| |
| | | Year : 2010 | Volume : 21 | Issue : 2 | Page : 185-188 | | | Evaluation of telomerase expression in chronic periodontitis | | | TM Balaji1, V Vettriselvi2, Solomon FD Paul2, Suresh R Rao1
1 Sri Ramachandra Dental College, Department of Periodontics, Porur, Chennai - 600 116, India
2 Department of Human Genetics, Sri Ramachandra University, Porur, Chennai - 600 116, India
Click here for correspondence address and email | Date of Submission | 11-Nov-2008 | | Date of Decision | 02-Jul-2009 | | Date of Acceptance | 01-Oct-2009 | | Date of Web Publication | 22-Jul-2010 | | |      | |  Abstract | | | Background : Human telomerase is a multi subunit ribonucleoprotein enzyme concerned with telomeric lengthening and homeostasis in man. This enzyme has been found to be elevated in inflammatory conditions like rheumatoid arthritis and silica injury lung. Since chronic periodontitis is also an inflammatory condition where immune cells and cytokines mediate tissue destruction, we set out to evaluate telomerase in gingival tissue samples from healthy subjects and chronic periodontitis patients by reverse transcriptase polymerase chain reaction.
Materials and Methods : Gingival biopsies were obtained from eight healthy subjects and eight chronic periodontitis patients. Reverse transcriptase polymerase chain reaction (RTPCR) was carried out to evaluate telomerase gene expression in the samples.
Results : None of the healthy gingival tissue samples expressed the telomerase gene while all the chronic periodontitis samples expressed it. The severe chronic periodontitis samples expressed the gene more intensely than the moderate chronic periodontitis samples.
Conclusion : Various mechanisms have been explained to account for telomerase elevation in chronic periodontitis .This study helps us understand the role of telomerase in the pathogenesis of periodontal disease. It could be concluded that telomerase could be used as a marker to assess the severity of inflammation in chronic periodontitis. Keywords: Chronic periodontitis, inflammation, telomere, telomerase How to cite this article:
Balaji T M, Vettriselvi V, Paul SF, Rao SR. Evaluation of telomerase expression in chronic periodontitis. Indian J Dent Res 2010;21:185-8 |
How to cite this URL:
Balaji T M, Vettriselvi V, Paul SF, Rao SR. Evaluation of telomerase expression in chronic periodontitis. Indian J Dent Res [serial online] 2010 [cited 2014 Mar 6];21:185-8. Available from: http://www.ijdr.in/text.asp?2010/21/2/185/66632 | The anatomically complex and unique periodontal tissues are affected by numerous diseases, the most common and prevalent amongst them being chronic periodontitis. Chronic periodontitis is an inflammatory condition of microbial etiology, initiated and perpetuated by pathogenic bacteria in dental plaque, which causes progressive destruction of the periodontal ligament and alveolar bone, resulting clinically in pocket formation and tooth mobility. The host immune response is an important feature of this disease. All somatic cells of the human body, including the cells of the immune system, have a life span and restricted doubling potential, decided by the telomeres them posses. Telomeres, which are specialized structures capping the end of all eukaryotic chromosomes are synthesized by telomerase, a multi sub-unit ribonucleoprotein enzyme. Even though this enzyme is present constitutively in normal human cells and organs with a high turnover rate, like the testes, spleen, stomach and thymus, [1] it is present in high amounts, in activated T and B lymphocytes. [2],[3] Transient elevation in telomerase levels has been found in inflammatory conditions like silica injury lung [4] and rheumatoid arthritis. [5] In rheumatoid arthritis, telomerase levels have been found to serve as a predictor of the aggressiveness and severity of the condition. [6] Since chronic periodontitis is similar to rheumatoid arthritis in the fact, that inflammatory cells in union with cytokines mediate tissue destruction, this study was undertaken to assess the levels of telomerase in periodontal inflammation by reverse transcriptase polymerase chain reaction.
