Shortened telomere length is demonstrated in T-cell subsets together with a pronounced increased telomerase activity in CD4 positive T cells from blood of patients with mycosis fungoides and parapsoriasis

We have recently demonstrated that telomerase activity is increased and telomere length shortened in lymphocytes from peripheral blood of patients with cutaneous T-cell lymphoma. In order to determine which cell type has increased telomerase activity and shortened telomere length, CD4+, CD8+, CLA+ CD3+ and CLA- CD3+ T cells were isolated from peripheral blood of 25 patients, including 15 patients with mycosis fungoides and 10 patients with parapsoriasis. Eleven healthy individuals were used as controls; CD19+ B cells were separated from each individual as an internal control. The results showed that the increased telomerase activity was significantly predominating in the CD4+ T-cell subset. Significantly shortened telomere length was found in CD4+ and CD8+ T-cell subsets from the patients compared with the same cell subsets obtained from healthy individuals. However, no difference was observed between the subsets; CD19+ B cells collected from patients and healthy control individuals had similar telomerase activity and telomere length which was significantly different from the values found in T cells. The telomere length was significantly shorter in CLA+ CD3+ subset than in CLA- CD3+ subset. Interestingly, increased telomerase activity and shortened telomere length was also detected in CD4+ T cells from patients with parapsoriasis indicating that alteration of telomerase activity and telomere length in CD4+ T cells is an early event in the pathogenesis of cutaneous T-cell lymphoma. Thus, the results indicate that a significant high level of telomerase activity and shortened telomere length frequently occur in T cells of patients with CTCL and may reflect tumorigenesis.     

The effect of the long-term cultivation on telomere length and morphology of cultured epidermis

BACKGROUND: Cultured epidermis has been successfully used in clinical treatment such as burns and pigmentary disorders. Although the generation of wide cultured epidermis for clinical use may require repeated passages, especially for allografts, the effects of long-term cultivation on its quality and cell viability are not well known. OBJECTIVES: To investigate the changes in morphology, telomere length, and telomerase activity during the passages of cultured epidermis and keratinocytes up to the passage limit, and to examine the usefulness of telomere length as a performance criterion for cultured epidermis. METHODS: The keratinocytes obtained from five patients were used to generate cultured epidermis. At the early passage and after cultivation up to the passage limit, morphology, telomere length and telomerase activity were investigated by using microscopes, southern blot analysis and telomeric repeat amplification protocol assay, respectively. RESULTS: The cultured cells started to show morphological changes when each passage was close to its limit and the cell sheets assumed an irregular stratification with various sizes of cytoplasm and nuclei. At the passage limit, the telomere length had decreased approximately 80-85%, and the average telomerase activity had declined under serum-free culture conditions. CONCLUSION: The results of this study showed the morphological change and telomere length reduction by long-term cultivation on cultured epidermis. Although the reduction in telomere length and telomerase activity may not be the major cause of the senescence, they could provide a useful information for the quality of the cultured epidermis.     

先天性角化不良与端粒酶、端粒相关基因的研究进展

先天性角化不良是一种具有遗传异质性的皮肤遗传病,其临床特征为黏膜白斑、甲营养不良、皮肤异色症、骨髓衰竭、肿瘤易感性以及其他系统损害.目前研究表明,其发病与端粒的长度缩短相关.端粒酶组分的基因突变可导致端粒酶活性的降低,使端粒缩短.概述引起端粒酶活性降低、端粒缩短的端粒酶组分的多个相关基因的研究进展,进一步阐明先天性角化不良的发病机制.

Abstract：

Dyskeratosis congenita (DC) is a rare skin disorder with heterogeneity, which is characterized by mucosal leukoplakia, nail dystrophy, abnormal skin pigmentation, bone marrow failure, cancer predisposition and other system damage. Currently, it is revealed that the pathogenesis of DC is related to the shortening of telomere length. Gene mutation of telomerase complex may result in a decline in telomerase activity and shortening of telomere length. This paper presents the advances in researches of telomerase complex genes responsible for reduction in telomerase activity and shortening of telomerase length, which may facilitate further elucidation of DC pathogenesis.     

Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target

Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments.     

Telomere length of the skin in association with chronological aging and photoaging

BACKGROUND: Telomere shortening has been implicated in cellular senescence, which may cause certain aging phenotypes. OBJECTIVE: To reveal whether telomere shortening is associated with chronological aging and/or photoaging of the skin, we measured telomere length in the epidermis and in the dermis from sun-protected and from sun-exposed sites of the skin. METHODS: Seventy-six specimens of epidermis from sun-protected sites and 24 specimens of epidermis from sun-exposed sites were analyzed. Sixty specimens of the dermis were also analyzed. In six cases, epidermal specimens from sun-protected and from sun-exposed sites of the same individual were analyzed. RESULTS: Comparison of telomere lengths revealed that the epidermis has shorter telomeres than the dermis. Telomere length in the epidermis and in the dermis was reduced with age, and average telomere shortening rates in the epidermis and in the dermis were 9 and 11 bp/yr, respectively. Unexpectedly, telomere length was not significantly different between epidermis from sun-exposed sites and from sun-protected sites. CONCLUSION: We could not show the evidence that telomere shortening is associated with photoaging of the skin.     

Augmented telomerase activity and reduced telomere length in parthenium‐induced contact dermatitis

Background Parthenium dermatitis is a common chronic inflammatory disease with activated T lymphocytes that recognize the antigens, which leads to proliferation and differentiation. Telomeres and telomerase play an important role in the regulation of life span of the cell. Telomere length maintained by telomerase, are specialized repeats present at the end of chromosomes which protect it from degradation, end‐to‐end fusion and are important for integrity of chromosomes. Objectives The aim of this study was to measure telomerase activity and telomere length in Peripheral blood mononuclear cell (PBMC), CD4⁺ and CD8⁺ T lymphocytes from parthenium dermatitis patients. Methods The study includes 50 patients of parthenium dermatitis confirmed by patch testing and 50 healthy controls. Telomerase activity was measured using the telomere repeat amplification protocol using PCR–ELISA kit. Telomere length was measured by using Telo TAGGG Telomere Length Assay Kit. Results Significantly elevated levels of telomerase activity was observed in PBMC, CD4⁺ and CD8⁺ T cells of parthenium dermatitis patients as compared with healthy controls. However, significantly reduced telomere length in PBMC, CD4⁺ and CD8⁺ T cells have been found in patients than healthy subjects, but there was no difference between CD4⁺ and CD8⁺ T cells in patients. Conclusion This study might have provided insight into the role of telomerase in parthenium dermatitis that is characterized by the recruitment of T lymphocytes, which play an important role in this inflammatory disease. The augmented telomerase activity and reduced terminal restriction fragment length might be explored as a potential diagnostic/prognostic marker for parthenium dermatitis in future.     

Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.     

The role of telomeres in the ageing of human skin

Skin is a self-renewing tissue that is required to go through extensive proliferation throughout the lifespan of an organism. Telomere shortening acts as a mitotic clock that prevents aberrant proliferation such as cancer. A consequence of this protection is cellular senescence and ageing. The telomerase enzyme complex maintains telomere length in germline cells and in cancer cells. Telomerase is also active in certain somatic cells such as those in the epidermis but is almost undetectable in the dermis. Increasing evidence indicates that telomerase plays a significant role in maintenance of skin function and proliferation. Mutations in telomerase component genes in the disease dyskeratosis congenita result in numerous epidermal abnormalities. Studies also indicate that telomerase activity in epidermal stem cells might have roles that go beyond telomere elongation. Telomeres in skin cells may be particularly susceptible to accelerated shortening because of both proliferation and DNA-damaging agents such as reactive oxygen species. Skin might present an accessible tissue for manipulation of telomerase activity and telomere length with the potential of ameliorating skin diseases associated with ageing.     

Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes

Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9–69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3–12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p < 0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p < 0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of keratinocytes but not fibroblasts. Telomerase activity is an important criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression.     

