OBJECTIVETo observe the effect of Angelica sinensis polysaccharides (ASP) on the length of telomere, the activity of telomerase and the expression of P53 protein in mice hematopoietic stem cells (HSCs), and explore ASP's potential mechanism for regulating HSC aging.

METHODC57BL/6J mice were randomly divided into the normal group, the aging group and the intervention group. The aging group was radiated with X ray to establish the mice aging HSC model. The intervention group was orally administered with ASP during X-ray irradiation, while the normal group was orally administered with NS. Their HSCs were isolated by immunomagnetic beads. Cell cycles analysis and senescence-associated beta-galactosidase (SA-beta-Gal) staining were used to detect changes in aging HSCs. The expression of P53 was determined by western blot analysis. The length of telomere and the vitality of telomerase were analyzed by southern blot and TRAP-PCR, respectively.

RESULTCompared with the normal group, X-ray irradiation could significantly increase the cell ratio of in HSC G1 stage, rate of SA-beta-Gal positive cells and expression of P53 protein, and reduce the length of telomere and the vitality of telomerase. Compared with the aging group, ASP could significantly inhibit the cell ratio of in HSC G1 stage and the increase in the number of SA-beta-Gal positive cells, down-regulate the expression of P53 protein, and increase the length of telomere and the vitality of telomerase in HSCs.

CONCLUSIONASP could antagonize X-ray-induced aging of HSCs, which may be related to the increase in the length of telomere and the activity of telomerase, as well as the down-regulation of the expression of P53 protein.

Angelica sinensis ; chemistry ; Animals ; Cell Cycle ; drug effects ; physiology ; Cellular Senescence ; drug effects ; physiology ; Female ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; isolation & purification ; pharmacology ; Telomerase ; biosynthesis ; metabolism ; Telomere ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; biosynthesis ; metabolism

OBJECTIVETo construct a RNA interference expression vector targeting human telomerase reverse transcriptase gene (hTERT) gene and investigate its effects on telomerase activity and proliferation in breast cancer cells in vitro.

METHODSThe shRNA sequences targeting hTERT gene were designed and recombined into pSuper-retro-puro vector. The breast cancer cell lines MCF-7 and MDA-MB231 were transfected with the recombined vector, and the telomerase activity of the cells was tested by telomerase repeat sequence amplification-enzyme linked immunosorbent assay (TRAP-ELISA). The proliferation of the transfected cells was assessed using MTT and soft-agar clone formation assays.

RESULTSThe recombinant plasmids pSuper-retro-puro-TERT RNAi#1 and #2 were successfully constructed as confirmed by enzymatic digestion and DNA sequencing. The telomerase activity in the transfected breast cancer cells were down-regulated significantly as compared with that in negative control cells (Plt;0.005). The transfection resulted in significant inhibition of the proliferation of both MCF-7 and MDA-MB231 cells as detected by MTT assay (Plt;0.05) and soft agar clone formation assay (Plt;0.001).

CONCLUSIONTransfection with the recombinant plasmid containing the shRNA targeting hTERT gene can down-regulate telomerase activity and inhibit proliferation of breast cancer cells in vitro, suggesting the potential of gene therapy targeting telomerase in the treatment of breast cancer.

Breast Neoplasms ; enzymology ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Female ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; metabolism ; Telomerase ; biosynthesis ; genetics ; metabolism ; Transfection

Animals ; Cell Culture Techniques ; methods ; Cell Line ; Cell Proliferation ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver ; cytology ; Liver Diseases ; therapy ; Liver, Artificial ; Mice ; Recombination, Genetic ; Simian virus 40 ; genetics ; Telomerase ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis

OBJECTIVETo construct a recombinant lentivirus RNA interference (RNAi) vector carrying hTERT gene, and to obtain the titer of the lentiviral stock for investigating the expression in the eukaryotic cells and the effect on the hTERT gene silencing in the eukaryotic cells.

METHODSTwo complimentary oligos of small interference RNA (siRNA) with hairpin structures targeting the hTERT gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into T293 cells, viral supernatant was harvested to determine the titer. U87 cells infected by virus were harvested and the expression of hTERT, telomerase activity and apoptosis were detected by reverse transcription-PCR(RT-PCR), TRAP assay and flow cytometry separately.

RESULTSSequencing data showed that the constructed plasmids contained the correct sequences of hTERT siRNA transcript templates. A vector producing cell line T293 was established, and the titer for transfection was obtained. RT-PCR and TRAP flow cytometry analyses demonstrated that hTERT shRNA expression construct could suppress the expression of hTERT and telomerase activity and induce apoptosis.

CONCLUSIONA lentivirus RNAi vector targeting hTERT gene was successfully constructed, which decreased the expression of hTERT and telomerase activity effectively and induced apoptosis. It has set up a research platform for the gene therapy of tumors which take hTERT as the target.

Base Sequence ; Cell Line ; Flow Cytometry ; Gene Knockdown Techniques ; methods ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Molecular Sequence Data ; Oligodeoxyribonucleotides ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; biosynthesis ; genetics

The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E. coli BL21 solublely, and purified by Ni Sepharose affinity chromatography which, however, is not fit for large scale protein purification. The gene of LITS-L was then PCR amplified to remove the 6 x His tag, and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E. coli B21, and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly, similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study, a technique for preparation of LPTS-L protein in a large scale is established.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Telomerase ; antagonists & inhibitors ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo construct an eukaryotic expression vector of human telomerase reverse transcriptase (hTERT) gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with gamma-irradiation on cell survival and telomerase activity.

METHODSAccording to the coding sequence of hTERT mRNA, the target of RNAi was designed, and recombinant expression plasmid pshRNA-hTERT was constructed. The vector was transfected into Hep-2 cells. The radiosensitivity of Hep-2 cells was determined by clonogenic assay. Telomeric repeat amplification protocol (TRAP-PCR-ELISA) was used to observe the telomerase activity in each group. Results Recombinant expression vector pshRNA-hTERT was successfully transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60. 8%. pshRNA-hTERT not only inhibited telomerase activity of Hep-2, but also inhibited the raise of telomerase activity induced by gamma-irradiation. Exposure of Hep-2 cells to pshRNA-hTERT for 24 hrs before irradiation resulted in a decrease in mean surviving fraction of Hep-2 cells compared with cells of group with irradiation alone (67. 7% vs 85. 7%, P <0. 05) .

CONCLUSIONRNAi showed a significant inhibitory effect to the expression of hTERT. The results indicate that pshRNA-hTERT can effectively inhibit telomerase activity of Hep-2 cells treated or untreated with 2 Gy gamma-irradiation and significantly enhance the radiosensitivity of Hep-2 cells in vitro. The role of radiosensitization of pshRNA-hTERT may be related with the inhibition of telomerase activity.

Carcinoma, Squamous Cell ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Survival ; genetics ; radiation effects ; Cobalt Radioisotopes ; Enzyme-Linked Immunosorbent Assay ; Gamma Rays ; Humans ; Laryngeal Neoplasms ; enzymology ; genetics ; pathology ; Plasmids ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo explore the relationship between human papillomavirus (HPV) 16 infection and the expression of p33(ING1b), human telomerase reverse transcriptase (hTERT) in cervical squamous cell carcinoma of Uygur Female in Xinjiang Uygur Autonomous Region.

METHODSPolymerase chain reaction (PCR) and immunohistochemical methods were used to detect HPV16 infection and the expression of p33(ING1b) and hTERT in the normal control group (n=12), the patients with cervical intraepithelial neoplasm (CIN) (n=34), and the patients with cervical squamous cell carcinoma (SCC) (n=50).

RESULTSIn the cervical tissues of Uygur female, the HPV16 infection rate was 0 in control group, 22.2% in the CIN 1 group, 44.0% in CIN 2 & CIN 3 group, and 74.0% in SCC group (P = 0.000). The expression rate of p33(ING1b) decreased was 91.7% in control group, 77.7% in CIN 1 group, 68.0% in CIN 2 & CIN 3 group, and 36.0% in SCC group (P = 0. 000). The expression rate of hTERT was 50.0% in control group, 66.6% in CIN 1 group, 88.0% in CIN 2 & CIN 3 group, and 94.0% in SCC group (P = 0.000). In the cervical tissues of Uygur female, the HPV16 infection rate was negatively correlated with the expression of p33(ING1b) (r = -0.294, P = 0.004), and was positively correlated with the expression of hTERT (r = 0.286, P = 0.005). The expression of p33(ING1b) was negatively correlated with the expression of hTERT (r = -0.361, P = 0.000).

CONCLUSIONThe infection of HPV 16 correlates with the decreased expression of p33(ING1b) and increased expression of hTERT in the cervical squamous cell carcinoma of Uygur female in Xinjiang.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; China ; Female ; Human papillomavirus 16 ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; Nuclear Proteins ; biosynthesis ; Papillomavirus Infections ; metabolism ; virology ; Telomerase ; biosynthesis ; Tumor Suppressor Proteins ; biosynthesis ; Uterine Cervical Neoplasms ; metabolism ; virology

OBJECTIVEThe aim of this study was designed to investigate the effect of TSA on human umbilical vein endothelial cells and to reveal its possible mechanisms and relationship between apoptosis and activity of telomerase reverse transcriptase.

METHODSsulforhodamine B method was employed to determine the growth rate of umbilical vein endothelial cells. The cell apoptotic rate was measured by flow cytometry (FCM). The hTERT and p21(Waf1) mRNA expression before and after TSA treatment were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The quantitative of hTERT protein expression in cells were detected by flow cytometry. After transfection, the cell telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAP-ELISA) and early apoptosis was measured by Annexin V/PI stain and flow cytometry.

RESULTSAfter being treated with TSA, the proliferation of umbilical vein endothelial cells was inhibited. Slight apoptosis and cell cycle arrest were detected. However, the same concentration of TSA induced serious apoptosis in HeLa cells. Up-regulation of hTERT mRNA expression was observed within 48 h after TSA treatment, but the change of p21(Waf1) expression was not significant. The umbilical vein endothelial cells hTERT protein expression level was increased within 24 h. After transfection of the dominant negative, wild type and control hTERT plasmid, a significant difference of telomerase activity in these cells was observed by PCR-TRAP-ELISA assay. WT-hTERT-transfected cells were more resistant to apoptosis induced by trichostatin A.

CONCLUSIONHuman umbilical vein endothelial cells could be resistant to apoptosis induced by high concentrate TSA, and hTERT might play an important role in this process.

Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; methods ; Flow Cytometry ; Gene Expression ; drug effects ; HeLa Cells ; Humans ; Hydroxamic Acids ; pharmacology ; Plasmids ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Telomerase ; genetics ; metabolism ; Transfection ; Umbilical Veins ; cytology

Telomere length maintenance is regarded as a fundamental step in tumorigenesis, as most human brain tumors, including meningiomas, stabilize the ends of their chromosomes using telomerase. This investigation represents an introduction to telomerase expression in canine and feline meningiomas. Twenty-five archived cases (14 dogs and 11 cats) were immunohistochemically tested for human-telomerase reverse transcriptase (h-TERT), scored, and quantified; furthermore, mitoses were counted on sections stained with a modified toluidine blue. The h-TERT antibody immunolabelled the nucleus and nucleolus of meningeal neoplastic cells, with an intensity ranging from mild to strong and a speckled distribution; a significantly higher expression in cats was noted, while no significant association between h-TERT immunolabelling and sex or histotype was evident in dogs or cats. The telomerase enzyme represents a fundamental parameter of potential malignant transformation, which may occur independently of the signal to proliferate, thereby supplying the cells with unlimited growth capabilities. Telomerase expression could be a prognostic indicator independent of the kinetic parameters, although this should be evaluated using a larger dataset with available clinical information.
Animals ; Brain Neoplasms/enzymology/pathology/*veterinary ; Cat Diseases/*enzymology/pathology ; Cats ; Dog Diseases/*enzymology/pathology ; Dogs ; Female ; Immunohistochemistry/veterinary ; Male ; Meningioma/enzymology/pathology/*veterinary ; Mitosis/physiology ; Regression Analysis ; Telomerase/*biosynthesis

The study was aimed to investigate the expression level of TRF1 protein in human acute leukemia and relationship between expression level of TRF1 protein and activity of telomerase. A quantitative Western blot technique was developed using anti-TRF1(33 - 277) monoclonal antibody and GST-TRF1 fusion protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. 20 cases of acute leukemias were studied when 11 normal volunteer's bone marrow was used as control. The results showed that the expression level of TRF1 protein in normal bone marrow (2.217 +/- 0.461 microg/microl) was significantly higher than that in bone marrow of acute leukemia patients (0.754 +/- 0.343 microg/microl) (P < 0.01). There was no remarkable difference of expression level of TRF1 protein between ALL and ANLL (0.628 +/- 0.281 microg/microl vs 0.844 +/- 0.360 microg/microl, P > 0.05). After chemotherapy, TRF1 expression level of patients with complete remission raised (0.772 +/- 0.307 microg/microl vs 1.683 +/- 0.344 microg/microl, P < 0.01), but lower than that of normal (2.217 +/- 0.461 microg/microl, P < 0.01). TRF1 expression level of patients without complete remission was not remarkable different after chemotherapy (0.726 +/- 0.443 microg/microl vs 0.894 +/- 0.338 microg/microl, P > 0.05). TRF1 expression level of patients with complete remission was higher than that in patients without complete remession (1.683 +/- 0.344 microg/microl vs 0.894 +/- 0.338 microg/microl, P < 0.01). For all sample the telomerase activity was determined. It was confirmed that the activity of telomerase in normal bone marrow was lower than that in bone marrow of acute leukemia patients (0.125 +/- 0.078 microg/microl vs 0.765 +/- 0.284 microg/microl, P < 0.01). There was no significantly difference of expression level of TRF1 protein between ALL and ANLL (0.897 +/- 0.290 microg/microl vs 0.677 +/- 0.268 microg/microl, P > 0.05). After chemotherapy, telomerase activity of patients with complete remission reduced (0.393 +/- 0.125 microg/microl), but higher than that of normal (0.125 +/- 0.078 microg/microl, P < 0.01). It is concluded that expression level of TRF1 protein in AL patients is significantly decrese and associated with therapeutic efficaciousness and the activity of telomerase (P < 0.001).
Adolescent ; Adult ; Aged ; Bone Marrow Cells ; enzymology ; metabolism ; Child ; Female ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; metabolism ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; enzymology ; metabolism ; Telomerase ; metabolism ; Telomeric Repeat Binding Protein 1 ; biosynthesis ; genetics