| Materials and Methods | |  |
Selection of subjects and sample collection
A total of 16 patients attending the outpatient clinic of the Department of Periodontology, at Sri Ramachandra Dental College were recruited for this study after obtaining their informed consent. The study was approved by the Institutional Ethics Committee of the Sri Ramachandra University. The study comprised of two groups - healthy control (n=8) and chronic periodontitis (n=8). Gingival tissue samples were obtained from healthy control subjects and chronic periodontitis patients according to the method of Tamilselvan et al. [7] Healthy control gingival tissue samples were obtained from subjects undergoing orthodontic treatment while chronic periodontitis samples were obtained from patients undergoing flap surgery in the Department of Periodontics. The control subjects were systemically and periodontally healthy. Gingival tissue samples were obtained from the control subjects from sites without clinical evidence of gingival inflammation or bleeding on probing and with a probing depth of < 4mm. All chronic periodontitis samples expressed clinical signs of inflammation and were taken from sites with probing depth > 6mm. Patients free of systemic disorders were included in the study. Pregnant women, smokers and patients on anti inflammatory and antibiotic therapy up to six months before were excluded from the study. Chronic periodontitis samples were graded on the basis of severity grading given by the World Workshop of Periodontics disease classification, according to which sites with 1-2 mm attachment loss are graded slight, 3-4 mm attachment loss graded moderate, and >5mm attachment loss, graded severe. The gingival tissue samples, after collection were rinsed, and transferred to sterile screw capped vials containing absolute ethanol (Merck) and stored at -20°C till further processing.
Total RNA isolation and reverse transcriptase polymerase chain reaction
Total RNA was isolated from the tissue samples using a one-step RNA trizol reagent (Medox Biotech, India). Reverse transcriptase polymerase chain reaction was performed using the one-step RTPCR kit (ABgene, Surrey UK). The human telomerase reverse transcriptase (hTERT) gene was amplified using the primer sequence: sense: AGA GTG TCT GGA GCA AGT TGC, antisense: CGT AGT CCA TGT TCA CAA TCG. The house keeping gene, glyceraldehydes-3-phosphate dehydrogenase (GADPH) was co-amplified with hTERT using the primer sequence: sense: GAA GGT GAA GGT CGG AGT C, antisense: GAA GAT GGT GAT GGG ATT TC. The PCR cycling conditions used for the study were: first strand synthesis: 1 cycle, 47 o C- 1 hour, denaturation: 1 cycle, 94 o C, 20 seconds, annealing: 40 cycles, 61 o C, 45 seconds, extension: 72 o C, 1 min/Kb and final extension: 1 cycle, 72 o C, 5 minutes. After PCR, the final product was subjected to agarose gel electrophoresis. The gel was placed in the UV station (Ultralum, USA) for scanning and viewing. Gel documentation was performed using commercially available Sigma gel software (Sigma SPSS Inc, North Michigan, and USA).
| Results | | |
Eight healthy control gingival tissue samples and eight chronic periodontitis samples were subjected to RTPCR analysis. Four chronic periodontitis samples were obtained from moderate sites and four from severe sites according to the severity grading mentioned earlier. None of the control samples expressed the telomerase (h TERT) gene [Figure 1], while all the chronic periodontitis samples expressed the gene with a mean value of 74.905 ± 6.98% [Figure 2]. Mean hTERT expression (%) in moderate chronic periodontitis (n=4) was 69.70±2.76, and severe chronic periodontitis (n=4) was 80.75 ± 4.71. Since only the periodontitis samples had the telomerase gene expression, the values were statistically significant when compared to the control. Also, the severe chronic periodontitis samples had significantly more telomerase expression, compared to the moderate chronic periodontitis samples.
