OBJECTIVETo quantitatively measure plasma level of human telomerase reverse transcriptase (hTERT) mRNA in patients with nasopharyngeal carcinoma (NPC) and explore its implications for NPC diagnosis and treatment.

METHODSWith 24 healthy volunteers serving as controls, the plasma level of hTERT mRNA was detected in 33 NPC patients by real-time PCR before and after treatments with chemotherapy or radiotherapy, and its association with the clinicopathological parameters of the patients were analyzed.

RESULTSThe NPC patients showed a significantly higher mean plasma level of hTERT mRNA than the healthy volunteers (10.75 ± 4.29 vs 0.95 ± 0.37, P<0.05). The plasma hTERT mRNA level in the NPC patients was significantly correlated with clinical staging, tumor size, and degree of nodal metastasis (P<0.05) but with gender or age (P>0.05). In patients with stage I and II NPC, the plasma hTERT mRNA level decreased significantly after radiotherapy (5.60 ± 2.33 vs 3.43 ± 1.42); in patients in advanced stages (III and IV), plasma hTERT mRNA level decreased significantly from 12.68 ± 3.08 to 10.68 ± 2.48 (P<0.05) after chemotherapy and to 3.13 ± 1.69 (P<0.05) after radiotherapy.

CONCLUSIONRadiotherapy and chemotherapy can effectively suppress elevated plasma hTERT mRNA levels in NPC patients. Plasma hTERT mRNA level is closely related to the clinicopathological factors and provides important information for early diagnosis and therapeutic effect evaluation of NPC.

Carcinoma ; Case-Control Studies ; Humans ; Nasopharyngeal Neoplasms ; blood ; RNA, Messenger ; blood ; Telomerase ; blood

BACKGROUND: Background Telomerase activation and human telomerase RNA (hTR) expression are known to be related to the preservation of the "stemness" of stem cells. In this study, we have inhibited the expression of hTR to find the relationship between the telomerase activity and differentiation of normal hematopoietic stem cells. METHODS: We used cord blood collected from 10 full term pregnant women. We classified the CD34+ hematopoietic stem cells from the same donor into three groups: the Ad-OA group was treated with the recombinant adenoviral (Ad) vector Ad-OA using telomerase antisense, the Ad-M6 group was treated with a mutant version of the Ad-OA without telomerase antisense, and a control group without any treatment. RESULTS: The mean number of colony-forming cells (CFCs) were 110+/-38 for the Ad-OA groups, 540+/-56 for the Ad-M6 groups, and 650+/-72 for the control groups. Thus, CFCs in the Ad-OA group were lower than in the Ad-M6 group (P<0.01). The myeloid portion of the CFCs in the Ad-OA group was higher than the Ad-M6 and control groups (P<0.01). The Ad-OA group showed a higher percentage of granulocytes suggesting more of a tendency for myeloid differentiation than the Ad-M6 and control groups (P<0.01). We found that the suppression of telomerase activity by the antisense telomerase adenovirus induced the differentiation of hematopoietic stem cells confirmed by differential cell count and cytochemical staining. CONCLUSION: These findings suggest that the activity of the telomerase may play a role in the differentiation of normal CD34+ hematopoietic stem cells into mature cells.
Adenoviridae ; Cell Count ; Female ; Fetal Blood ; Granulocytes ; Hematopoietic Stem Cells ; Humans* ; Pregnant Women ; RNA* ; Stem Cells ; Telomerase* ; Tissue Donors

The present study was aimed to investigate the effect of different altitudes on telomere length of rat peripheral blood leukocyte and possible mechanism. Sixty male rats were randomly divided into three groups, lower altitude control group (10 m), moderate altitude group (2 260 m) and very high altitude group (simulated 5 000 m). The moderate altitude group and very high altitude group rats were transported to Xining and hypobaric chamber in Qinghai University, respectively. The peripheral blood specimens were extracted 30 d after the transportation. By means of real-time PCR, automatic blood cell analyzer, ELISA, TBA and WST-1 methods, the telomere lengths of blood leukocyte, the hemoglobin (Hb) contents, the plasma levels of telomerase reverse transcriptase (TERT) and hypoxia-inducible factor 1α (HIF-1α), the plasma content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured, respectively. The results showed that the telomere lengths of peripheral blood leukocyte in moderate altitude group were longer than those in control group and very high altitude group. The changes of TERT were compatible with the telomere length of peripheral blood leukocyte under different altitudes. The levels of HIF-1α in moderate altitude group and very high altitude group were higher than that of control group. The very high altitude group showed decreased SOD activities and increased level of MDA, compared with the other two groups. These results suggest that the telomere lengths of rat peripheral blood leukocyte in moderate altitude are elongated, and that the telomere-elongating effect is lost under very high altitude. The changes of HIF-1α, TERT and oxidative stress damage are the main mechanisms of telomere length changes. Moderate altitude living might be beneficial to increasing the life span in mammals.
Altitude ; Animals ; Hemoglobins ; metabolism ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit ; blood ; Leukocytes ; physiology ; Male ; Malondialdehyde ; blood ; Oxidative Stress ; Rats ; Superoxide Dismutase ; metabolism ; Telomerase ; blood ; Telomere ; physiology

