Difference In Telomerase Activity Research Articles

Honeybee trophocytes and fat cells as target cells for cellular senescence studies

Trophocytes and fat cells are distributed around the abdominal segments in honeybees (Apis mellifera). Whether these cells are a good model for cellular senescence studies is unknown. Here we used histochemical, biochemical, and genetic techniques to investigate the fluctuation of age-related molecules in trophocytes and fat cells of newly emerged and old worker bees. Histochemical studies revealed that old worker trophocytes and fat cells exhibited more senescence-associated β-galactosidase, lipofuscin granules, and non-homogeneous cellular morphology compared to the same cells in newly emerged workers. Biochemical assays demonstrated that trophocytes and fat cells of old workers expressed more lipid peroxidation and protein oxidation than those of newly emerged workers. Molecular genetic analyses detected no difference in telomerase activity or telomere length in trophocytes and fat cells between newly emerged and old workers, showing that these cells do not divide in adulthood. These results suggest that the expression of age-related molecules in trophocytes and fat cells is associated with the senescence of the cells and that honeybee trophocytes and fat cells can serve as a mode for cellular senescence.

Inhibition of poly (ADP-Ribose) polymerase-1 in telomerase deficient mouse embryonic fibroblasts increases arsenite-induced genome instability

BackgroundThe telomerase enzyme is a viable target for anti-cancer therapy given the innate differences in telomerase activity between tumour cells and normal somatic cells. However, the time lag between telomerase inhibition and telomeres becoming critically short to trigger cell death, allows cancer cells to acquire drug resistance. Inhibition of DNA repair pathways along with telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. Poly (ADP-Ribose) Polymerase-1 (PARP-1), an important DNA damage sensor and a DNA repair factor, has important roles in maintaining telomeres and chromosomal stability. In this study, the effects of combined inhibition of PARP-1 and telomerase in mouse embryonic fibroblasts (MEFs) following sodium arsenite exposure (a carcinogen and potent DNA damaging agent), were evaluated.ResultsInhibition of PARP in telomerase deficient MEFs induced an increase in arsenite-induced DNA damage as compared to control cells. Combined inhibition also resulted in enhanced genomic instability, demonstrated by elevated micronuclei induction and chromosomal aberrations with decreased cell survival. In addition, telomerase inhibition in PARP-1 deficient MEFs led to greater telomere shortening and increased genomic instability.ConclusionsOur study demonstrated that the co-inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of combined inhibition of PARP-1 and telomerase as a strategy to minimise the problems associated with long-term telomerase inhibition in cancer therapeutics.

Telomerase activity is not related to life history stage in the jellyfish Cassiopea sp.

The polyp (scyphistoma) of the jellyfish Cassiopea sp. can be maintained in culture for a long time, as polyps repeatedly reproduce asexually via formation of vegetative buds or propagules. The medusa, which is the sexually reproducing stage, typically has a relatively short life span. As a first step to understand the difference in life spans of the polyp and medusa stages of Cassiopea sp., we measured telomerase activity in different life cycle stages. We found telomerase activity in tissues of aposymbiotic polyps and propagules and symbiotic ephyrae (newly budded medusae) and adult medusae. No significant difference in telomerase activity was found between polyps and the bell region of the medusae. The cloned elongation products of the stretch PCR contained the TTAGGG repeats suggesting that the jellyfish has the ‘vertebrate’ telomere motif (TTAGGG) n . This is the first study to show that somatic tissues of both polyp and medusa stages of a cnidarian had telomerase activity. Telomerase activity in somatic tissues may be related to the presence of multipotent interstitial cells and high regenerative capacity of cnidarians.

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in prostate cancer cells (PC3). Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-alphatreatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-alpha treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-alpha-induced apoptosis of PC3 cells.

