. For this reason, the recent availability of potent and specific histone deacetylase inhibitors has provoked a great deal of interest amongst cancer biologists, oncologists and pharmacologists. Within the past 2- 3 years, several novel histone deacetylase inhibitors have been reported, many of which have already been tested in vivo in mouse models of cancer. In this review we focus on the rationale behind the use of histone deacetylase inhibitors as anticancer agents. Moreover, we review some of the recent findings pertaining to the use of these compounds as anticancer agents.

Epithelial expression of human papillomavirus type 16 E7 protein results in peripheral CD8 T‐cell suppression mediated by CD4+CD25+ T cells

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‐β 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‐2Db 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.

Interferon-γ as a regulator of squamous differentiation

Interferon-gamma (IFN-gamma) is a potent immunomodulatory molecule. Recent studies demonstrate that IFN-gamma can induce growth arrest and differentiation in epithelial cells. The signalling pathways controlling growth and differentiation in epithelial cells appears to be different to those regulating immune functions in non-epithelial cells and appear to impact on key cell cycle regulatory genes such as cdk1 and E2F1. In addition, studies with IFN-gamma have highlighted the complexity of the signalling pathways regulating the expression of differentiation markers in squamous differentiating epithelia. Given the actions of IFN-gamma upon epithelial cell growth and differentiation it should be considered a potential regulator of both immune and epithelial cell targets in various inflammatory pathologies such as psoriasis.

Expression vectors encoding human growth hormone (hGH) controlled by human muscle-specific promoters: prospects for regulated production of hGH delivered by myoblast transfer or intravenous injection

We report here the construction of vectors that produce and secrete human growth hormone (hGH) in a muscle-specific manner. The promoter regions of the genes encoding human skeletal alpha-actin (HSA) and troponin I slow (HTnIs) were linked to the hGH-encoding gene. These vectors were designated pHSA2000GH and pHTnIs4200GH, respectively. The HSA and HTnIs promoters linked to the cat gene have previously been shown to be necessary and sufficient for developmentally regulated muscle-specific expression. Furthermore, these promoters function in a fibre-type-specific manner in transgenic animals. Transient and stable transfection analyses with pHSA2000GH and pHTnIs4200GH indicated that: (i) these vectors efficiently synthesized hGH in a muscle-specific manner; (ii) the myogenic master regulatory gene, myoD, a determinant of cell fate, trans-activated expression of hGH in pluripotential non-muscle cells; and (iii) these hGH expression vectors were developmentally regulated during myogenic differentiation. These regulated tissue/fibre-type-specific hGH-containing plasmids are suitable vectors for the delivery and stable production of GH in livestock and GH-deficient hosts by either transgenesis, myoblast transfer or liposome-mediated intravenous injection.