A transgenic mouse model with cyclin D1 overexpression results in cell cycle, epidermal growth factor receptor, and p53 abnormalities.

The cyclin D1 oncogene is critical in the progression of the cell cycle through the G1 phase. It is frequently overexpressed in squamous cell carcinomas originating from the head/neck and esophagus. Yet, the functional consequences of aberrant cyclin D1 overexpression are not entirely understood apart from increased cell proliferation. To address this question, we have developed a transgenic mouse model in which the EBV ED-L2 promoter targets cyclin D1 to the stratified squamous epithelium in a tissue-specific fashion to the tongue and esophagus, thereby resulting in a dysplastic phenotype. We now demonstrate that the dysplastic phenotype is associated with increased cell proliferation based on proliferating cell nuclear antigen overexpression and abnormalities in cyclin-dependent kinase 4, epidermal growth factor receptor, and p53. In aggregate, these studies suggest that alterations in certain oncogenes and tumor suppressor genes occur early during head/neck and esophageal carcinogenesis.

The association of Epstein-Barr virus DNA with esophageal squamous cell carcinoma.

The Epstein-Barr virus (EBV), a member of the herpesviruses, is a double stranded 170 kilobase DNA virus important in many human benign and malignant conditions. It has been implicated in the pathogenesis of proliferative diseases of lymphocytes and tumors of epithelial derivation. The etiology and pathogenesis of esophageal squamous cell carcinoma (ESCC) is thought to involve a combination of genetic and environmental events which lead to epithelial cell transformation. The aim of this study was to determine whether an association exists between EBV and ESCC. DNA was extracted from 16 human ESCC cell lines and microdissected tumor specimens from 60 patients. The polymerase chain reaction was used to amplify a 400 base pair fragment corresponding to the BamH1W fragment repeat sequence of EBV. Southern blotting, utilizing an oligonucleotide probe specific for the BamH1W sequence, was used to confirm positive results and increase sensitivity of detection. 5/60 tumor samples and 1/16 ESCC cell lines were positive for the EBV sequence. Positive tumor samples were estimated to contain one copy of EBV per 20 cellular genomes. Given the role of EBV in other tumors of epithelial derivation, it is possible that EBV may contribute to the molecular pathogenesis of ESCC.

Cyclin D1 overexpression combined with N-nitrosomethylbenzylamine increases dysplasia and cellular proliferation in murine esophageal squamous epithelium.

We previously described the oral-esophageal tissue-specific expression of cyclin D1 with the Epstein-Barr virus ED-L2 promoter in transgenic mice, and resulting dysplasia. Given the evidence for an interplay between environmental and genetic factors in esophageal squamous carcinogenesis, the aim of this study was to determine the potential cooperation of the nitrosamine compound N-nitrosomethylbenzylamine (NMBA), an esophageal specific carcinogen, in the cyclin D1 transgenic mice. NMBA was first demonstrated to induce dysplasia in two strains of inbred mice, C57BL/6 and FVB/N. Subcutaneous NMBA was then administrated to wild type and transgenic mice beginning at 4 weeks of age. Mice were monitored for the duration of the study for general appearance, activity and weight, and were euthanized at 12 and 15 months. Histopathologic analysis revealed increased severity of dysplasia in cyclin D1 mice treated with NMBA compared with treated age-matched wild-type mice and untreated mice. There was also increased proliferating cell nuclear antigen (PCNA) expression in the esophagi of NMBA treated cyclin D1 mice. Taken together, these findings suggest that a genetic alteration, specifically cyclin D1 overexpression and a chemical carinogen, NMBA, may cooperate to increase the severity of esophageal squamous dysplasia, a prominent precursor to carcinoma.

The Krüppel-like transcriptional factors Zf9 and GKLF coactivate the human keratin 4 promoter and physically interact.

Zf9/CPBP/KLF6 is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines. Additionally, Zf9 localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, Zf9 transactivates the human keratin 4 (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of Zf9 and GKLF/KLF4, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between GKLF and Zf9, a novel finding for Krüppel-like family members.

Adenocarcinoma of the esophagogastric junction.

Adenocarcinoma of the esophagogastric junction (EGJ) has increased rapidly in incidence in the latter half of the twentieth century. The increase in incidence has affected white men between the ages of 40 and 60 disproportionately. Understanding the etiology and improving treatment requires careful classification of EGJ tumors. A recent consensus conference recognized three types of EGJ adenocarcinomas: distal esophageal, cardia, and subcardia gastric. Distal esophageal adenocarcinomas are associated with Barrett's esophagus. Helicobacter pylori infection may play a role in some adenocarcinomas of the subcardia, but the association is unproven. Therapy for all types of EGJ tumors is surgical, but multimodal forms of treatment are commonly used because of the advanced stage at which these tumors often present. Several endoscopic options exist for primary therapy of early-stage tumors and for palliation.

The keratinocyte-specific Epstein-Barr virus ED-L2 promoter is regulated by phorbol 12-myristate 13-acetate through two cis-regulatory elements containing E-box and Krüppel-like factor motifs.

We previously employed 782 base pairs of the Epstein-Barr virus ED-L2 early lytic cycle promoter in a transgenic mouse model to target cyclin D1 to the stratified squamous epithelium of the tongue and esophagus. This promoter is located 5' to the transcriptional start site of a short open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (LMP-1 oncogene) open reading frame. We studied transcriptional regulation of the ED-L2 promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The transcriptional activity of the ED-L2 promoter was stimulated 40-fold by PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter demonstrated that the sequences necessary for PMA-stimulated trans-activation were located in two separate cis-regulatory regions of the promoter: -187 to -164 and -144 to -114 base pairs from the transcription start site of BNLF-2A. Importantly, mutation of critical base pairs in each region was sufficient to abolish PMA-stimulated trans-activation in the native ED-L2 promoter. Region -187 to -164 contains a CACCTG (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regions do not, however, demonstrate enhancer characteristics when tested in a heterologous minimal promoter system. Variations of the CACACCC motif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Krüppel-like family of transcription factors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demonstrated that one complex binding the -187 to -164 region containing the CACCTG nucleotides has characteristics of the helix-loop-helix protein upstream stimulatory factor, whereas a factor binding the CACACCC motif may be a member of the Krüppel-like family. These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-L2 promoter in squamous epithelial cells.

Transcriptional regulation of the differentiation-linked human K4 promoter is dependent upon esophageal-specific nuclear factors.

The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.

Transactivation of the human keratin 4 and Epstein-Barr virus ED-L2 promoters by gut-enriched Krüppel-like factor.

The Krüppel-like family of transcription factors comprises genes that appear to have tissue-restricted functions. Expression of gut-enriched Krüppel-like factor (GKLF) may be important in the switch from proliferation to differentiation in the squamous epithelium. We sought to determine transcriptionally mediated effects of GKLF on two promoters active in the esophageal squamous epithelium, namely the Epstein-Barr virus ED-L2 and human keratin 4 promoters. Both promoters contain a CACCC-like motif previously shown to bind GKLF. To determine whether GKLF regulates genes containing this element, we first demonstrated expression and then cloned the full-length human GKLF from an esophageal squamous carcinoma cell line. In a transient transfection system, GKLF increased the activity of both promoters >25-fold, localized to regions containing the CACCC-like element. Recombinant GKLF specifically binds the CACCC-like motif in both promoters. GKLF epitope-tagged protein leads to the formation of two proteins of 65 and 34 kDa. The chromatographically purified 65-kDa protein binds the CACCC-like element from both Epstein-Barr virus ED-L2 and keratin 4 promoters, which is not attenuated by the 34-kDa protein. In summary, GKLF is expressed in esophageal squamous epithelial cells and transcriptionally activates two esophageal epithelial promoters important at the transition toward differentiation.

A study of long-attending psychiatric day-patients and the services provided for them.

A year's follow-up study of day-hospital long-attenders underlined doubts as to the adequacy of service provision in urban areas of England and Wales for the increasing numbers of chronic psychiatric patients living in the community. Very few of the 82 men and 53 women were married or capable of employment, and both psychiatric and physical morbidity were high; 2 young men committed suicide and 3 elderly men died within a year of discharge. Over the age of 45 men tended to be socially isolated and over the age of 60 they were also physically ill; a number probably required better residential care. Few women under 45 attended, possibly because the services offered were unacceptable.

Mouse keratin 4 is necessary for internal epithelial integrity.

Keratins are intermediate filaments of epithelial cells. Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes.

Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age.