GERD is associated with shortened telomeres in the squamous epithelium of the distal esophagus

Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortened by repeated cell divisions and by oxidative DNA damage, and cells with critically shortened telomeres cannot divide. We hypothesized that chronic gastroesophageal reflux disease (GERD)-induced injury of the esophageal squamous epithelium results in progressive telomeric shortening that eventually might interfere with mucosal healing. To address our hypothesis, we compared telomere length and telomerase activity in biopsy specimens of esophageal squamous epithelium from GERD patients and control patients. Endoscopic biopsies were taken from the esophageal squamous epithelium of 38 patients with GERD [10 long-segment Barrett's esophagus (LSBE), 15 short-segment (SSBE), 13 GERD without Barrett's esophagus] and 16 control patients without GERD. Telomere length was assessed using the terminal restriction fragment assay, and telomerase activity was studied by the PCR-based telomeric repeat amplification protocol assay. Patients with GERD had significantly shorter telomeres in the distal esophagus than controls [8.3 +/- 0.5 vs. 10.9 +/- 1.5 (SE) Kbp, P = 0.043]. Among the patients with GERD, telomere length in the distal esophagus did not differ significantly in those with and without Barrett's esophagus (LSBE 7.9 +/- 0.8, SSBE 8.6 +/- 0.9, GERD without BE 8.7 +/- 1.0 Kbp). No significant differences in telomerase activity in the distal esophagus were noted between patients with GERD and controls (4.0 +/- 0.39 vs. 5.2 +/- 0.53 RIUs). Telomeres in the squamous epithelium of the distal esophagus of patients who have GERD, with and without Barrett's esophagus, are significantly shorter than those of patients without GERD despite similar levels of telomerase activity.

Telomerase activity in the vaginal margins of radical hysterectomy in patients with carcinoma of the cervix: correlation with histology and human papillomavirus

This study was undertaken to evaluate the telomerase activity both in the tumor and in the vaginal margins of radical hysterectomy in patients with squamous cell carcinoma (SCC) of the cervix. Thirty-three patients with SCC of the cervix (study group) and 13 patients with uterine myoma (control group) were prospectively studied. Tissue samples were taken from the tumor or cervix, anterior vaginal margin (AVM), and posterior vaginal margin (PVM). The specimens were analyzed by histopathology, by a telomerase PCR-TRAP-ELISA kit, and by polymerase chain reaction using human papillomavirus (HPV) DNA. The telomerase activity was significantly higher in the tumor than in the benign cervix (P < 0.001). There was no difference in telomerase activity in the AVM and PVM in patients with cervical carcinoma compared to the control group. Telomerase activity was associated with the presence of histologic malignancy in the PVM of patients submitted to radical hysterectomy (P= 0.03). This association was not observed with the presence of HPV in AVM or PVM in the study group. Telomerase activity is a marker of histologic malignancy in patients with SCC of the cervix. There was no association between the telomerase activity and the presence of HPV in vaginal margins of patients submitted to radical hysterectomy.

Smoking is associated with increased telomerase activity in short-term cultures of human bronchial epithelial cells

Telomerase plays an important role in the maintenance of telomere ends in normal and tumor cells and ectopic expression can immortalize human bronchial epithelial (HBE) cells. We assessed telomerase activation, growth properties and methylation status in the hTERT promoter in a panel of HBE cell cultures in relation to smoking and previous lung cancer history. HBE cells were obtained from a total of 26 subjects, six of whom were lifelong non-smokers, while 20 subjects had a smoking history, including seven who had lung carcinoma. Telomerase activity was determined using the telomeric repeat amplification protocol (TRAP). Maximum passage number and time to senescence were also determined through extended culturing. The distribution of the telomerase activity between ever-smokers and never-smokers was significantly different ( P=0.03, F-test), and there was a strong correlation between telomerase activity and the number of pack-years smoked ( P=0.0012, F-test for slope). A small difference in telomerase activity was observed according to lung cancer status ( P=0.02, F-test). Telomerase activity was not correlated with maximum passage number after extended culturing or with time to senescence. None of the HBE cultures demonstrated methylation of the hTERT promoter. Our results indicate an association between tobacco carcinogen exposure and telomerase activity in normal bronchial epithelium, although a causative role of tobacco smoking in the (re)activation of telomerase can not be proven. An increase in telomerase activity in normal bronchial epithelium might extend the lifespan of cells at risk for malignant transformation, and thus contribute to lung carcinogenesis.

