Constitutive Overexpression of Human Telomerase Reverse Transcriptase but Not c- myc Blocks Terminal Differentiation In Human HaCaT Skin Keratinocytes  Ana Cerezo, Hans-Jürgen Stark, Sharareh Moshir, Petra Boukamp  Journal of Investigative Dermatology  Volume 121, Issue 1, Pages 110-119 (July 2003) DOI: 10.1046/j.1523-1747.2003.12304.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 hTERT expression and telomerase activity. (A) hTERT expression was investigated by reverse transcription–PCR using primers that are specific for detecting the four major splice variants: full length (fl), α-, β-, and α/β splice form (TERT-HT2026F and TERT-HT2482R;Kilian et al, 1997). With the exception of HaCaT-TERT cells (2 μL), 5 μL of each PCR product was loaded per lane. Fibroblasts and normal human keratinocytes (NHK) were included for comparison and GAPDH as internal control. NHK sample was subjected to 35 cycles of PCR amplification for comparison of the expression level, and subjected to 40 cycles of amplification in order to detect alternative splice variants. (B) Telomerase activity was measured by TRAPeze (Oncor). Fifty nanograms of protein extract were used in each assay. HaCaT, HaCaT-myc, and HaCaT-TERT extracts (mass culture) were serially diluted (1:1=undiluted, 1:2, 1:5, and 1:10, as indicated) with telomerase-negative fibroblasts to guarantee an unaltered protein concentration. Telomerase activity from the undiluted HaCaT extracts was taken as 100% and the dilutions were calculated accordingly. An RNase inactivated HaCaT sample (+) and a fibroblast sample (F) were included as negative controls. IC=internal control. (C) Southern blot analysis of HaCaT-and HaCaT variants using RsaI for DNA digestion and a telomere-specific DNA probe (Oncor) for hybridization. Marker=λ/HindIII. The mean telomere repeat fragment length (TRFL) are indicated. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Proliferation characteristics of HaCaT, HaCaT-myc, and HaCaT-TERT cells in conventional cultures. (A) Growth curve of HaCaT, HaCaT-myc, and HaCaT-TERT cells during a 96 h growth period. Cells (105) were initially seeded and triplicate cultures counted at daily intervals. (B) BrdU incorporation analysis of the cells grown in conventional cultures on plastic. BrdU labeling index of HaCaT-myc and HaCaT-TERT cells is expressed as a percentage of the parental HaCaT cells. (C) Representative immunofluorescence micrographs of TUNEL stained cultures from HaCaT, HaCaT-myc, and HaCaT-TERT cells 48 h after plating. (D) Colony forming efficiency of HaCaT, HaCaT-myc, and HaCaT-TERT cells. Representative 2 wk old cultures with a seeding density of 500 cells are shown. Colony number and size distribution represent the mean value of four experiments. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 c-Myc expression analysis. (A) Reverse transcription–PCR showing c-myc mRNA expression in HaCaT, HaCaT-myc, and HaCaT-TERT cells. Fibroblasts and normal human keratinocytes were included for comparison. GAPDH served as loading control. (B) Comparison of c-Myc expression in HaCaT, HaCaT-myc, and HaCaT-TERT cells by western blot analysis. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Growth behavior of HaCaT, HaCaT-myc, and HaCaT-TERT cells in organotypic cocultures. (A) Hematoxylin/eosin stained histologic sections of HaCaT, HaCaT-myc, and HaCaT-TERT cells after 1 and 3 wk of growth in organotypic cocultures. (B) Percentage of BrdU-labeled cells in 1, 2, and 3 wk old epithelia. Results are expressed as percentage of total cell number. (C) TUNEL staining of organotypic cocultures 1 and 3 wk after plating. Note the different localization of TUNEL-positive cells in HaCaT and HaCaT-myc cultures versus cultures from HaCaT-TERT cells. Bar=25 μm. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of early and late differentiation markers in 3 wk old organotypic cocultures of HaCaT, HaCaT-myc, and HaCaT-TERT cells. (A) Immunofluorescence staining of the basement membrane component type IV collagen (green) and the keratin pair K1/K10 (red), (B) integrin α6, and (C) involucrin as early markers and (D) filaggrin, (E) loricrin, and (F) transglutaminase 1 as late markers in HaCaT, HaCaT-myc and HaCaT-TERT cultures. Discontinuous lines indicate in (B) the uppermost part of the epithelium and in (C, F) the position of the basement membrane zone. Bar=25 μm. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of hTERT in the epidermis and 3 wk old organotypic cocultures of HaCaT and HaCaT-TERT cells. Immunofluorescence staining of hTERT (red nuclear staining) and basement membrane component nidogen (green) in normal epidermis, HaCaT, and HaCaT-TERT organotypic cocultures. The nuclei are counterstained with Hoechst. Bar=25 μm. Journal of Investigative Dermatology 2003 121, 110-119DOI: (10.1046/j.1523-1747.2003.12304.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions