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• W2024659633 abstract "Human telomerase RNA (hTER) expression in skin was examined by in situ hybridization analysis. All newborn foreskins examined (n = 5) expressed hTER in epidermal basal cells at moderate levels. Telomerase RNA was not detectable in most adult specimens from sun protected areas (six of seven), whereas all samples obtained from sun exposed areas (n = 8) showed moderate hTER signals in epidermal basal cells. Telomerase RNA was also detected at moderate to strong levels in basal cells of psoriasis, contact dermatitis, and the proliferative cells of the anagen hair bulb. Basal cell carcinoma samples (14 of 15) had moderate to high hTER expression throughout the entire tumor, whereas squamous cell carcinomas (seven of eight) showed variable intensities of hTER expression but only in the cells at the periphery of tumor nests. All melanomas examined (n = 5) had moderate hTER expression in all tumor cells. The hTER signal intensities in skin tumors did not correlate with the age or sex of the donors, the clinical history of the lesions, or the histologic subtypes. To address whether hTER expression correlated with the proliferative state, sequential sections were stained with anti-Ki-67 antibody, a proliferation marker. In newborn foreskins, squamous cell carcinomas, and basal cell carcinomas, the distributions of hTER and Ki-67 were similar but not always identical. Telomerase RNA was more abundant than Ki-67 in the basal and suprabasal layer of newborn foreskins, suggesting that hTER expression is present both in actively cycling and in resting cells. Human telomerase RNA (hTER) expression in skin was examined by in situ hybridization analysis. All newborn foreskins examined (n = 5) expressed hTER in epidermal basal cells at moderate levels. Telomerase RNA was not detectable in most adult specimens from sun protected areas (six of seven), whereas all samples obtained from sun exposed areas (n = 8) showed moderate hTER signals in epidermal basal cells. Telomerase RNA was also detected at moderate to strong levels in basal cells of psoriasis, contact dermatitis, and the proliferative cells of the anagen hair bulb. Basal cell carcinoma samples (14 of 15) had moderate to high hTER expression throughout the entire tumor, whereas squamous cell carcinomas (seven of eight) showed variable intensities of hTER expression but only in the cells at the periphery of tumor nests. All melanomas examined (n = 5) had moderate hTER expression in all tumor cells. The hTER signal intensities in skin tumors did not correlate with the age or sex of the donors, the clinical history of the lesions, or the histologic subtypes. To address whether hTER expression correlated with the proliferative state, sequential sections were stained with anti-Ki-67 antibody, a proliferation marker. In newborn foreskins, squamous cell carcinomas, and basal cell carcinomas, the distributions of hTER and Ki-67 were similar but not always identical. Telomerase RNA was more abundant than Ki-67 in the basal and suprabasal layer of newborn foreskins, suggesting that hTER expression is present both in actively cycling and in resting cells. basal cell carcinoma human telomerase RNA component In normal human somatic cells, the end region of chromosomes or telomeres gradually shorten with each cell division (Harley et al., 1990Harley C.B. Futcher A.B. Greider C.W. Telomeres shorten during aging of human fibroblasts.Nature. 1990; 345: 458-460Crossref PubMed Scopus (4368) Google Scholar; Hastie et al., 1990Hastie N.D. Dempster M. Dunlop M.G. Thompson A.M. Green D.K. Allshire R.C. Telomere reduction in human colorectal carcinoma and with aging.Nature. 1990; 346: 866-868Crossref PubMed Scopus (1443) Google Scholar; Blackburn, 1991Blackburn E.H. Structure and function of telomeres.Nature. 1991; 350: 569-573Crossref PubMed Scopus (2897) Google Scholar). The shortening of telomeres to a critical length is thought to be the signal inducing cells into a state of irreversible growth arrest (Allsopp et al., 1992Allsopp R.C. Vaziri H. Patterson C. et al.Telomere length predicts replicative capacity of human fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10114-10118Crossref PubMed Scopus (1881) Google Scholar; Allsopp and Harley, 1995Allsopp R.C. Harley C.B. Evidence for a critical telomere length in senescent human fibroblasts.Exp Cell Res. 1995; 219: 130-136Crossref PubMed Scopus (341) Google Scholar). In contrast, proliferative male germline cells can maintain stable telomere lengths. Unlike somatic cells, these proliferative cells constitutively express high levels of telomerase, a reverse transcriptase, which is capable of adding new telomeric repeats to chromosomes (Greider and Blackburn, 1985Greider C.W. Blackburn E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.Cell. 1985; 43: 405-413Abstract Full Text PDF PubMed Scopus (2442) Google Scholar; Mantell and Greider, 1994Mantell L.L. Greider C.W. Telomerase activity in germline and embryonic cells of Xenopus.EMBO J. 1994; 13: 3211-3217Crossref PubMed Scopus (116) Google Scholar). Therefore, telomerase is considered important for continuous cell divisions. Consistent with this hypothesis, most tumor tissues and immortal cells (Counter et al., 1992Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1877) Google Scholar, Counter et al., 1994aCounter C.M. Botelho F.M. Wang P. Harley C.B. Bacchetti S. Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.J Virol. 1994; 68: 3410-3414Crossref PubMed Google Scholar, Counter et al., 1994bCounter C.M. Hirte H.W. Bacchetti S. Harley C.B. Telomerase activity in human ovarian carcinoma.Proc Natl Acad Sci USA. 1994; 91: 2900-2904Crossref PubMed Scopus (714) Google Scholar; Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6287) Google Scholar; Broccoli et al., 1995Broccoli D. de Young J.W. Lange T. Telomerase activity in normal and malignant hematopoietic cells.Proc Natl Acad Sci USA. 1995; 92: 9082-9086Crossref PubMed Scopus (707) Google Scholar; Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar), as well as regenerative cells, such as stem cells of skin (Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar), intestine (Hiyama et al., 1996Hiyama E. Hiyama K. Tatsumoto N. Kodama T. Shay J.W. Yokoyama T. Telomerase activity in human intestine.Int J Oncol. 1996; 9: 453-458PubMed Google Scholar), and blood (Hiyama et al., 1995Hiyama K. Hirai Y. Kyoizumi S. et al.Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.J Immunol. 1995; 155: 3711-3715PubMed Google Scholar), possess telomerase activity, whereas most normal somatic tissues contain minimal or no detectable telomerase activity. There are, however, some exceptions, such as normal activated lymphocytes, which exhibit some detectable telomerase activity (Hiyama et al., 1995Hiyama K. Hirai Y. Kyoizumi S. et al.Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.J Immunol. 1995; 155: 3711-3715PubMed Google Scholar; Buchkovich and Greider, 1996Buchkovich K.J. Greider C.W. Telomerase regulation during entry into the cell cycle in normal human T cells.Mol Biol Cell. 1996; 7: 1443-1454Crossref PubMed Scopus (201) Google Scholar; Ogoshi et al., 1997Ogoshi M. Takashima A. Taylor R.S. Mechanisms regulating telomerase activity in murine T cells.J Immunol. 1997; 158: 622-628PubMed Google Scholar). Telomerase activities in these studies were determined using the telomeric repeat amplification protocol (Piatyszek et al., 1995Piatyszek M.A. Kim N.W. Weinrich S.L. Hiyama K. Hiyama E. Wright W.E. Shay J.W. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP).Meth Cell Sci. 1995; 17: 1-15Crossref Scopus (390) Google Scholar). The telomeric repeat amplification protocol is a functional assay using fresh or fresh frozen cell or tissue extracts, and does not identify which cells in tissue samples express telomerase activity (Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6287) Google Scholar; Piatyszek et al., 1995Piatyszek M.A. Kim N.W. Weinrich S.L. Hiyama K. Hiyama E. Wright W.E. Shay J.W. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP).Meth Cell Sci. 1995; 17: 1-15Crossref Scopus (390) Google Scholar; Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar). Therefore, it remains unclear which cutaneous cell subpopulations express telomerase activity. Telomerase is composed of RNA and protein components. The human telomerase RNA component (hTER) has been recently cloned and shown to be critical for telomerase activity (Feng et al., 1995Feng J. Funk W.D. Wang S.-S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2014) Google Scholar). Although telomerase RNA expression levels do not always correlate with telomerase activity by northern analysis (Avilion et al., 1996Avilion A.A. Piatyszek M.A. Gupta J. Shay J.W. Bacchetti S. Greider C.W. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.Cancer Res. 1996; 56: 645-650PubMed Google Scholar), this discrepancy might be due to intra-tumor heterogeneity in large tumors (Avilion et al., 1996Avilion A.A. Piatyszek M.A. Gupta J. Shay J.W. Bacchetti S. Greider C.W. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.Cancer Res. 1996; 56: 645-650PubMed Google Scholar). Therefore, it is important to determine hTER expression and activity of individual cells in tissues and tumors. The histologic distributions of hTER were examined by in situ hybridization in lung tumors (Soder et al., 1997Soder A.I. Hoare S.F. Muir S. Going J.J. Parkinson E.K. Keith W.N. Amplification, increased dosage and in situ expression of telomerase RNA gene in human cancer.Oncogene. 