EFFECTS OF SENSE TANKYRASE TRANSFECTION ON SMOOTH MUSCLE CELLS OF CORPUS CAVERNOSUM IN RAT

This study was conducted to construct the eukaryotic expression vectors for sense tankyrase and to investigate the effects of tankyrase transfection on smooth muscle cells of corpus cavernosum in the rat. After the eukaryotic expression vectors for sense tankyrase were constructed and identified smooth muscle cells of rat corpus cavernosum were transfected with the recombinant plasmids of sense tankyrase (pcDNA-TNKS). Levels of DNA and RNA were then evaluated. Measurement of telomerase activity was conducted by TRAP-ELISA assay, the length of telomere by Southern blot and the growth curve by MTT assay. We have found that the eukaryotic expression vectors for sense tankyrase were constructed and the recombinant plasmids of sense tankyrase were transfected into smooth muscle cells of rat corpus cavernosum successfully; no significant differences in telomerase activity were observed between TNKS-transfected cells (SMC-TANKS), zero-load- transfected cells (SMC-Zeo), and non-transfected cells (SMC) (P > 0.05). The length of telomere in SMC-TANKS was longer than that in SMC-Zeo or SMC (P < 0.01), and the OD value of TNKS-transfected cell was significantly higher than that of the non-transfected cells (P < 0.01). These results suggested that the eukaryotic expression vectors for sense tankyrase were constructed successfully, which provides the basis for gene therapy. Transfection of recombinant plasmids of sense tankyrase helps change the telomerase length of smooth muscle cells of corpus cavernosum and extend the cell life span.

Telomerase activity in the vaginal margins of radical hysterectomy in patients with carcinoma of the cervix: correlation with histology and human papillomavirus

This study was undertaken to evaluate the telomerase activity both in the tumor and in the vaginal margins of radical hysterectomy in patients with squamous cell carcinoma (SCC) of the cervix. Thirty-three patients with SCC of the cervix (study group) and 13 patients with uterine myoma (control group) were prospectively studied. Tissue samples were taken from the tumor or cervix, anterior vaginal margin (AVM), and posterior vaginal margin (PVM). The specimens were analyzed by histopathology, by a telomerase PCR-TRAP-ELISA kit, and by polymerase chain reaction using human papillomavirus (HPV) DNA. The telomerase activity was significantly higher in the tumor than in the benign cervix (P &lt; 0.001). There was no difference in telomerase activity in the AVM and PVM in patients with cervical carcinoma compared to the control group. Telomerase activity was associated with the presence of histologic malignancy in the PVM of patients submitted to radical hysterectomy (P = 0.03). This association was not observed with the presence of HPV in AVM or PVM in the study group. Telomerase activity is a marker of histologic malignancy in patients with SCC of the cervix. There was no association between the telomerase activity and the presence of HPV in vaginal margins of patients submitted to radical hysterectomy.

Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene (TERT) in Human and Mouse Cells

The transcriptional regulation of the human telomerase catalytic subunit (hTERT) plays a critical role in telomerase activity. Approximately 200 bp of the proximal core promoter is responsible for basic hTERT expression; however, the function of the distal regulatory elements remains unclear. The transcription factor activator protein 1 (AP-1) is involved in cellular proliferation, differentiation, carcinogenesis, and apoptosis and is expressed broadly in both cancer and normal cells. There are several putative AP-1 sites in the hTERT promoter, but their functions are unknown. The present study examined the regulatory role of AP-1 in hTERT gene transcription. Overexpression of AP-1 leads to transcriptional suppression of hTERT in cancer cells. The combination of c-Fos and c-Jun or c-Fos and JunD strongly suppresses hTERT promoter activity in transient-expression analyses. The hTERT promoter region between -2000 and -378 is responsible for this function. Gel shift and supershift analyses, as well as ChIP, show binding of JunD and c-Jun on two putative AP-1 sites within this region. Mutations in the AP-1 binding sites rescued suppressions caused by AP-1, suggesting this is a direct regulation of the hTERT promoter. In contrast, there was no effect on mTERT expression or mTERT promoter activity by AP-1 overexpression in mouse fibroblasts. The species-specific function of AP-1 in TERT expression may in part help explain the difference in telomerase activity between normal human and mouse cells.

Comparative Functional Study of the Viral Telomerase RNA Based on Natural Mutations

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. We previously characterized the first identified viral telomerase RNA (vTR) encoded by the Marek's disease virus (MDV) (Fragnet, L., Blasco, M. A., Klapper, W., and Rasschaert, D. (2003) J. Virol. 77, 5985-5996). This avian herpesvirus induces T-lymphomas. We demonstrated that the vTR subunit of the oncogenic MDV-RB1B strain is functional and would be more efficient than its chicken counterpart, cTR, which is 88% homologous. We take advantage of the functionality of those natural mutant TRs to investigate the involvement of the mutations of vTR on its efficiency in a heterologous murine cell system and in a homologous in vitro system using the recombinant chicken telomerase reverse transcriptase. The P2 helix of the pseudoknot seems to be more stable in vTR than in cTR, and this may account for the higher activity of vTR than cTR. Moreover, the five adenines just upstream from the P3 helix of vTR may also play an important role in its efficiency. We also established that the substitution of a single nucleotide at the 3'-extremity of the H-box of the vaccine MDV-Rispens strain vTR resulted in a lack of its accumulation within the cell, especially in the nucleus, correlated with a decrease in telomerase activity.

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.

Quantitative Assessment of Telomerase Activity and Human Telomerase Reverse Transcriptase Messenger RNA Levels in Pancreatic Juice Samples for the Diagnosis of Pancreatic Cancer

Measurement of telomerase activity is a promising diagnostic tool for pancreatic cancer. Detection of mRNA for human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is also a diagnostic candidate. In the present study, we developed a telomeric repeat amplification protocol assay with real-time PCR and a protocol for quantification of hTERT mRNA with real-time PCR. To evaluate the feasibility of these methods for diagnosis of pancreatic cancer, we measured telomerase activity and hTERT expression in pancreatic cancer cell lines, pancreatic tissues, and pancreatic juice samples from patients with different pancreatic diseases. There were significant correlations between telomerase activity and hTERT expression in cell lines, tissues, and juice samples. The levels of telomerase activity and hTERT expression were significantly higher in tumoral tissues than in nontumoral tissues. In pancreatic juice specimens, some carcinoma samples showed remarkably high expression of hTERT. However, there were no significant differences in hTERT expression between patients with carcinoma and those with benign diseases, although significant differences in telomerase activity were observed. Our present results suggest that the combined assessment of hTERT and telomerase activities in pancreatic juice provides a potent diagnostic method for pancreatic cancer.

Telomerase, hTERT and splice variants in Barrett??s oesophagus and oesophageal adenocarcinoma

The enzyme telomerase is re-activated in most cancers but its mechanism of regulation in oesophageal carcinogenesis is unclear. The aim of this study was to determine the roles of human telomerase reverse transcriptase (hTERT) mRNA expression and hTERT mRNA splicing in the regulation of telomerase enzyme activity in Barrett's oesophagus and oesophageal adenocarcinoma. Paired samples from oesophageal adenocarcinoma (n=21) and adjacent macroscopically normal mucosa, and paired samples from Barrett's oesophagus (n=16) and adjacent cardia mucosa were obtained. Telomerase activity was measured by the telomeric repeat amplification protocol assay. hTERT mRNA was quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Splice variants within the hTERT reverse transcriptase domain (alpha, beta, alpha beta) were detected by RT-PCR. In oesophageal adenocarcinoma, compared to adjacent mucosa, median telomerase activity increased significantly (from 5 to 229 total product generated (tpg), P=0.0002), but median hTERT mRNA levels were not significantly different. Similarly, median telomerase activity was significantly higher in oesophageal adenocarcinoma compared to Barrett's oesophagus (229 vs 20 tpg, P=0.001), but hTERT mRNA levels were not significantly different. There was no significant difference in telomerase activity and hTERT mRNA levels between Barrett's oesophagus and adjacent cardia. The frequency of detection of all variants increased from cardia to Barrett's oesophagus to oesophageal adenocarcinoma (P<0.05). A major increase in telomerase activity occurs after the Barrett's oesophagus stage in oesophageal carcinogenesis. Levels of hTERT mRNA and hTERT mRNA splicing patterns did not correlate with telomerase activity and do not appear to regulate enzyme activity. In this cancer, an important clinical diagnostic role for the transcripts of the telomerase gene is improbable.

Expression of telomerase activity and oxidative stress in human hepatocellular carcinoma with cirrhosis.

To study the expression and significance of telomerase activity and oxidative stress in hepatocellular carcinoma (HCC) with cirrhosis. In this study, TRAP-ELISA assay was used to determine telomerase activity in 21 cases of HCC as well as in 23 cases of hepatic cirrhosis. Malondialdehyde (MDA), glutathione S-transferase (GST) and total anti-oxidative capacity (T-AOC) were also examined in the same samples with human MDA, GST and T-AOC kits. Eighteen of 21 cases of HCC were found to have increased telomerase activity, whereas only three of the 23 non-cancerous cirrhotic samples were found to have weak telomerase activity, and the difference was significant (P<0.001). No significant difference in telomerase activity was detected according to different tumor size, tumor stage, histological grade, HBsAg, contents of albumin, bilirubin, ALT, AFP, r-GT and platelet. There were significant differences between HCC and cirrhosis in the expression of MDA, GST and T-AOC respectively. Telomerase activity correlated positively with the content of MDA (P<0.05). Telomerase activation is the early event of carcinogenesis, which is not correlated with clinicopathological factors of HCC. The dysfunction of the anti-oxidative system is closely correlated with the progression from cirrhosis to hepatocellular carcinoma. Oxidative stress may contribute partly to telomerase activation.

Telomerase activity in human leukemic cells with or without monosomy 7 or 7q-.

BackgroundIn bone marrow material from patients with various leukemias we noted that samples with either a deletion on the long arm of one chromosome 7 (7q-) or a monosomy 7 had a higher telomerase activity. Considering that introduction of a chromosome 7 into a cancer cell line had been reported to eliminate telomerase activity, that 7q- is a common negative prognostic finding in cancers, and that the deleted segment (band 7q31) contains an unidentified tumor suppressor gene, we wondered if this gene might be a telomerase inhibitor.ResultsWe found no significant difference in telomerase activity between the three groups of patient samples. In contrast to reports on tumor cell lines we observed no amplification of the telomerase genes.MethodsWe analyzed telomerase activity and copy number of the telomerase genes hTERT and hTR in frozen archival bone marrow samples from leukemia patients with a referral diagnosis of AML, and either a monosomy for chromosome 7, a deletion on the long arm of chromosome 7 (7q-), or none of these aberrations. Telomerase activity was measured with a commercially available kit, and the copy number of the telomerase genes was tested by FISH.ConclusionsWe found no evidence of a telomerase inhibitor in band 7q31. The lack of telomerase gene amplification found in cell lines from solid tumors could reflect that this amplification is a property of solid tumors, not of hematological cancers.

Telomerase activity and expression of telomerase reverse transcriptase correlated with cell proliferation in meningiomas and malignant brain tumors in vivo.

Telomerase activity was examined in intracranial tumors and compared with gene expression of the two core components of telomerase--the reverse transcriptase subunit (hTERT) and the RNA subunit (hTR)--and the proliferative index. We investigated 32 tumors across three to five sampled regions (20 meningiomas, 1 acoustic schwannoma, 1 pituitary adenoma, 8 gliomas, and 2 medulloblastomas). Telomerase activity was demonstrated by the telomeric repeat amplification protocol (TRAP) assay in seven (22%) intracranial tumors (four malignant brain tumors, two atypical meningiomas and one ependymoma) but could not be detected in the 18 (100%) benign meningiomas. hTERT and hTR mRNA were detected using reverse-transcription polymerase chain reaction (RT-PCR). hTERT mRNA was present in 20 (63%) intracranial tumors. Whereas hTERT mRNA transcripts were consistently low or absent in meningiomas, malignant brain tumors exhibited elevated hTERT mRNAs. Multiple regions of glioblastomas showed differences in telomerase activity and in the presence of hTERT mRNA. RT-PCR analysis revealed, for the first time in intracranial tumors, the presence of hTERT mRNA spliced products, corresponding to full-length mRNA as well as spliced mRNAs with critical reverse transcriptase motifs deleted. Only tumors with marked telomerase activity showed all hTERT spliced messages simultaneously. The absence of a positive correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT spliced variants. We found a significant correlation between telomerase activity scores and Ki-67 proliferation index. A positive association is also seen between Ki-67 staining and the degree of hTERT mRNA expression. This shows that there seems to be a relationship between telomerase activity or the degree of hTERT expression and proliferation rate in intracranial tumors.

Cutting edge: telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but is associated with hTERT phosphorylation and nuclear translocation.

Capacity for cellular replication is critically important for lymphocyte function and can be regulated by telomerase-dependent maintenance of telomere length. In contrast to most normal human somatic cells that do not express telomerase due to the failure to transcribe telomerase reverse transcriptase (hTERT), lymphocytes express telomerase in a highly regulated fashion yet constitutively transcribe hTERT during development and activation. Here, we report that hTERT protein is present in both thymocytes and blood T cells at equivalent levels despite their substantial differences in telomerase activity, and that induction of telomerase activity in resting CD4(+) T cells is not dependent on net hTERT protein increase. Moreover, hTERT is phosphorylated and translocated from cytoplasm to nucleus during CD4(+) T cell activation. Thus, human T lymphocytes regulate telomerase function through novel events independent of hTERT protein levels, and hTERT phosphorylation and nuclear translocation may play a role in regulation of telomerase function in lymphocytes.

Comparison of telomerase levels before and after differentiation of two cell lines of human neuroblastoma.

Telomerase is the enzyme that is responsible for maintaining telomere length in human germ cells, tumor cells, and immortalized cells. Its specific role in the immortilization process is unknown. This study was performed to determined whether the level of telomerase activity in human neuroblastoma cell lines correlated with their level of differentiation. We proposed that as neuroblastoma cells differentiated into more mature or benign cells, the levels of telomerase expression would decrease. Two human neuroblastoma cells lines, SK-N-AS and SK-N-DZ, were differentiated using retinoic acid. These cells were assayed for telomerase activity by the telomere repeat amplification protocol (TRAP) before, during, and after treatment with retinoic acid for 8 days. Untreated cells were used for control and were compared to the retinoic acid-treated cells. Differentiation of the cell lines was confirmed by assaying expression of ret mRNA using the reverse-transcriptase polymerase chain reaction (RT-PCR) and gel electrophoresis of the radiolabeled products. No statistical difference in telomerase activity was noted between control and treated groups. While telomerase activity has been shown by others to correlate with tumor aggressiveness in human neuroblastoma cells, the mechanism that is involved appears to be separate from cellular differentiation.

Age-related changes in activation of telomerase in the bone marrow of normal and thymectomized mice.

Age-related and thymus-dependent regulation of telomerase activity was studied in the bone marrow of normal (physiological aging) and thymectomized (experimental aging) mice. There was no strong correlation between the age and telomerase activity in bone marrow cells of normal mice. We observed only small individual differences in telomerase activity. Thymectomy 2-fold increased telomerase expression in young (2-month-old) and old (24-month-old) animals. Individual differences in telomerase activity in the bone marrow of thymectomized mice were more pronounced and did not depend on the age. The role of the thymus in cell aging is discussed.