| Discussion | | |
Somatic human cells demonstrate a strictly limited growth potential, and enter senescence after a determined number of cell divisions, due to progressive telomere shortening. Telomeric lengthening by telomerase is the predominant mechanism of telomere homeostasis in man. Even though the telomerase enzyme is composed of many components, there is mounting evidence to indicate that human telomerase reverse transcriptase (hTERT) is the most functional part of telomerase. [8],[9] Hence we set out, to measure hTERT expression in healthy gingival tissues and gingival tissue samples from chronic periodontitis patients. The result of our study showed that there was no expression of the telomerase gene in healthy gingival tissue samples (n=8), while all the chronic periodontitis samples (n=8) expressed the gene with a mean value of 74.905±6.98%. Among the cells of the human body, stem cells, T and B lymphocytes and gonads have detectable telomerase. This enzyme is absent or repressed in somatic tissues. [10],[11] Meyerson et al.,[10] found telomerase to be absent in human heart, kidney and liver tissue while Kalluri et al.,[12] found absence of telomerase in human fibroblasts from donors of 62, 71 and 93 years of age. Our study also could not detect the telomerase gene in an adult somatic tissue (viz.) the gingiva. Like all mucosal tissues, the gingiva is composed of epithelium, and connective tissue densely populated by fibroblasts. [13] This tissue has a very high turnover rate and is believed to have progenitor cells that contribute to tissue homeostasis. The plausible reason for the absence of the telomerase in healthy gingiva could be the presence of abundant numbers of fibroblasts, which are telomerase negative cells, [12] and the reduced presence of stem cells which are telomerase positive cells. [14],[15] Our results are concurrent with the findings of Sumida et al.[16] Miyoshi et al.[17] and Shimamoto. [18] who have also reported the absence of the telomerase gene expression in healthy buccal mucosa samples.
As mentioned earlier, all the chronic periodontitis samples tested in our study expressed telomerase with a mean value of 74.905 ± 6.98%. Chronic periodontitis represents an inflammatory condition of microbial etiology. The periodontopathic bacteria produce toxins and lipopolysaccharide antigenic determinants, which instigate the host response. The initial acute inflammation of periodontal tissues is brief and dominated by neutrophils and mast cells, which are shortlived. On the other hand, chronic inflammation, which is a feature of periodontitis, is characterized by the participation of antigen presenting cells like dendritic cells, T and B lymphocytes which have a long life span and posses detectable telomerase. [2],[3],[19] Since these cells have been demonstrated to infiltrate the gingival tissue of chronic periodontitis patients, [20],[21] we could possibly detect the telomerase gene, in all the eight chronic periodontitis samples tested. Moreover, activation of the T cell receptor and B cell receptor by mitogens and phytohaemagglutinins increases the telomerase levels multifold, as suggested by studies by Weng et al., [22] Hu BT et al.,[23] and Hideya et al.[24] Since sustained activation of T and B cells occur in chronic periodontitis, this could serve as a reason for telomerase upregulation.
The participation of lymphocytes and monocytes in chronic periodontitis results in the production of an array of cytokines. IL-2 and IL-6 have been demonstrated in elevated levels, in the gingival crevicular fluid of chronic periodontitis patients. These cytokines have actually been found to up regulate telomerase, through the IL-2 receptor and STAT 3 signaling mechanisms in T lymphocytes and monocytes respectively. [25]
The most implicated pathogen associated with the microbial etiology of periodontitis is Porphyromonas gingivalis. This pathogen is capable of intracellular invasion into buccal epithelial cells. [26] It can down regulate p 53, conferring an anti apoptotic phenotype to epithelial cells. A study by Rahman et al., [27] recently found that p53 down regulation is associated with telomerase up regulation. It is possible that intracellular colonization of Porphyromonas gingivalis  ral and sulcular epithelial cells in chronic periodontitis may indirectly upregulate telomerase. Recently, viruses, especially herpes viruses have been implicated as an etiological factor in Chronic Periodontitis. [28],[29],[30] Viral transfection has also been found to upregulate telomerase in lymphoma and hepatoma cell lines. [31],[32] The presence of viruses, in periodontal pockets could also act as a trigger factor in up regulating telomerase.
Microbial attack coupled with the host response in chronic periodontitis, culminates in pocket formation. Periodontal pockets have lowered oxygen tension, and are more hypoxic than the healthy gingival sulcus. Anderson et al.,[33] found that hypoxia can stimulate telomerase expression by unknown mechanisms, in an experiment on solid tumors of the gastrointestinal tract. A similar hypoxic state in periodontal pockets may be the reason for increased telomerase expression. Chronic periodontitis is a healing lesion, characterized by periods of exacerbation and remission. Even though there might be active destruction of alveolar bone and attachment loss occurring, the superficial gingival tissues may undergo fibrosis due to long standing inflammation. Telomerase expression has been found to be elevated in fibrotic conditions like bleomycin lung injury. [4] mediated by cytokines like IL-4.
In periodontitis too, the chronically inflamed fibrotic gingiva may express more telomerase in the above mentioned manner. The telomerase gene has also been found to be elevated in other inflammatory states like rheumatoid arthritis. Yamanishi et al.,[6] have suggested that telomerase activity is a predictor of the severity of the condition. In our study too, the severe chronic periodontitis samples expressed more telomerase than the moderate chronic periodontitis samples and thereby can serve as a marker of periodontal inflammation. However, further studies need to be conducted to justify the role of telomerase in chronic periodontitis [Table 1] and [Table 2].
| References | | |
| 1. | Mei F, Zhang B, Tang ZW, Hou L. Expression of telomerase associated gene in normal, atrophic and tumorous testes. Chin Med Sci J 2005;20:217-20.  [PUBMED] | | 2. | Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711-95. [PUBMED] [FULLTEXT] | | 3. | Yamada O, Motoji T, Mizoguichi H. Upregulation of telomerase activity in human lymphocytes. Biochem Biophy Acta 1996;1314:260-6. | | 4. | Liu T, Nozaki Y, Phan SH. Regulation of telomerase activity in rat lung fibroblasts. Am J Respir Cell Mol Biol 2002;26:534-40. [PUBMED] [FULLTEXT] | | 5. | Yudoh K, Matsuno H. The role of telomerase in joint deterioration in rheumatoid arthritis. Drugs Today (Barc) 2001;3:595-606. | | 6. | Yamanishi Y, Hiyama K, Maeda H, Ishioka S, Murakami T, Hiyama E, et al. Telomerase activity in rheumatoid synovium correlates with mononuclear cell infiltration and disease aggressiveness of rheumatoid arthritis. J Rheumatol 1998;25:214-20. [PUBMED] | | 7. | Tamilselvan S, Raju SN, Loganathan D, Kamatchiammal S, Abraham G, Suresh R. Endothelin 1 and its receptors ETa and ETb in drug induced gingival overgrowth. J Periodontol 2007;78:290-5. [PUBMED] [FULLTEXT] | | 8. | Counter CM, Hahn WC, Wei W, Caddle SD, Beijersbergen RL, Lansdorp PM, et al. Dissociation among in vitro telomerase activity, telomere maintenance and cellular immortalization. Proc Natl Acad Sci U S A 1998;95:14723-8. [PUBMED] [FULLTEXT] | | 9. | Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended life span. Curr Biol 1998;854:318-27. | | 10. | Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST 2, the putative human telomerase catalytic subunit gene, is up regulated in tumor cells and during immortalization. Cell 1995;90:785-95. | | 11. | Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277:955-9. [PUBMED] [FULLTEXT] | | 12. | Rao KS. Telomere (telomerase) hypothesis of aging and immortalization. Indian J Biochem Biophys 1996;33:88-92. [PUBMED] | | 13. | Schroeder HE, Mόnzel-Pedrazzoli S, Page R. Correlated morphometric and biochemical analysis of gingival tissue in early chronic gingivitis in man. Arch Oral Biol 1973;18:899-923. | | 14. | Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711-5. [PUBMED] [FULLTEXT] | | 15. | Chiu CP, Dragowska W, Kim NW, Vaziri H, Yui J, Thomas TE, et al. Differential expression of telomerase activity in hematopoietic progenitor cells from adult bone marrow. Stem cells 1996;14;239-48. | | 16. | Sumida T, Hamakawa H, Sogawa K, Sugita A, Tanioka H, Ueda N. Telomerase components as a diagnostic tool in human oral lesions. Int J Cancer 1999;80:1-4. [PUBMED] [FULLTEXT] | | 17. | Miyoshi Y, Tsukinoki K, Imaizumi T, Yamada Y, Ishizaki T, Watanabe Y, et al. Telomerase activity in oral cancer. Oral Oncol 1999;35:283-9. [PUBMED] [FULLTEXT] | | 18. | Shimamoto H. Telomerase activity in oral squamous cell carcinoma and leukoplakia. Kokubyo Gakkai Zasshi 2001;68:25-33. | | 19. | Ping L, Asai A, Okada A, Isobe K, Nakajima H. Dramatic increase of telomerase activity during dendritic cell differentiation and maturation. J Leukoc Biol 2003;74:270-6. [PUBMED] [FULLTEXT] | | 20. | Jotwani R, Palucka AK, Al-Quotub M, Nouri-Shirazi M, Kim J, Bell D, et al. Mature dendritic cells infiltrate the T cell rich region of the oral mucosa in chronic periodontitis. J Immunol 2001;167:4693-700. [PUBMED] [FULLTEXT] | | 21. | Reinhardus WA, Cloning TT. Characterization and antigen specify of T cells subsets extracted from gingiva of chronic periodontitis. Infect Immun 1995;62:2147-53. | | 22. | Weng NP, Granger L, Hodes RJ. Telomere lengthening and telomerase activation during B cell differentiation. Proc Natl Acad Sci U S A 1997;94:10827-32. [PUBMED] [FULLTEXT] | | 23. | Hu BT, Insel RA. Upregulation of telomerase in human B cells occurs independently of cellular proliferation and with expression of the telomerase catalytic sub-unit. Eur J Immunol 1999;29:3745-53. [PUBMED] [FULLTEXT] | | 24. | Igarashi H, Sakaguchi N. Telomerase activity is induced in human peripheral B lymphocytes by the stimulation to the antigen receptor. Blood 1997;89:1299-307. [PUBMED] [FULLTEXT] | | 25. | Konnikova L, Simeone MC, Kruger MM, Kotecki M, Cochran BH. Signal transducer and activator of transcription 3 (STAT 3) regulates human telomerase reverse transcriptase in human cancer and primary cells. Cancer Res 2005;65:6516-20. [PUBMED] [FULLTEXT] | | 26. | Rudney JD, Chen R, Sedgewick GJ. Intracelular Aa and P. Gingivalis in buccal epithelial cells collected from human subjects. Infect Immun 2001;69:2700-7. [PUBMED] [FULLTEXT] | | 27. | Rahman R, Latonen L, Wiman KG. hTERT antagonizes p53-induced apoptosis independently of telomerase activity. Oncogene 2005;24:1320-7. [PUBMED] [FULLTEXT] | | 28. | Contreras A, Slots J. Herpes virus in human periodontal disease. J Periodontal Res 2000;35;3-16. | | 29. | Kamma JJ, Contreras A, Slots J. Herpes viruses and periodontopathic bacteria in early onset periodontitis. J Clin Periodontol 2001;28:879-85. [PUBMED] [FULLTEXT] | | 30. | Saygun I, Kubar A, Ozdemir A, Slots J. Periodontitis lesions are a source of salivary EBV and cytomegalovirus. J Periodontal Res 2005;40:187-91. [PUBMED] [FULLTEXT] | | 31. | Mochida A, Gotoh E, Senpuku H, Harada S, Kitamura R, Takahashi T, et al. Telomere size and telomerase activity in EBV virus positive and negative Burkitt lymphoma lines. Arch Virol 2005;150:2139-50. [PUBMED] [FULLTEXT] | | 32. | Zhang X, Dong N, Zhang H, You J, Wang H, Ye L. Effects of hepatitis B virus X protein on human telomerase expression and activity in hepatoma cells. J Lab Clin Med 2005;145:98-104. [PUBMED] [FULLTEXT] | | 33. | Anderson CJ, Hoare SF, Ashcroft M, Bilsland AE, Keith WN. Hypoxic regulation of telomerase gene by transcriptional and post translational mechanisms. Oncogene 2006;25:61-9. [PUBMED] [FULLTEXT] |
Correspondence Address:
Suresh R Rao
Sri Ramachandra Dental College, Department of Periodontics, Porur, Chennai - 600 116
India
DOI: 10.4103/0970-9290.66632 PMID: 20657085
[Figure 1], [Figure 2]
[Table 1], [Table 2] | |
| | | | | | | | | | | |
| Article Access Statistics | | | Viewed | 1877 | | | Printed | 68 | | | Emailed | 2 | | | PDF Downloaded | 188 | | | Comments | [Add] | | | | 
|
Users online: 60 
