Stuart H. Yuspa

Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6

The BNLF-1 gene of Epstein-Barr virus (EBV) encodes the latent membrane protein (LMP), one of the putative oncogene products of the virus. This gene has been expressed from two different enhancer-promoter constructs in transgenic mice, to determine its biological activity and possible contribution to oncogenesis. While transgenic mice expressing LMP in many tissues demonstrated poor viability, expression of LMP specifically in the epidermis induces a phenotype of hyperplastic dermatosis. Concomitant with the expression of LMP in this tissue (and in the esophagus) is an induction of the expression of a hyperproliferative keratin, K6, at aberrant locations within the epidermis. The epithelial hyperplastic phenotype caused by the LMP-encoding transgenes implies that the LMP plays a role in the acanthotic condition of the tongue epithelium in the human EBV- and HIV-associated syndrome oral hairy leukoplakia, as well as possibly predisposing the nasopharyngeal epithelium to carcinogenesis.

Subunit structure of the mouse epidermal keratin filament

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.

Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progressionxa0in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled Membrane Channels and Transporters in Cancers.

Sequential Changes in Gene Expression during Epidermal Differentiation

Genes encoding the major proteins expressed in mouse epidermis have been isolated and characterized. Using a combination of in situ hybridization with RNA probes, which are specific for individual mRNA’s, and indirect immunofluorescence with specific antisera, which were elicited with synthetic peptides corresponding to unique sequences within each protein, it is possible to show that these genes belong to at least four subsets: those expressed predominantly in the proliferating basal layer of the epidermis (keratins 5 and 14); those expressed predominantly in the differentiated suprabasal spinous layer and to a less extent the granular layer (keratins 1 and 10); those initially expressed in the upper spinous layer but most prominently in the granular layer (the gene encoding the precursor for filaggrin, a protein thought to promote keratin filament aggregation and the gene encoding a major component of the comified envelope) and those only expressed under hyperproliferative conditions (keratin 6). In order to identify sequences regulating the expression of these genes, transgenic mice have been produced which contain one of the human differentiation-associated keratin genes (K1). The human K1 gene is located within a 12 kilobase (Kb) fragment and is flanked by 2 Kb upstream and 3 Kb downstream. This DNA fragment contains sufficient sequence information for tissue-specific, developmental-specific and differentiation-specific expression.

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene.

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.

Stem Cells in Nonmelanoma Skin Cancer

Mosaic pattern analysis and genetic mutations common to all cells of a cancer show that squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as squamous dysplasias, are clonal, whereas focal hyperplasias are polyclonal. One compartment of putative stem cells in the skin is located in the bulge of the hair follicle. Cells in this compartment are multipotent and can give rise to progeny that differentiate into any of the epidermal cells or adnexal organs. The interfollicular epidermal proliferative unit (EPU) in normal skin is a columnar group of differentiating cells overlying 10–12 basal cells and is believed to be derived from a single, centrally located stem cell with a more limited potential than the follicular stem cell. Stem cells in the skin cycle slowly and are identified by retaining a pulsed DNA marker for extended periods. Other markers include increased expression of β31 or β4 integrins; decreased expression of the transferrin receptor or connexin 43; and unique expression of keratins 15,17, and 19. BCCs appear to arise from follicular bulge stem cells and are associated with genetic changes in the Sonic Hedgehog developmental pathway. SCCs can arise from stem cells in the interfollicular EPU and infundibulum of the hair follicle as well as the bulge. Benign squamous neoplasms may also arise from the more differentiated cell populations. Alterations in the ras pathway have been implicated in both experimental and human squamous cell carcinogenesis. Genetic or epigenetic changes in stem cell markers that have been associated with squamous cell neoplasms include alterations in integrins, telomerase, c-myc, and p63.