Apoptosis ; physiology ; Cell Cycle ; physiology ; Humans ; Neoplasms ; etiology ; metabolism ; Tankyrases ; genetics ; physiology ; Telomerase ; metabolism ; physiology ; Telomere ; genetics ; metabolism ; Telomere-Binding Proteins ; genetics ; physiology ; Telomeric Repeat Binding Protein 1 ; genetics ; physiology ; Telomeric Repeat Binding Protein 2 ; genetics ; physiology

Humans ; Telomere ; metabolism ; Telomere-Binding Proteins ; metabolism

<b>OBJECTIVEb>To observe the distribution pattern of human telomere repeat binding factor 1(TRF1) in the telomerase-positive (HeLa) and telomerase-negative cells (WI38-2RA) and to investigate its expression level during the cell cycle.

<b>METHODSb>The full-length sequences of TRF1(TRF1FL) and its mutant with N and C terminus deletion (TRF1DeltaNC) were generated by PCR amplification, the resulting fragments were cloned into pEGFP-C2 mammalian expression vector. GFP-tagged proteins were verified by Western blotting with rabbit anti-TRF1 and mouse anti-GFP antibodies after cell transfection. Immunofluorescence staining were performed to detect the TRF1 localization in HeLa and WI38-2RA cells. Metaphase spreads from HeLa cells were also prepared to observe TRF1 localization in chromosomes. HeLa cells were arrested by thymidine and nocodazole at different cell stages. Cell cycles were analyzed by flow cytometry and TRF1 levels were evaluated by semi-quantitative Western blotting.

<b>RESULTSb>TRF1FL and TRF1PNC fragments were sized about 1.3 kb and 0.95 kb. GFP-tagged TRF1FL and TRF1DeltaNC proteins were 80 kD and 60 kD, respectively. In both HeLa and WI38-2RA cells, TRF1FL had a speckled distribution in the nuclei,however, TRF1FL did not coincide with promyelocytic leukemia (PML) nuclear body in HeLa cells while it exclusively did in WI38-2RA cells. Moreover, TRF1FL was exactly localized at the termini of metaphase spreads in HeLa cells. In contrast, TRF1PNC was diffusely distributed throughout the nuclei. Analysis by semi-quantitative Western blotting indicated that TRF1 levels increased with cell cycle progression, which reached the zenith at the M phase and went down to the nadir at G1/S point. The TRF1 level at M phase was about 3.9 times than that at G1/S point(t=12.92iP<0.01).

<b>CONCLUSIONb>TRF1 has a different localization in telomerase-positive and telomerase-negative cells, which suggests TRF1 might exert different functions in these cells. TRF1 level is regulated with cell cycle.

Cell Cycle ; HeLa Cells ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Mutation ; Telomerase ; metabolism ; Telomere-Binding Proteins ; biosynthesis ; genetics ; metabolism ; Telomeric Repeat Binding Protein 1 ; biosynthesis ; genetics ; metabolism ; Tumor Cells, Cultured

As an important telomere binding protein, TPP1 protects the ends of telomeres and maintains the stability and integrity of its structure and function by interacting with other five essential core proteins (POT1, TRF1, TRF2, TIN2, and RAP1) to form a complex called Shelterin. Recently, researchers have discovered that TPP1 participates in protection of telomeres and regulation of telomerase activity. The relationship between TPP1 and tumorigenesis, tumor progression and treatment has also been investigated. This paper reviews the latest findings of TPP1 regarding to its structure, function and interaction with other proteins involved in tumorigenesis.
Chromosomal Instability ; DNA Damage ; Humans ; Neoplasms ; genetics ; Telomere ; Telomere-Binding Proteins ; chemistry ; physiology

<b>OBJECTIVEb>To analysze genotype and measure telomere length in two Chinese patients with dyskeratosis congenita(DC).

<b>METHODSb>The peripleral blood DNA was extracted in two patients characterized by mucocutaneous abnormalities (abnormal nails, lacy reticulated skin pigmentation, and oral leukoplakia), bone marrow failure, DC genes were amplified by polymerase chain reaction (PCR), including DKC1, TERT, TERC, TINF2, NOP10, NHP2, then DNA sequencing was performed for abnormal exons. Lymphocyte telomere length was measured by flow cytometry-fluorescence in situ hybridization(Flow-FISH).

<b>RESULTSb>Abnormal peaks were found in exon 6 of TINF2 gene of the two patients and a 811C→T transition in TINF2 gene in one patient. DNA sequencing showed a 848C→A transition in TINF2 gene in another patient. Relative telomere length was remarkable less than that of normal children with same age.

<b>CONCLUSIONSb>Physician should think about DC if the young patients with mucocutaneous abnormalities and marrow failure. Early detection of related genes and measurernant of tolomere length may contribute to avoid misdiagnosis. TINF2 c.811C→T (Q271X) and TINF2 c.848C→A (P283H) exist in the two patients, it is reported in China for the first time.

Base Sequence ; Bone Marrow ; China ; Dyskeratosis Congenita ; Exons ; Genotype ; Humans ; In Situ Hybridization, Fluorescence ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Telomere ; Telomere-Binding Proteins

<b>OBJECTIVEb>To analysis the clinic and genotype in two Chinese patients with Dyskeratosis congenita (DC).

<b>METHODSb>The two patients were characterized by mucocutaneous abnormalities (abnormal nails, lacey reticular pigmentation, and oral leukoplakia), bone marrow failure. They were diagnosed with DC. DC genes were amplified by polymerase chain reaction (PCR), including DKC1, TERT, TERC, TINF2, NOP10, NHP2, then DNA sequencing was performed for abnormal exons.

<b>RESULTSb>An abnormal peak was found in exon 6 of TINF2 gene of the two patients. DNA sequencing showed a 845G→A transition in TINF2 gene in the two patients.

<b>CONCLUSIONb>We should think about DC if the young patients with mucocutaneous abnormalities and marrow failure. TINF2 c.845G→A(R282H) does exist in the two patients. It is reported in China for the first time.

Base Sequence ; Child, Preschool ; DNA Mutational Analysis ; Dyskeratosis Congenita ; diagnosis ; genetics ; Exons ; Female ; Humans ; Infant ; Male ; Telomere-Binding Proteins ; genetics

This study was purposed to explore the relationship between the mRNA expression of telomere protection protein TIN2 and POT1 and the pathogenesis of myelodysplastic syndrome (MDS). The expression of TIN2 and POT1 genes at the mRNA levels were detected by real-time fluorescence quantitative PCR in 51 patients with MDS and 10 normal controls. The results showed that the mRNA expressions of TIN2 in RA/RARS/RCMD/MDS-U, RAEB-1 and RAEB-2 groups according to the World Health Organization criteria were significantly higher than that in the controls (P < 0.05); the mRNA expressions of POT1 in RA/RARS/RCMD/MDS-U, RAEB-1 and RAEB-2 groups were significantly lower than that in the controls (P < 0.05). The mRNA expressions of TIN2 in high-risk group, inter risk-2 group and inter risk-1 group according to the international prognostic scoring system criteria were significantly higher than that in controls (P < 0.05). There was no significant difference between low risk group and the control group. The mRNA expressions of POT1 in high risk group, inter-risk-2 group and inter-risk-1 group were significantly lower than the controls (P < 0.05). There was no significant difference between low risk group and the control group. The mRNA expression of TIN2 in normal chromosome group was significantly lower than that in abnormal chromosome group (P < 0.05). There was no significant difference between normal chromosome group and the control group. The mRNA expression of POT1 in normal chromosome group was significantly higher than that in abnormal chromosome group (P < 0.05). There was no significant difference between normal chromosome group and the control group. It is concluded that the abnormal mRNA expression of TIN2 and POT1 may be involved in the regulation of telomere dynamics of MDS patients, the regulatory mechanism may be related to the telomere length and the pathogenesis of MDS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; metabolism ; pathology ; Case-Control Studies ; Cell Adhesion Molecules ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Telomere ; metabolism ; Telomere-Binding Proteins ; genetics ; metabolism ; Young Adult

<b>OBJECTIVEb>By testing the changes of telomere binding protein in malignant transformation BEAS-2B cells induced by coal tar pitch smoke extracts, to study the role of protection of telomeres 1 (POT1), telomeric repeat binding factor 1 (TRF1) and TRF2 in tumorgenesis that contact with coal tar pitch.

<b>METHODSb>The BEAS-2B cells were induced by coal tar pitch smoke extracts to form malignant transformation cell model in vitro. The gene expression levels of mRNA were assessed by real-time quantitative RT-PCR, the protein expression variations were determined by cell culture overslip of immunohistochemical methods.

<b>RESULTSb>In malignant transformation cells, the mRNA expression level (POT1: 0.63 ± 0.04, TRF1: 0.36 ± 0.01) and the protein expression level (POT1: 0.36 ± 0.05, TRF1: 0.09 ± 0.03) of POT1 and TRF1 was statistically significant decreased compared to that of BEAS-2B group (mRNA: POT1: 1.00 ± 0.04, TRF1: 1.01 ± 0.16; protein: POT1: 0.55 ± 0.07, TRF1: 0.27 ± 0.07) and DMSO group (mRNA: POT1: 0.89 ± 0.12, TRF1: 0.90 ± 0.08; protein: POT1: 0.55 ± 0.10, TRF1: 0.26 ± 0.04) (P < 0.05); mRNA expression level (1.45 ± 0.07) and the protein expression level (0.88 ± 0.06) of TRF2 was increased compared to that of BEAS-2B group (mRNA: 1.00 ± 0.07, protein: 0.48 ± 0.06) and DMSO group (mRNA: 1.00 ± 0.06, protein: 0.50 ± 0.06) (P < 0.05).

<b>CONCLUSIONb>The change of gene and protein expression level in POT1, TRF1, and TRF2 involved in the process that evolved into malignant transformation in bronchial epithelial cells BEAS-2B induced by coal tar pitch smoke extracts.

Cell Line ; Cell Transformation, Neoplastic ; metabolism ; Coal Tar ; toxicity ; Epithelial Cells ; cytology ; metabolism ; pathology ; Humans ; Repetitive Sequences, Nucleic Acid ; Telomere-Binding Proteins ; genetics ; metabolism

<b>OBJECTIVEb>To investigate the role of telomerase and telomere repeat binding factors (TRF) in apoptosis.

<b>METHODSb>The proliferative activity of Tca8113 cells was assessed by methyl thiazolyl tetrazolium (MTT) assay. After Tca8113 cells were treated with adriamycin at 5 mg/L, apoptotic morphology was observed under microscope with Giemsa staining and apoptosis examined by flow cytometry; analysis of telomerase activity was performed by TRAP-enzyme-linked immunosorbent assay; expression and expression level of TRF proteins were detected with immunohistochemical staining and immunofluorescence label assay, respectively.

<b>RESULTSb>After Tca8113 cells were treated with adriamycin at 5 mg/L for 5 days and 7 days, the cells apoptosis was found. Telomerase activity dropped in time-dependent manner. Expression of TRF proteins appeared in nucleus of the cells. No statistical difference in expression levels of TRF was observed between the treated and untreated cells.

<b>CONCLUSIONSb>Tca8113 cells apoptosis induced by adriamycin decreased telomerase activity, but did not influence the expression level of TRF proteins.

Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Humans ; Telomerase ; metabolism ; Telomere-Binding Proteins ; metabolism ; Tongue Neoplasms ; metabolism ; pathology

<b>OBJECTIVEb>To study interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A.

<b>METHODSb>Nek2A305-446 protein was expressed and purified in E.coli and TACP1 protein was expressed in transfected 293T cells. Pull-down assay was used to examine the interaction between Nek2A305-446 and TACP1. TACP1 and Nek2A complex was tested by co-immunoprecipitation assay with polyclonal anti-TACP1 antibody. The localization of those two proteins in Hela cells was verified by immunofluorescence.

<b>RESULTSb>TACP1 was pulled down by Nek2A305-446 protein but not by GST control. Nek2A was co-precipitated with TACP1 protein by polyclonal anti-TACP1 antibody but not by pre-immunization serum. The Immunofluorescence test showed that these two proteins formed a complex at centrosome during mitosis.

<b>CONCLUSIONb>Centrosomal protein TACP1 is a novel interacting protein with Nek2A, both of which are localized in centrosome during mitosis.

Cell Line ; Centrosome ; metabolism ; Escherichia coli ; genetics ; Fluorescent Antibody Technique ; HeLa Cells ; Humans ; Immunoprecipitation ; Mitosis ; NIMA-Related Kinases ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Telomere-Binding Proteins ; genetics ; metabolism ; Transfection