Telomerase play a key role in the maintenance of telomere length and chromosome integrity. We have evaluated the association between telomerase activity and the risk of lung cancer in peripheral blood. Telomerase activity in peripheral blood mononuclear cells was measured by a PCR-designed telomeric repeat amplification protocol in 63 lung cancer patients and 190 healthy controls that were matched for age, gender, and smoking status. Telomerase activity was significantly lower in the lung cancer patients than in controls (mean +/- standard deviation; 1.32 +/- 1.65 vs 2.60 +/- 3.09, P < 1 x 10(-4)). When telomerase activity was categorized into quartiles based on telomerase activity in the controls, the risk of lung cancer increased as telomerase activity reduced (Ptrend = 1 x 10(-4)). Moreover, when the subjects were categorized based on the median value of telomerase activity, subjects with low telomerase activity were at a significantly increased risk of lung cancer compared to subjects with high telomerase activity (adjusted odds ratio = 3.05, 95% confidence interval = 1.60-5.82, P = 7 x 10-4). These findings suggest that telomerase activity may affect telomere maintenance, thereby contributing to susceptibility to lung cancer.
Age Factors ; Aged ; Case-Control Studies ; Female ; Humans ; Leukocytes, Mononuclear/enzymology/immunology ; Lung Neoplasms/*enzymology/*etiology ; Male ; Middle Aged ; Odds Ratio ; Risk Factors ; Sex Factors ; Smoking ; Telomerase/*blood

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.
AC133 Antigen ; Antigens, CD ; Antigens, CD34 ; analysis ; Cell Cycle ; Cells, Cultured ; Fetal Blood ; cytology ; Glycoproteins ; analysis ; Hematopoietic Stem Cells ; physiology ; Humans ; Immunophenotyping ; Peptides ; analysis ; Telomerase ; metabolism

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.
Antigens, CD34 ; blood ; Blotting, Western ; Cell Differentiation ; DNA-Binding Proteins ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; enzymology ; Humans ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

STUDY DESIGN: In vitro cell culture. PURPOSE: The purpose of the study was to investigate the effect of high glucose on premature stress-induced senescence of rat notochordal cells. OVERVIEW OF LITERATURE: Glucose-mediated increase of oxidative stress is a major causative factor for the development of diseases associated with diabetes mellitus such as senescence. However, no information is available for the effect of high glucose on premature stress-induced senescence of rat notochordal cells. METHODS: Notochordal cells were isolated from 4-week-old rats, cultured and placed in either 10% fetal bovine serum (FBS, normal control) or 10% FBS plus two high glucose concentrations (0.1 M and 0.2 M, experimental conditions) for 1 and 3 days. We identified and quantified the mitochondrial damage (mitochondrial transmembrane potential), reactive oxygen species (ROS) and antioxidants, such as manganese superoxide dismutase (MnSOD) and catalase, for each condition. We also identified and quantified senescence and telomerase activity. Finally, we determined the expression of proteins related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB) pathways. RESULTS: Two high glucose concentrations enhanced the disruption of mitochondrial transmembrane potential and excessive generation of ROS in notochordal cells for 1 and 3 days, respectively. The expressions of MnSOD and catalase were increased in notochordal cells treated with both high glucose concentrations at 1 and 3 days. The telomerase activity declined at 1 and 3 days. Two high glucose concentrations increased the occurrence of stress-induced senescence of notochordal cells by p16-pRB pathways at 1 and 3 days. CONCLUSIONS: Despite compensatory expression of antioxidants, high glucose-induced oxidative stress accelerates stress-induced senescence in rat notochordal cells. This may result in dysfunction of notochordal cells, leading to accelerated premature disc degeneration. The prevention of excessive generation of oxidative stress by strict blood glucose control is important to prevent or to delay premature disc degeneration in young patients with diabetes mellitus.
Aging* ; Animals ; Antioxidants ; Blood Glucose ; Catalase ; Cell Aging ; Cell Culture Techniques ; Diabetes Mellitus ; Glucose* ; Humans ; Intervertebral Disc Degeneration ; Membrane Potentials ; Notochord* ; Oxidative Stress ; Rats* ; Reactive Oxygen Species ; Superoxide Dismutase ; Telomerase

Cancer stem cells have stem cell-like features, such as the ability for self-renewal and differentiation but show unlimited growth because they have the lost normal regulation of cell growth. Cancer stem cells and normal stem cells have similar features. They show high motility, diversity of progeny, robust proliferative potential, association with blood vessels, immature expression profiles, nestin expression, epidermal growth factor (EGF)-receptor expression, phosphatase and tensin homolog (PTEN) expression, hedgehog pathway activity, telomerase activity, and Wnt pathway activity. On the other hand, with cancer cells, some of these signaling pathways are abnormally modified. In 1875, Cohnheim suggested the concept of cancer stem cells. Recently, evidence for the existence of cancer stem cells was identified. In 1994, the cancer stem cells'specific cell surface marker for leukemia was identified. Since then, other specific cell surface markers for cancer stem cells in solid tumors (e.g. breast and colon cancer) have been identified. In oral cancer, studies on cancer stem cells have been performed mainly with squamous cell carcinomas. Oral cancer specific cell surface markers, which are genes strongly expressed in oral cancer and cancer stem cell specific side populations, have been identified. Cancer stem cells are resistant to radiotherapy and chemotherapy. Therefore, to eliminate malignant tumors efficiently and reduce the recurrence rate, therapy targeting cancer stem cells needs to be performed. Currently, studies targeting the cancer stem cells'specific signaling pathways, telomerase and tumor vasculatures are being done.
Antigens, Surface ; Blood Vessels ; Breast ; Carcinoma, Squamous Cell ; Colon ; Epidermal Growth Factor ; Hand ; Hedgehogs ; Intermediate Filament Proteins ; Leukemia ; Microfilament Proteins ; Mouth Neoplasms ; Neoplastic Stem Cells ; Nerve Tissue Proteins ; Recurrence ; Signal Transduction ; Stem Cells ; Telomerase ; Wnt Signaling Pathway

OBJECTIVETo explore the regulatory effects of hTERT and TEP1 on telomerase activity in hematopoiesis.

METHODSThe hTERT and TEP1 mRNA expression was detected by RT-PCR and the telomerase activi-ty by TRAP.

RESULTSIn mononuclear cells (MNC) and CD(34)(-) cells, no detectable telomerase activity and hTERT mRNA expression were found. CD(34)(+) cells showed hTERT expression and a low level telomerase activity. TEP1 mRNA was detected in MNC, CD(34)(-) and CD(34)(+) cells with no significant difference in the expression level. In the CD(34)(+) cells cultured in vitro with growth factors for 7 days, the telomerase activity and the expression of hTERT mRNA were upregulated, but were downregulated in the long time culture. No significant changes in TEP1 expression was observed.

CONCLUSIONIn the course of hematopoiesis, hTERT mRNA expression was in accordance with telomerase activity, hTERT gene plays a crucial role in the expression of telomerase activity, while TEP1 gene plays, if any, a much smaller role.

Antigens, CD34 ; immunology ; Carrier Proteins ; genetics ; DNA-Binding Proteins ; Fetal Blood ; cytology ; metabolism ; Gene Expression ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Telomerase ; genetics

OBJECTIVETo investigate the associations between Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) infections and the methylation levels of PTEN and hTERT genes and explore their roles in children with acute lymphoblastic leukemia (ALL).

METHODSBlood samples from 100 children with newly diagnosed acute lymphoblastic leukemia were centrifuged for serological detection of EBV and HCMV, and the patients were divided accordingly into EBV-infected group (n=20), HCMV-infected group (n=14), EBV and HCMV co-infected group (n=41), and non-infected group (control group, n=15). DNA was extracted from peripheral blood mononuclear cells (PBMCs) and modified with bisulfite ammonia sodium. The methylation levels of the promoters of PTEN and hTERT genes were detected with methylation-specific polymerase chain reaction (MS-PCR).

RESULTSCompared with those in non-infected group and EBV- or HCMV-infected group, the methylation levels of PTEN gene in the co-infected group were significantly decreased (P<0.05) while the methylation levels of hTERT gene significantly increased (P<0.05).

CONCLUSIONIn children with acute lymphoblastic leukemia, EBV and HCMV co-infection cause changes in the methylation levels of PTEN and hTERT. These results may be associated with epigenetic changes caused by viral infections, and further studies are needed to further verify this hypothesis.

Child ; Coinfection ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; DNA Methylation ; Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; isolation & purification ; Humans ; PTEN Phosphohydrolase ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; genetics ; virology ; Promoter Regions, Genetic ; Telomerase ; genetics ; metabolism