Constitutive Overexpression of Human Telomerase Reverse Transcriptase but Not c-myc Blocks Terminal Differentiation In Human HaCaT Skin Keratinocytes

Abstract

Formation of a well structured epidermis strictly depends on a tight balance between proliferation and differentiation. Accordingly, telomerase, which is restricted to proliferating cells, is downregulated with differentiation. It is unclear, however, whether this inhibition is essential to or only a consequence of the differentiation process. By studying different variants of the HaCaT skin keratinocytes we now show that constitutive overexpression of human telomerase reverse transcriptase (hTERT) in HaCaT-TERT cells (lacking its own differentiation-sensitive promoter) and constitutive expression of the c-myc gene in HaCaT-myc cells caused increased proliferation in conventional cultures; however, this proliferative advantage was not maintained in tissue-like organotypic cocultures. Despite reduced stratification, HaCaT-myc cells were still able to develop a fully differentiated epithelium. HaCaT-TERT cultures, on the other hand, expressed all markers of early but not of terminal differentiation. The failure to differentiate terminally was observed in hTERT mass cultures and individual clones and correlated with an intense nuclear hTERT staining of the uppermost cells of the HaCaT-TERT epithelia. Thus, our data suggest that constitutive overexpression of hTERT does not interfere with epidermal differentiation per se but blocks the terminal stage of differentiation and therefore indicates that hTERT/telomerase plays an active part in the regulatory pathway of epidermal differentiation.

Gain of 11q/cyclin D1 overexpression is an essential early step in skin cancer development and causes abnormal tissue organization and differentiation

“…Sections of 1-, 2-and 4-week-old cultures were stained with antibodies against the basement membrane component type IV collagen (coll IV) as well as characteristic early (keratin K1/10 and involucrin) and late (filaggrin and loricrin) epidermal differentiation markers. As demonstrated in Figure 7 for 4-week-old cultures, Hneo control cells exhibited a similar distribution of differentiation markers as described previously for the parental HaCaT cells (Cerezo et al, 2003). Keratin 1 and 10 were expressed in all suprabasal layers (Figure 7a), the honeycomb-like involucrin staining started in the second suprabasal layer (Figure 7b) and filaggrin was present in the uppermost living cell layer (Figure 7c).…”

“…HaCaT cells are particularly suited because they exhibit a number of chromosomal aberrations characteristic for skin SCCs but lack gain of 11q, are non-tumorigenic, and still possess the ability to form a normally differentiated epidermis-like epithelium under appropriate conditions (Boukamp et al, 1988(Boukamp et al, , 1997Lehman et al, 1993;Schoop et al, 1999;Cerezo et al, 2003). Figure 2 Cyclin D1 is overexpressed in keratoacanthomas (KAs) and squamous cell carcinomas (SCCs).…”

“…To test this, we performed organotypic co-culture (OTC) with the two Hneo clones and the three Hcyclin clones and analysed them in at least two independent experiments. Growing on a collagen gel with integrated fibroblasts at the air-medium interphase, HaCaT cells are able to form a stratified and well-differentiated epidermis-like epithelium within 4 weeks (Schoop et al, 1999;Cerezo et al, 2003). Accordingly, after 14 days of cultivation, both vector-transfected control cells had formed an only poorly stratified (mostly only three cell layers) epithelium, whereas after 4 weeks, the epidermis-like epithelium was fully differentiated and consisted of distinct basal, spinous and granular, as well as parakeratotic cornified layers.…”

Cigarette smoke components inhibited intercellular communication and differentiation in human pancreatic ductal epithelial cells

“…75,76 As stem and progenitor cells become more differentiated-or when tumor cells are induced to differentiate, the activity of this enzyme sharply decreases. 71,77,78 In essence, telomerase activity is an inverse marker of differentiation. Consistent with this telomerase phenomenon, the induction of differentiation in the H6c7 pancreatic cells with the adenylate cyclase stimulator, forskolin and IBMX, induced a sharp decline in telomerase activity.…”

Improving cell therapy—experiments using transplanted telomerase-immortalized cells in immunodeficient mice

“…hTERT-expressing cells show extensive changes in gene expression patterns (Smith et al, 2003;Cerezo et al, 2003). The ability of hTERT to exert a variety of effects that counteract cell death is striking (Sung et al, 2005) and these effects have been reported also in whole animals overexpressing TERT; in mice this protects against experimental heart failure ( Oh et al, 2001) and has many other effects in the cardiovascular system (Serrano and Andres, 2004).…”