Telomerase is Not an Epidermal Stem Cell Marker and is Downregulated by Calcium  Jackie R. Bickenbach  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1045-1052 (December 1998) DOI: 10.1046/j.1523-1747.1998.00420.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cellular localization of telomerase activity within the basal layer of the epidermis. (A) Immunofluorescence of LRC in mouse ear epidermis. Sections of ear tissue from mice labeled 4 wk previously with BrdU were double-stained with antibodies to BrdU (green) and to keratin K14 (red), which highlights the epidermis. Only the slowly cycling LRC (yellow) retain the BrdU label for 4 wk. Note that although the K14 gene is only expressed in basal cells, the K14 protein is highly resistant to degradation and remains detectable in cells even after they have differentiated.Scale bar: 50 μm. (B, C) Telomerase activity in mouse epidermis. (B) TRAP assay was performed from extracts from epidermal cells freshly isolated from the basal layer of adult mouse ear epidermis and selected by differential adhesion to collagen type IV. Extracts were prepared from 105 AC (lanes 1–4) and NAC (lanes 5–8) and mouse PDV tumor cell line (Fuseniget al. 1978) (as positive control,lanes 9–12). Cells were either pretreated with RNase (+) or not (–) before assaying for telomerase. Ladders represent 6 bp repeats starting with 40 bp. Samples represent 1000-, 100-, and 10-cell equivalents. (C) AC and NAC were diluted with telomerase-deficient fibroblasts (1:1, 1:5, 1:10, 1:50, 1:100) and assayed for telomerase activity. Note that telomerase activity is detectable in the NAC up to a dilution of 1:50, whereas activity is already significantly diluted in the 1:10 dilution of the AC. Journal of Investigative Dermatology 1998 111, 1045-1052DOI: (10.1046/j.1523-1747.1998.00420.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 In situ hybridization of normal human skin. Skin hybridized with an anti-sense (A) and sense (B) probe of hTR, the RNA component of the telomerase complex. Hybridization signals of varying levels are seen in patches of basal cells mostly along the lower parts of the rete ridges of the epidermis (e) (marked witharrows). Expression in the dermis (d) possibly represents that from blood vessels, whereas hybridization in the upper granular layer is likely to be artifactual.Insert: higher magnification of an hRT expressing rete ride. (B) Similarly, the control sense probe shows some background hybridization in the upper granular layer of the epidermis, whereas the basal layer of the epidermis and the dermis show no hybridization. (C) Labeling of a skin section with an anti-sense probe of histone-3, which indicates proliferation (S-phase cells, marked witharrowheads), marks single cells, which are more evenly distributed throughout the basal layer of the epidermis.Scale bar: 100 μm. Journal of Investigative Dermatology 1998 111, 1045-1052DOI: (10.1046/j.1523-1747.1998.00420.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ca++-dependent regulation of differentiation and telomerase activity in human skin keratinocytes. (A) Keratinocyte cultures treated with 2 μM calcium ionophore A23187 for 24 h and then cultured in regular medium were analyzed by phase contrast microscopy for the induction of differentiation visualized here by the formation of cornified envelopes. Compared with the control cells (c) the calcium ionophore-treated keratinocytes already contain some large cornified envelopes (arrows) after 1 d with a maximal number by 3 d (3d). After 5 d, proliferating small polygonal cells reappeared (5d, left-hand side), and had repopulated the culture by day 7 (7d).Scale bar: 100 μm. (B) Cell extracts were assayed for telomerase activity before calcium ionophore A23187 treatment (K 0d,lanes 2–4), after 1 d when the calcium ionophore containing medium was replaced (Cal 1d,lanes 7–8) and at 2–5 d (Cal 2d to 5d,lanes 9–16). Negative controls were lysis buffer (c,lane 1) and RNase treated extracts (+,lanes 2 and 19) and positive controls HaCaT cells (lane 19). As a further control keratinocytes were treated only with the ionophore solvent dimethylsulfoxide and telomerase activity was determined after 1 d (D 1d,lanes 5–6)and 5 d (D 5d,lanes 17–18). Note that the first reduction of telomerase activity was already seen after 1 d with virtually complete inhibition by day 2. Journal of Investigative Dermatology 1998 111, 1045-1052DOI: (10.1046/j.1523-1747.1998.00420.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Sensitivity of telomerase activity to Ca++. Telomerase-containing lysates were isolated from normal human keratinocytes (K) and HaCaT cells (H). Sensitivity to Ca++ was measured by using the TRAP assay (A) and increasing the concentrations of Ca++ from 0 to 2 mM, with a standard Mg++ concentration of 1.5 mM (keratinocytes,lanes 2–8; HaCaT cells,lanes 9–13), or by increasing the concentrations of Mg++ from 1 to 3.5 mM (keratinocytes,lanes 14–16; HaCaT cells,lanes 17–18) during the telomere repeat amplification step. Controls were lysis buffer (C,lane 1) and normal keratinocytes run under standard conditions (1.5 mM Mg++ with no additional Ca++) (lane 19). (B) As further controls various combinations of Ca++ or Mg++ concentrations (numbers below the lines represent mM) were tested in the telomerase reaction (first step) and PCR reaction (second step).Lanes 1 and 12 are lysis buffer controls. Inlanes 2–7 increasing concentrations of Mg++ were added to the first step with no changes in the second step. Inlanes 8–11 the Mg++ or Ca++ concentrations in the first step were kept unchanged but in the second step increasing amounts of Mg++ or Ca++ were added. Similarly, increasing concentrations of Mg++ or Ca++ during PCR reaction either individually (lanes 15–19) or in combination (lanes 23–24) did not lead to PCR amplification artifacts (nonspecific inhibition). Only increasing concentrations of Ca++ during telomerase reaction reproducibly caused inhibition of primer elongation (lanes 21–22). Journal of Investigative Dermatology 1998 111, 1045-1052DOI: (10.1046/j.1523-1747.1998.00420.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The non-PCR-based (direct) telomerase assay was performed with partly purified extracts from HeLa cells in the presence of increasing concentrations of Ca++ (0–2 mM Ca++,lanes 2–7; C = buffer control,lane 1) showing the same dose-dependent inhibition of telomerase with increasing Ca++ concentrations. The reversible effect of Ca++ inhibition by increasing the EGTA concentration is demonstrated inlanes 8–11. Control reactions with 1 mM EGTA and no additional Ca++ allowed full activity (lane 8). This activity was completely abolished by addition of 2 mM Ca++ (lane 9), but could be restored by the addition of 2 mM EGTA when added either during the telomere repeat elongation step (20 min after incubation without EGTA [*],lane 10) or when the assay was started in the presence of 2 mM EGTA (lane 11). Journal of Investigative Dermatology 1998 111, 1045-1052DOI: (10.1046/j.1523-1747.1998.00420.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions