Advance in research on the function of telomeric shelterin component TPP1 and its relationship with characteristics of tumors / 中华医学遗传学杂志

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.

Pontos de venda de alimentos e associação com sobrepeso/obesidade em escolares de Florianópolis, Santa Catarina, Brasil / Retail food outlets and the association with overweight/obesity in schoolchildren from Florianópolis, Santa Catarina State, Brazil / Puntos de venta de alimentos y asociación con sobrepeso/obesidad en escolares de Florianópolis, Santa Catarina, Brasil

O estudo descreve os pontos de venda de alimentos e sua associação com sobrepeso/obesidade em escolares de Florianópolis, Santa Catarina, Brasil. Desenho transversal com amostra probabilística de 2.506 escolares de escolas públicas (n = 19) e privadas (n = 11). O sobrepeso/obesidade foi classificado pela referência da Organização Mundial da Saúde de 2007. Foram realizadas análises brutas e ajustadas por meio de regressão de Poisson. A prevalência de sobrepeso/obesidade foi de 34,2%. Na rede pública, foram verificados 19,6% de sobrepeso e 13,5% de obesidade. Na rede privada, observaram-se 22,4% de sobrepeso e 11,1% de obesidade. Na rede pública, foi encontrada associação entre sobrepeso/obesidade e utilização da padaria (p = 0,004). Na rede privada, observou-se que os escolares de famílias que utilizaram o supermercado apresentaram 26% menos de sobrepeso/obesidade do que os escolares que não utilizam esses pontos de venda de alimentos (p = 0,003). Os dados encontrados evidenciam a existência de associação entre a utilização de alguns tipos de pontos de venda de alimentos (supermercado e padaria) e a prevalência de sobrepeso/obesidade na população escolar.

The study analyzes retail food outlets and their association with overweight/obesity in schoolchildren from Florianópolis, Santa Catarina State, Brazil. The study used a cross-sectional design with a random sample of 2,506 schoolchildren from public (n = 19) and private schools (n = 11). Overweight and obesity were classified according to World Health Organization guidelines for 2007, and crude and adjusted analyses were performed using Poisson regression. Prevalence of overweight/obesity was 34.2%. In public schools, 19.6% of the children were overweight and 13.5% were obese, as compared to 22.4% and 11.1% in private schools. An association was found in the public school system between overweight/obesity and the use of bakeries for food purchases (p = 0.004). In the private school system, children of families that bought groceries at the supermarket showed 26% less overweight/obesity compared to those who did not (p = 0.003). The data show an association between some types of food outlets (supermarkets and bakeries) and prevalence of overweight/obesity in the school-age population.

El estudio describe los puntos de venta de alimentos y su asociación con el sobrepeso/obesidad en escolares de Florianópolis, Santa Catarina, Brasil. Se trata de un estudio transversal con una muestra aleatoria de 2.506 escolares de las escuelas públicas (n = 19) y privadas (n = 11). El sobrepeso/obesidad se clasifica, en función de la OMS en 2007, con análisis ajustados y crudos que se realizaron mediante la regresión de Poisson. La prevalencia de sobrepeso/obesidad fue de un 34,2%. En el sistema público el resultado fue de un 19,6% sobrepeso y un 13,5% obesidad. En el privado se observó un 22,4% de sobrepeso y 11,1% obesidad. En el primero se encontró una correlación entre el sobrepeso/obesidad y el consumo de bollería (p = 0,004). En las escuelas privadas se observó que los escolares de familias que habían utilizado el supermercado tenían un 26% menos de sobrepeso/ obesidad que los niños en edad escolar que no utilizaron este punto de venta de alimentos (p = 0,003). En el momento del estudio existe una asociación entre el uso de algunos tipos de punto de venta de alimentos (supermercado y panadería) y la prevalencia de sobrepeso/obesidad en escolares.

Genotype analysis and telomere length measure in patients with dyskeratosis congenita / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analysze genotype and measure telomere length in two Chinese patients with dyskeratosis congenita(DC).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

mRNA expression of telomere protection protein TIN2 and POT1 in bone marrow of patients with myelodysplastic syndrome / 中国实验血液学杂志

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.

Dyskeratosis congenital: clinical features and genotype analysis in two Chinese patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To analysis the clinic and genotype in two Chinese patients with Dyskeratosis congenita (DC).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

The change of telomere protein in BEAS-2B malignant transformation cell induced by coal tar pitch smoke extracts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer

Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1) in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC). The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS) and nuclear export signal (NES) are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30) patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

Interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Centrosomal protein TACP1 is a novel interacting protein with Nek2A, both of which are localized in centrosome during mitosis.</p>

Human pot1 gene exon12 mutation screening in cultured human carcinoma cell strains (lines) / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the exon12 mutation of pot1 gene in cultured human carcinoma cell strains (lines).</p><p><b>METHODS</b>The chromosomal DNA was extracted from 27 cultured carcinoma cell strains (lines). The exon 12 of pot1 gene was amplified by PCR, and the product was purified and screened. The screening results were compared with the data of GenBank and NCBI and the exon 12 mutations in cultured human carcinoma cell strains (lines) analyzed.</p><p><b>RESULTS</b>The exon12 sequence of pot1 could be specifically amplified using the designed primers. Direct sequence analysis of the PCR products after purification showed that 4 of the 5 carcinoma cell lines of the female genital system such as Hela and HO8910-PM cells shared the same transition (G17722-->C) in exon12 of human pot1 gene resulting in a conversion of G1385-->C in the cDNA and amino acid change of Leu454-->Phe in the translated polypeptide. The rest of the 23 cell strains (lines) from different origins showed no such mutation.</p><p><b>CONCLUSION</b>The exon12 (17,722 bp) is a mutant region specific for female genital system tumor.</p>

Effects of apoptosis of Tca8113 cells induced by adriamycin on telomerase and telomere repeat binding factor proteins / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the role of telomerase and telomere repeat binding factors (TRF) in apoptosis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>Tca8113 cells apoptosis induced by adriamycin decreased telomerase activity, but did not influence the expression level of TRF proteins.</p>

Study on the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell lines / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>Detecting the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell line cells on the base of determining its genomic structure and its four pseudogenes to clarify if hTRF1 mutation is one of the factors of the activation of telomerase.</p><p><b>METHODS</b>hTRF1cDNA sequences were obtained from GenBank, its genome structure and pseudogenes were forecasted by BLAST and other biology information programs and then testified by sequencing. Real-time RT-PCR was used to detect the expression of hTRF1mRNA in 10 cell line cells, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji. Telomerase activities of cells were detected by using telomeric repeat amplification (TRAP)-ELISA protocol. PCR and sequencing were used to detect mutation of each exon of hTRF1 in 10 cell line cells.</p><p><b>RESULTS</b>hTRF1 gene, mapped to 8q13, was divided into 10 exons and spans 38.6 kb. Four processed pseudogenes of hTRF1 located on chromosome 13, 18, 21 and X respectively, was named as PsihTRF1-13, PsihTRF1-18, PsihTRF1-21 and PsihTRF1-X respectively. All cell line cells showed positive telomerase activity. The expression of hTRF1 was significantly lower in malignant hematopoietic cell lines cells (0.0338, 0.0108-0.0749) than in normal mononuclear cells (0.0493, 0.0369-0.128) (P=0.004). But no significant mutation was found in all exons of hTRF1 in 10 cell line cells. Four variants were found in part of intron 1, 2 and 8 of hTRF1. Their infection on gene function is unknown and needs further studies.</p><p><b>CONCLUSION</b>hTRF1 mutation is probably not one of the main factors for telomerase activation in malignant hematopoietic disease.</p>

Localization of human telomere repeat binding factor 1 in telomerase-positive and-negative cells and its expression during cell cycle / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Study on the mutation of human telomeric repeat binding factor 1 gene in malignant hematopoietic cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect mutations of human telomeric repeat binding factor 1 (TERF1) gene in 11 malignant hematopoietic cell lines, which have positive telomerase activity, and evaluate the significance of the mutations.</p><p><b>METHODS</b>Genome structure of TERF1 was predicted by using biology information program, and verified by PCR and sequencing. Telomerase activity was detected by telomeric repeat amplification (TRAP)-ELISA. PCR and sequencing were used to detect mutation of each exon of TERF1 in 11 cell lines, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji and MDS-RAEB cell line MUTZ-1. Five DNA samples from healthy volunteers were detected as normal controls.</p><p><b>RESULTS</b>TERF1 gene has 10 exons and spans 38.6 kb. All the 11 cell lines showed positive telomerase activity. No mutation was found in all exons of TERF1 in the 11 cell lines. However, 4 variants were found in intron1, 2 and 8 near exon1, exon2 and exon9, respectively. The variants in MUTZ-1 was different from those in leukemia cell lines; but no difference was found between the variants in myelogenous and lymphoblastic leukemia cell lines.</p><p><b>CONCLUSION</b>TERF1 mutation is probably not among the main factors of the gene dysfunction in malignant hematopoietic diseases.</p>