砷化物抑制银屑病角质形成细胞增殖的研究

银屑病主要表现为角质形成细胞过度增殖及凋亡异常,其中角质形成细胞过度增殖是由于细胞凋亡功能障碍或失调所致,凋亡缺陷在银屑病的发病机制巾发挥着重要作用。研究表明,砷化物可以通过对Fas/Fas配体、端粒和端粒酶的影响以及联合阻断JNK等多种途径诱导角质形成细胞凋亡,从而抑制其过度增殖。因此,砷化物有望成为一种局部治疗银屑病的新型药物。     

Telomeres rather than telomerase a key target for anti‐cancer therapy?

It was in the 1930s that telomeres (from the Greek telos = end and meros = part) were first recognized as essential structures at the ends of the chromosomes and were shown to be important for chromosomal stability (Muller HJ: The remaking of chromosomes. The Collecting Net-Woods Hole 1938: 13: 181-198, McClintock B, The stability of broken ends of chromosomes in Zea mays. Genetics 1041: 26: 234-282). However, it was only in 1978 that the first telomeric sequence was identified -- in the protocoa Tetrahymena, a single cell organism that at a certain stage of development has many identical minichromosomes with twice as many telomeres (Blackburn EH and Gall JG. A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 1978: 120: 33-53.). Today we know that telomeres form specialized, three-dimensional DNA-protein structures and fulfil important capping functions. Besides, telomeric DNA is essential as 'access DNA' for those cells that are not able to counteract loss of DNA during replication because they do not express telomerase, the enzyme responsible for telomere length maintenance. Since telomerase is mostly found in tumor cells and inhibition correlates with telomere shortening and finally growth inhibition, telomerase and lately also the telomeres themselves have become attractive targets for anti-cancer therapy. This review aims to critically throw light on different therapeutical approaches and comes to the conclusion that telomeres may be the better targets for cancer therapeutics.     

皮肤老化和光老化中端粒-端粒酶机制研究进展

单纯南时间所致的人皮肤程序性或内源性老化及改变,与由环境损伤所致的多莺性损伤或外源性老化加速之间有着明显的不同,后者的进程主要由日光中的紫外线所介导,故称为光老化.端粒及端粒酶在细胞的生命周期中起着不容忽视的作用,与细胞的老化密切相关.皮肤内源性老化主要由细胞周期中进行性的端粒缩短所致,而在光暴露的皮肤,内源性老化的进程被环境因素所放大.     

Quantitative in situ evaluation of telomeres in fluorescence in situ hybridization-processed sections of cutaneous melanocytic lesions and correlation with telomerase activity

BACKGROUND: Telomere length is correlated with cellular ageing and immortalization processes. In some human cancers telomere length measurement has proved to be of diagnostic and prognostic value. Results comparable with the traditional terminal restriction fragment length determination by Southern blotting have been obtained in metaphase and interphase cells in some studies by fluorescence in situ hybridization (FISH) analysis; FISH additionally allows for the quantification of telomeres at the cellular level. OBJECTIVES: In this study, 32 melanocytic lesions were analysed by FISH, aiming at investigating possible telomere differences among various benign and malignant lesions and correlation with telomerase activity (TA) level. METHODS: FISH was performed on paraffin sections from six common naevi, eight Spitz naevi, 12 melanomas, six melanoma metastases and nine control samples of normal skin. Telomere mean maximum diameter (Feret max), area and number per nuclear area were calculated by image analysis on fluorescent images elaborated through KS400 and in situ imaging system (ISIS) for FISH analysis programs. Mean TA level was also calculated in all lesions and correlated with telomere parameters. RESULTS: Telomere number per nuclear area was significantly lower in melanomas and metastases than in benign common and Spitz naevi and in control skin (7 small middle dot24 +/- 3.3; 6.11 +/- 3 vs. 14.46 +/- 5.6; 16.92 +/- 7.8; and 12.59 +/- 3.4, respectively; P < 0 .001). No significant differences were found for the other telomere parameters. In common and Spitz naevi, telomere number was positively correlated with Feret max (P = 0.046 and P < 0.0001, respectively). TA was significantly higher in melanomas and metastases than in the other groups (70.18 +/- 25.2; 105.07 +/- 30 vs. 2.16 +/- 2.4; 2 .99 +/- 2.1; 2 +/- 1.2, respectively; P< or = 0. 001) and it was inversely correlated with telomere number per nuclear area in melanomas (P = 0.0041). No other significant correlations were found. CONCLUSIONS: Encouraging results have been obtained from quantitative telomere evaluation in the diagnosis of melanocytic lesions, although an analysis of a larger number of cases would be necessary to provide more reliable data. An extreme shortening of some telomeres probably results in the decrease of telomeric signals and the lower mean number of detectable telomeres in melanomas and metastases. In melanomas, telomere number per nuclear area is also inversely correlated with TA levels. Quantitative FISH of melanocytic lesions could give more specific information at the cellular level in telomere and telomerase fields of investigation.     

Increase in telomerase activity during progression of melanocytic cells from melanocytic naevi to malignant melanomas

Abstract During successive cell divisions of mortal cells the length of the telomeres (TTAGGG repeats in vertebrates) at the end of chromosomes decreases. It has been suggested that this process is responsible for cellular senescence. Expression of the ribonucleoprotein telomerase appears to prevent shortening of telomeres in germ-line cells and cancer cells. The purpose of this study was to investigate telomerase activity in melanocytic lesions and its possible role in the multistep tumor progression model of malignant melanoma. To quantify the level of telomerase activity both in cultured cells and in fresh tissue samples the TRAP (telomeric repeat amplification protocol) ELISA was used. Eight cell lines of malignant melanoma, 3 primary cultures of fibroblasts, 36 melanocytic naevi, 5 atypical melanocytic naevi, 3 Spitz’s naevi, 31 primary malignant melanomas and 13 metastases of malignant melanomas were investigated. Also 34 samples of skin (22 samples of perilesional skin and 12 samples of normal skin) were analysed. In our experiments all melanoma cell lines were strongly positive, whereas in fibroblasts telomerase activity could not be detected. Of the primary melanomas and metastatic melanomas, 90.3% and 92.3%, respectively, were strongly positive, and of the atypical melanocytic naevi, 80% were positive. Of the 36 common melanocytic naevi only 10 (27.7%) expressed weak telomerase activity and of the 34 samples of human skin, 4 (11.7%) expressed very weak telomerase activity. Our results indicate that telomerase activity increases from benign melanocytic naevi to atypical naevi and further to malignant melanoma and metastatic melanoma cells, and therefore may play a role in tumour initiation and progression. Received: 26 June 1998 / Received after revision: 28 October 1998 / Accepted: 2 November 1998     

Comparative analysis of telomere lengths and erosion with age in human epidermis and lingual epithelium

We investigated progressive telomere shortening in normal human epidermis and lingual epithelium during aging, and attempted, in particular, to ascertain whether the telomere shortening that accompanies aging occurs at the same rate in different tissues. We studied telomeric DNA integrity, and estimated annual telomere loss, in 52 specimens of epidermis and 48 specimens of lingual epithelium collected at autopsy from subjects who had died at ages between 0 and 101 y. Most of the DNA samples were measured twice by southern blot hybridization. In addition, the correlation between telomere lengths in the two types of tissues was examined. The telomere reduction rates in epidermis and lingual epithelium were 36 bp and 30 bp per y, respectively, and these were significantly different. The rates obtained by the second measurements in epidermis and lingual epithelium were 39 and 32 bp per y, respectively, and these were also significantly different. The mean telomere lengths in the epidermis of eight neonates and the lingual epithelium of seven neonates were 13.2+/-1.0 and 13.8+/-1.0 kb, respectively. Comparison of telomere lengths in the two tissues for 41 paired samples showed that the mean telomere length in the epidermis (10.7+/-2.3 kb) was less than that in the lingual epithelium (12.4+/-2.5 kb); however, statistical analysis revealed a very significant relationship between epidermal and lingual epithelial telomere length (r=0.842, p<0.0001). These results indicate that the telomeres in epidermis and lingual epithelium are characterized by tissue-specific loss rates.