1997; 14: 1013-1021Crossref PubMed Scopus (162) Google Scholar; Yashima et al., 1997bYashima K. Litzky L.A. Kaiser L. et al.Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.Cancer Res. 1997; 57: 2373-2377PubMed Google Scholar) and malignant and nonmalignant lymph nodes (Yashima et al., 1997aYashima K. Piatyszek M.A. Saboorian H.M. Virmani A.K. Brown D. Shay J.W. Gazdar A.F. Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.J Clin Pathol. 1997; 50: 110-117Crossref PubMed Scopus (100) Google Scholar). In lung, hTER was expressed in tumor but not normal adjacent tissue (Soder et al., 1997Soder A.I. Hoare S.F. Muir S. Going J.J. Parkinson E.K. Keith W.N. Amplification, increased dosage and in situ expression of telomerase RNA gene in human cancer.Oncogene. 1997; 14: 1013-1021Crossref PubMed Scopus (162) Google Scholar; Yashima et al., 1997bYashima K. Litzky L.A. Kaiser L. et al.Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.Cancer Res. 1997; 57: 2373-2377PubMed Google Scholar). In lymph nodes, hTER was expressed at high levels in tumor cells and at low levels in lymphocytes located in germinal centers (Yashima et al., 1997aYashima K. Piatyszek M.A. Saboorian H.M. Virmani A.K. Brown D. Shay J.W. Gazdar A.F. Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.J Clin Pathol. 1997; 50: 110-117Crossref PubMed Scopus (100) Google Scholar). These observations suggest that the detection of hTER is useful for the diagnosis of malignant cells in tissues. There is mounting evidence that telomerase activity is associated with cell proliferation and differentiation. In normal murine and human lymphocytes, telomerase activity is upregulated upon activation in parallel with cell proliferation (Hiyama et al., 1995Hiyama K. Hirai Y. Kyoizumi S. et al.Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.J Immunol. 1995; 155: 3711-3715PubMed Google Scholar; Ogoshi et al., 1997Ogoshi M. Takashima A. Taylor R.S. Mechanisms regulating telomerase activity in murine T cells.J Immunol. 1997; 158: 622-628PubMed Google Scholar). Conversely, immortal cells, which express telomerase activity, downregulate this activity when they are driven to quiescence by growth factor deprivation or contact inhibition (Holt et al., 1996Holt S.E. Wright W.E. Shay J.W. Regulation of telomerase activity in immortal cell lines.Mol Cell Biol. 1996; 16: 2932-2939Crossref PubMed Scopus (357) Google Scholar, Holt et al., 1997Holt S.E. Disner D.L. Shay J.W. Wright W.E. Lack of cell cycle regulation of telomerase activity in human cells.Proc Natl Acad Sci USA. 1997; 94: 10687-10692Crossref PubMed Scopus (146) Google Scholar). In addition, when cell lines expressing telomerase activity are induced to differentiate by diverse agents, downregulation of both telomerase activity and hTER is observed (Albanell et al., 1996Albanell J. Han W. Mellado B. Gunawardane R. Scher H.I. Dmitrovsky E. Moore M.A.S. Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines.Cancer Res. 1996; 56: 1503-1508PubMed Google Scholar). Therefore, using a probe for hTER, this study was designed to determine the distribution patterns of hTER in normal and diseased skin and to ascertain if hTER is regulated by the proliferative state of cells by comparing the distribution of hTER with Ki-67. A full length of hTER complementary DNA [cloned in pGEM-5Zf (Promega, Madison, WI)] was kindly provided by Geron (Menlo Park, CA) (Feng et al., 1995Feng J. Funk W.D. Wang S.-S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2014) Google Scholar). Anti-sense and sense (control) RNA strands were synthesized and labeled with [35S]-UTP (Amersham, Arlington, IL) using the SP6 (anti-sense) or T7 (sense) bacteriophage promoter as previously described (Yashima et al., 1997aYashima K. Piatyszek M.A. Saboorian H.M. Virmani A.K. Brown D. Shay J.W. Gazdar A.F. Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.J Clin Pathol. 1997; 50: 110-117Crossref PubMed Scopus (100) Google Scholar). The specific activity of the radiolabeled probes was ≈6 × 10 7 cpm per μg template DNA. After in vitro RNA synthesis, the probes were degraded to an average length of 200 nucleotides by alkali hydrolysis, purified using a G-50 column (Boehringer Mannheim, Indianapolis, IN), resuspended in 100 mM dithiothreitol, aliquoted, and stored at –80°C until use. Samples were obtained either from the archives of the Department of Dermatology, University Texas Southwestern Medical Center, or from surgical procedures, circumcisions, and biopsies. Details of in situ hybridization for hTER have been published previously (Yashima et al., 1997aYashima K. Piatyszek M.A. Saboorian H.M. Virmani A.K. Brown D. Shay J.W. Gazdar A.F. Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.J Clin Pathol. 1997; 50: 110-117Crossref PubMed Scopus (100) Google Scholar). Briefly, freshly obtained samples were fixed overnight at 4°C in 4% paraformaldehyde in phosphate buffer saline and then embedded in paraffin. Three micron thick sections were mounted on Superfrost+ slides and dried for 1 h at 80°C. The sections were deparaffinized, rehydrated, and treated with proteinase K (10 μg per ml) for 35 min. After postfixation with 4% paraformaldehyde/phosphate buffer saline, sections were acetylated in freshly prepared 0.25% acetic anhydrate/0.1 M triethanolamine for 10 min and then dehydrated. Each slide was hybridized overnight at 50°C with 1.5 × 106 cpm [35S] labeled RNA probe in the following mixture: 0.3 M NaCl, 0.02 M Tris pH 7.4, 5 mM ethylenediamine tetraacetic acid, 0.01 M sodium phosphate, 10% dextran sulfate, 0.5 mg yeast tRNA per ml, 0.02% wt/vol polyvynilpyrolidone, 0.02% wt/vol Ficoll, 0.02% wt/vol bovine serum albumin (fraction V), and 50% formamide. Post-hybridization washes included 2 × sodium citrate/chloride buffer/50% formamide/10 mM dithiothreitol at 65°C; 0.4 M NaCl/10 mM Tris/5 mM ethylenediamine tetraacetic acid with 20 μg Rnase per ml at 37°C; and 2 × 0.1 × sodium citrate/chloride buffer at 50°C. Slides were then dehydrated, dried, coated with Kodak NTB 2 emulsion, and stored in the dark at –70°C for 2 wk. The emulsion was developed with Kodak D19 developer and the slides were counterstained with hematoxylin and mounted in Permount medium before microscopic examination. In order to determine adequate RNA preservation, samples were hybridized with 28S rRNA specific probe cloned in pTRIOLEscript (Ambion, Austin, TX). Samples with poor or no detectable RNA were rejected from the analysis. Each slide was carefully studied by each observer prior to analysis. Regions of interest (epidermis or tumor cell clusters) were scrutinized for the most representative areas that were then used for analysis. Two different approaches were utilized. Because the emulsion overlying the tissue contained silver grains at different levels within the emulsion, the first approach utilized was a semiquantitative technique that attempted to measure all the grains detectable overlying individual cells. Utilizing an Olympus BX40 light microscope, the intensity of labeling was assessed by counting the number of silver grains within the nuclear outline at ×400 magnification. Grains were of differing sizes and therefore larger grains were counted as multiples. In other words, large grains were considered clumps of smaller grains and were assigned a value based on how many smaller grains could fit within the clump. One individual (MO) counted grains by focusing on different levels in the emulsion. The number of grains per nucleus for at least 100 morphologic or region specific cells per slide were counted and averaged. Values for grains per cell for nonspecific hybridization were obtained from sequential sections of the same samples hybridized with sense probe in the same fashion. The values from these control samples were subtracted from those obtained for specific hybridization. The specimens were scored as follows: –, undetectable/background (0–5 grains per nucleus);±, weak (6–10 grains per nucleus); +, moderate (11–15 grains per nucleus); + +, strong (16–20 grains per nucleus); and + + +, very strong (more than 21 grains per nucleus). A second quantitative technique was utilized by a second observer (TL) that attempted to control for nonspecific signal in the emulsion overlying samples hybridized with anti-sense probe. A ×400 photo micrograph was taken of a representative area of each sample with the microscope focused on the mid-level plane of the emulsion. This allowed visualization of the majority of the grains. T-MAX black and white 100 ASA (Kodak) print film was used and 4 × 6 inch prints were developed for analysis. Transparency film with a 5 × 5 mm grid imprinted on it was placed over the print in order to orient the observer. The number of grains overlying the nucleus were counted using a ×2 magnifying glass. All cells within a region of interest, whether positive for signal or not, were included in the analysis. Twenty morphologic or region specific cells were analyzed for each cell group per sample. Counts were averaged per cell group. Portions of the photograph not containing tissue were utilized to calculate background levels. A circle representing the average cell group nuclear circumference was superimposed over this area and grains were counted and averaged. This average background count was then subtracted from the average counts from the cell groups. Immunohistochemistry was performed on paraffin sections using a rabbit polyclonal anti-human Ki-67 antibody (Dako, Carpinteria, CA). Sections were deparaffinized, rehydrated, and treated in 0.1 M citric buffer acid (pH 6.0) for 5 min in a H2800 microwave processor at 800 W (Energy Bean Science, Agawarm, MA). After two cycles of microwave irradiation the sections were allowed to cool down to room temperature for 20 min. Subsequently, the slides were briefly washed with PBS Tween 20 and incubated with a 1:100 dilution of anti-Ki-67 antibody, biotin-conjugated goat anti-rabbit IgG, followed by the avidin-biotin-peroxidase detection kit (Vector, Burlingame, CA). Rabbit IgG served as a negative control. Sigma Plot software (Jandel Scientific, Corte Madera, CA) was used to perform paired comparisons utilizing the two tailed t test. The p values < 0.05 were considered statistically significant. Fresh newborn foreskins (n = 5) showed moderate levels of hTER in almost all basal and some low suprabasal cells (Figure 1a, brightfield and b, darkfield). hTER levels were significantly higher in the basal compared with suprabasalar levels (Figure 2; p = 0.01). Adult epidermis from sun protected areas (n = 7) showed weak hTER expression (Figure 1a,d), regardless of the age of the adult donors (Table 1). There was no difference in hTER levels in basal cells from these areas compared with suprabasal cells. In contrast, all adult specimens obtained from sun exposed epidermal areas (n = 8; Table 1 and Figure 2) showed moderate hTER expression in the basal cells (Figure 1a,f). This level of hTER was significantly higher than that of suprabasal cells (p = 0.01), and suggests a possible role of chronic UV radiation on hTER expression.Figure 2Quantitative analysis of hTER expression varied in different skin conditions. Subtracting background levels of silver grains from the number of grains overlying nuclear shadows, a calculated value was determined from black and white ×400 photomicrographs of tissue sections of different skin conditions. Values are expressed as the mean ± SEM. *, Counted grains over only the cells at the periphery of the tumor cell clusters.View Large Image Figure ViewerDownload (PPT)Table 1Semiquantitative analysis of hTER expression in normal, inflammatory, and malignant skin conditionsSexAgeSiteTelomerase RNAaTelomerase RNA expression: –, background/undetectable (0–5 grains per nucleus); ±, weak (6–10 grains); +, moderate (11–15 grains); ++, strong (16–20 grains); and +++, very strong (> 21 grains).Newborn foreskin, basal layer epidermal cells1M0Foreskin+2M0Foreskin+3M0Foreskin+4M0Foreskin+5M0Foreskin+Sun protected area, basal layer epidermal cells1M19Upper arm+2M19Upper arm+3M19Upper arm–4M37Upper arm+5M50Upper arm–6M51Back+7F36Upper arm+Sun exposed area, basal layer epidermal cells1M64Face+2M68Face+3M69Face+4M69Face+ +5M72Forearm+6M75Face+7M78Face+8F70Face+Inflammatory disease, basal layer epidermal cellsPsoriasisM60Back+PsoriasisM61Chest+PsoriasisF48Foot+ +Contact DM31Leg+Contact DM50Abdomen+Contact DF46Arm+Basal cell carcinoma cellsHistologic subtypesSolidM63Face+MorpheaM64Head+MorpheaM69Face–SolidM70Face+SolidM70Face+ +SolidM70Face+SolidM72Ear+SolidM72Head+ + +AdenoidM75Face+ +MorpheaM81Ear+ +SolidM81Face+SolidM83Ear+MorpheaF52Arm+SolidF55Neck+MorpheaF76Face+ +Squamous cell carcinoma cells1M40Face+2M48Face+ + +3M65Chest+ + +4M65Face–5M76Head+ + +6M76Head+ +7M76Face+8M79Ear+ +Melanoma cellsIn situM35Back+InvasiveM47Back+InvasiveM70Back+In situM74Back+InvasiveF55Arm+a Telomerase RNA expression: –, background/undetectable (0–5 grains per nucleus); ±, weak (6–10 grains); +, moderate (11–15 grains); ++, strong (16–20 grains); and +++, very strong (> 21 grains). Open table in a new tab Telomerase RNA was then examined in hair follicles of facial skin and was detected in the bulb as well as the outer root sheath cells along the entire length of the hair follicles (Figure 3a,b). The signal intensity of cells in the bulb (** in Figure 3) was much higher than that of the cells in the outer root sheath (* in Figure 3). This elevation of hTER expression in the outer root sheath is unlikely to be the result of chronic UV exposure, even though this sample was taken from a sun exposed area. We also observed similar hTER augmentation in the outer root sheath cells of sun protected area skin (data not shown). In addition, anagen hair follicle bulbs are situated in the deep dermis and subcutaneous fat, where UV should not have a direct effect. Therefore, hTER upregulation in hair follicles is unlikely due to the effects of UV radiation. Considering that basal epidermal cells and the cells in anagen bulbs are less differentiated and actively proliferating, hTER may be a reflection of the proliferative state of the keratinocytes in hair follicles. Enhanced hTER was observed in 14 of 15 basal cell carcinoma (BCC) cases (93.3%), and was expressed homogenously throughout all tumor cells (Figure 4a,b). Although the signal intensities varied among samples, they did not correlate with age, sex of the donors, clinical history of the lesions, or histologic subtype (Table 1). As a group, the number of grains per nuclei of the tumor cells was not statistically different from that measured for basal cells from sun exposed skin samples. In addition, more than half of the BCC samples (eight of 15) showed strong signals for hTER in the basal cells of the overlying epidermis, which was histologically normal or atrophic (data not shown). Seven of eight SCC samples (87.5%) were positive for hTER but only in cells at the periphery of the tumor nests (Figure 4a,d). This distribution pattern of hTER was more prominent in well-differentiated SCC, whereas in less differentiated SCC, hTER positive cells were scattered throughout the tumor nests (data not shown). Unlike BCC, these cells had higher levels of hTER than basal cells of sun exposed skin. Telomerase RNA was undetectable in one BCC and one SCC sample. The inability to detect hTER in these specimens was not due to RNA degradation because control 28S rRNA was positive for these two samples. All melanomas examined (two melanomas in situ, three melanomas with a thickness of > 0.6 mm) showed positive signals for telomerase RNA (Figure 4a,f). Levels of hTER expression were similar among all samples regardless of the invasive characteristics of the tumor (Table 1). Moderate to strong levels of hTER were present in cells in the expanded rete ridges of skin samples of active psoriasis (Figure 5a,b). Telomerase RNA was also expressed in the basal cells of contact dermatitis lesions at moderate levels. When grouped together, basal layer cells from these inflammatory conditions were found to have more hTER than basal cells in sun protected and sun exposed skin. Interestingly, infiltrating lymphocytes failed to show increased expression of hTER in active psoriasis and contact dermatitis lesions. Basal keratinocytes from psoriasis are actively proliferating and this further supports the hypothesis that hTER expression generally correlates with cell proliferation. To determine if expression of hTER correlates with the proliferative state, we performed immunohistochemical staining using a polyclonal anti-Ki-67 antibody (Smith et al., 1995Smith M.D. Healy E. Thompson Y. Morley A. Rees J.L. Use of in situ detection of histon mRNA in the assessment of epidermal proliferation: comparison with the Ki67 antigen and BrdU incorporation.Br J Dermatol. 1995; 132: 359-366Crossref PubMed Scopus (34) Google Scholar) on sequential paraffin sections prepared for hTER in situ hybridization. In newborn foreskins, hTER was expressed in all basal and some suprabasal keratinocytes Figure 6a, whereas Ki-67 positive cells were predominantly in the first suprabasal layer Figure 6a. In BCC, hTER was observed homogeneously among the tumor nest, but Ki-67 positive cells were scattered among the tumor nests (data not shown). In SCC, hTER positive cells and Ki-67 positive cells co-localized in the periphery of the tumor (data not shown). These findings show that the distributions of hTER and Ki-67 were similar but not always identical. The expression of hTER was more widespread than Ki-67. Previously, we reported that in newborn foreskins telomerase activity was concentrated in the epidermis (Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar). Our findings were corroborated by Harle-Bachor and Boukamp, who showed that this activity appeared to be localized to the proliferative basal keratinocytes (Harle-Bachor and Boukamp, 1996Harle-Bachor C. Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and immortal and carcinoma-derived skin keratinocytes.Proc Natl Acad Sci USA. 1996; 93: 6476-6481Crossref PubMed Scopus (452) Google Scholar). In this study, we show that in normal newborn foreskins, hTER co-localizes to the same basal keratinocytes as well as those cells just above the basal layer. In addition, results from Ki-67 staining studies reported here reveal that, whereas a few basal cells are positive for this proliferation marker, a greater number of suprabasal cells stain positive. It appears that both proliferating and nonproliferating nonmalignant epidermal cells can express hTER, but as they differentiate and move away from the basal layer they stop expressing hTER. Taken together these findings suggest that not only stem cells but also other basal or near basal cells, possibly the transient amplifying cells, are expressing hTER and could be responsible for the telomerase activity reported previously. On the other hand, the expression of hTER does not definitively implicate a specific subpopulation of cells as the telomerase activity competent population. It is possible that as the telomerase competent cells divide and differentiate, they downregulate the synthesis of the protein component of telomerase but continue to produce or retain hTER. Therefore, a portion of the hTER positive cells we detected could be telomerase incompetent cells that would ultimately become hTER negative as they enter terminal differentiation and move away from the basal layer. In adult epidermis, hTER expression was undetectable or weak in the basal cells from sun protected skin, but moderate in the basal keratinocytes of sun exposed skin, and in psoriatic and contact dermatitis lesions. Because all three psoriasis samples were taken from sun protected areas (back, foot, and chest), and only one patient received intermittent UV therapy for 1 y, it is unlikely that the increase in hTER of these psoriatic basal keratinocytes was due to UV radiation alone. We also previously reported that adult skin samples from sun protected areas contained minimal telomerase activity, whereas the samples from sun exposed areas, active psoriasis, and contact dermatitis lesions showed slightly elevated telomerase activity (Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar). These telomerase activity observations compared with the present hTER results suggest that the signal intensity of hTER correlates with telomerase activity in normal as well as inflammatory skin conditions. We also detect strong signals of hTER in anagen hair follicle bulb cells in the same areas where the majority of telomerase activity is localized (Ramirez et al., 1997Ramirez R.D. Wright W.E. Shay J.W. Taylor R.S. Telomerase activity concentrates in the mitotically active segments of human hair follicles.J Invest Dermatol. 1997; 108: 113-117Abstract Full Text PDF PubMed Scopus (151) Google Scholar). Taken together, distributions and signal intensity of hTER correlated with telomerase activity in these nonmalignant conditions. hTER expression was also found to be elevated in BCC, SCC, and melanoma in comparison with peritumoral epidermal cells. The exceptions were several BCC samples noted to have elevated hTER levels in normal epidermal cells in close proximity to tumor cell clusters. This phenomenon diminished the further away from the tumor cells one looked, suggesting the possibility of diffusable factors elaborated by the tumor cells that might be interfering with normal differentiation of adjacent nonmalignant epidermal cells resulting in their continued upregulation of hTER. When comparing the hTER expression profiles of common skin malignancies, we found that several patterns developed. BCC samples showed homogeneous hTER expression within the same tumor, but the signal intensities varied among different samples. SCC samples showed both intra-sample and inter-sample heterogeneity. Melanomas showed similar moderate levels of hTER expression in all samples, but the tumor cell populations were small. Ki-67 staining of BCC and SCC revealed a pattern of widespread hTER expression amongst all tumor cells, but a much smaller subpopulation of proliferating (Ki-67+) cells. This finding supports the concept that proliferating and nonproliferating cells can express hTER. Furthermore, it was found that the cells at the periphera of SCC clusters express more hTER than the more differentiated cells at the center mimicking the pattern seen in nonmalignant epidermis. This repeat in expression pattern suggests that not only the proliferative but also the differentiation state of the cell plays a role in hTER expression. It must also be noted that SCC, often a clinically more aggressive malignancy than BCC, had much higher levels of hTER expression than BCC. This suggests that the level of hTER may be a better indicator of malignancy than merely its presence in human keratinocytes. Our previous study showed that relative mean telomerase activity levels were highest in BCC, with lower levels in SCC and melanoma (Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar). hTER levels measured here do not by themselves assist in determining which tumor cell subpopulations are telomerase activity competent. On the other hand, other studies have shown that tumor cells expressing telomerase activity downregulate that activity after they are driven to differentiate or become quiescent (Holt et al., 1996Holt S.E. Wright W.E. Shay J.W. Regulation of telomerase activity in immortal cell lines.Mol Cell Biol. 1996; 16: 2932-2939Crossref PubMed Scopus (357) Google Scholar; Albanell et al., 1996Albanell J. Han W. Mellado B. Gunawardane R. Scher H.I. Dmitrovsky E. Moore M.A.S. Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines.Cancer Res. 1996; 56: 1503-1508PubMed Google Scholar). The hTER and Ki-67 expression pattern similarities between normal epidermis and SCC further supports the concept that cells downregulate hTER expression and possibly telomerase activity after they differentiate and stop proliferating. If this is true, then the increased hTER expression in suprabasal epidermal cells in psoriasis and contact dermatitis Figure 2 could be due to the migration of an expanded undifferentiate keratinocyte population into the suprabasalar compartment, as suggested by others (Bata-Csorgo et al., 1993Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.J Exp Med. 1993; 178: 1271-1281Crossref PubMed Scopus (113) Google Scholar). UV is a well-known carcinogen for skin, and it has been proposed that telomerase activation is one of the early events of photocarcinogenesis (Taylor et al., 1996Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. Detection of telomerase activity in malignant and nonmalignant skin conditions.J Invest Dermatol. 1996; 106: 759-765Crossref PubMed Scopus (265) Google Scholar; Ueda et al., 1997Ueda M. Ouhtit A. Bito T. et al.Evidence for UV-associated activation of telomerase in human skin.Cancer Res. 1997; 57: 370-374PubMed Google Scholar). Therefore, UV may elevate hTER expression in an early stage of the process. Psoriasis is a disorder of hyperproliferation of keratinocytes with an increase in stem cells and transient amplifying cells (Bata-Csorgo et al., 1995Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (180) Google Scholar). As described above, the hTER expression patterns in psoriasis correspond to the location of these proliferating cell populations. Precise mechanisms of psoriasis have not yet been defined, although an immunologic disorder is thought to drive this process (Valdimarssoh and Fry, 1986Valdimarssoh H. Fry L. Psoriasis a disease of abnormal keratinocyte proliferation induced by T lymphocytes.Immuno Today. 1986; 7: 256,Abstract Full Text PDF PubMed Scopus (272) Google Scholar). Various cytokines elaborated by infiltrating lymphocytes may increase the proliferative capacity of stem cells, and, therefore, may upregulate hTER expression. Recently, several biologic abnormalities, such as increased proliferation capacity (Kikuchi et al., 1993Kikuchi A. Sakuraoka K. Shimizu H. Nishikawa T. Immunohistochemical evaluation of epidermis overlying basal cell carcinomas.Br J Dermatol. 1993; 128: 644-649Crossref PubMed Scopus (15) Google Scholar), abnormal expression of involucrin (Said et al., 1984Said J.W. Sassoon A.F. Shintaku I.P. B-Schlegel S. Involucrin in squamous and basal cell carcinomas of the skin: An immunohistochemical study.J Invest Dermatol. 1984; 82: 449-452Abstract Full Text PDF PubMed Scopus (73) Google Scholar), or p53 mutations (Urano et al., 1995Urano Y. Asano T. Yoshimoto K. et al.Frequent p53 accumulation in the chronically sun exposed epidermis and clonal expansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma.J Invest Dermatol. 1995; 104: 928-932Crossref PubMed Scopus (62) Google Scholar), have been observed in the epidermis adjacent to BCC. It has been suggested that diffusible factors elaborated by malignant cells and/or activated dermal cells could be responsible for such alterations (Wolf and Bystryn, 1981Wolf D. Bystryn J.C. Alterations in antigenic properties of normal epidermis adjacent to basal cell carcinomas.J Invest Dermatol. 1981; 76: 442-444Abstract Full Text PDF PubMed Scopus (10) Google Scholar). Thus, hTER competent basal keratinocytes may increase their hTER expression through either the direct or the indirect effects of UV and/or cytokines. In general, the distributions of hTER were heterogeneous in nonmalignant and malignant skin. The presence of hTER expressing keratinocytes alone is not sufficient to detect malignant keratinocytes or telomerase activity competent cells. Both proliferating and resting keratinocytes can express hTER in vivo. Most hTER expressing keratinocytes are undifferentiated cells and likely include stem cell populations. In summary, this study supports the hTER in situ hybridization technique as being useful for determining the histologic distributions of hTER in skin. Once results can be correlated to results of the telomerase activity assay, it may facilitate new insights into the study of telomerase regulation and mechanisms of keratinocyte proliferation/differentiation. We thank Drs. Kazuo Yashima and Mark S. Gesell for technical assistance in the in situ hybridization, Drs. Woodring Wright and Paul Bergstresser for scientific discussions, Geron Corporation (Menlo Park, CA) for providing the telomerase RNA plasmid, Dr. Clay J. Cockerell and Mrs. Becki Ruser for providing specimens, and Ms. Becky Sheldon for secretarial assistance. This work was supported by the Dermatology Foundation and NIH Grant 5P30AR41940 and the Geron Corporation (Menlo Park, CA)." @default.
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