| Background and Objective Non-small cell lung cancer (NSCLC) is the most common human malignant tumor. The goals and hotpoints of current studies are the molecular mechanisms underlying NSCLC etiology. In recent years many factors associated with telomere control have been found. Telomeres are composed of tandem arrays of a short DNA sequence, 5'(TTAGGG)n3' in vertebrates, and associated proteins. They are essential genetic elements to stabilize the natural ends of linear eukaryotic chromosomes and protect the DNA ends from degradation and fusion. Telomeric DNA-binding proteins have an essential role both in regulation of the length of the telomeric DNA tract and in protection against chromosome end-to-end fusion. Telomeric repeat binding factor 1 (TRF1), one of the important telomeric binding proteins, consists of 438 amino acid and its molecular weight is about 60KD. In human cells, the changes of telomeric length have been implicated in the molecular clock controlling cell senescence and as a step in tumorigenesis. The maintenance of telomeric length is important for cancer cells to keep immortality. TRF1 plays pivotal roles in telomere protection and maintenance in mammalian cells. TRF1 cannegatively regulate telomeric length by inhibiting the interaction between telomerase and telomere. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA at the end of chromosomes and compensates for the end replication problems. It has been shown that telomerase is activated in a large majority of human cancer tissues, but not in most normal tissues and tissues adjacent to malignant or benign tumors. A similar protein-counting model was proposed for telomeric length homeostasis. The expression of a dominant negative allele of TRF1, which removes endogenous TRF1 from telomeres, leads to telomere elongation. In this system, TRF1 did not affect the activity of telomerase detectable in cell extracts, which suggests that TRF1 does not affect telomerase activity globally in the cell. Instead, TRF1 acts in cis as a negative length regulator at each individual telomere.Tankyrase(TANK1), originally identitied as a TRF1 binding protein, is a member of the growing family of poly(ADP-ribose) polymerase(PARPs). TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats in vitro. Ribosylation by TANK1 displaces TRF1 from telomeric DNA. This finding suggested that TANK1 might be a positive regulator of telomeric length in telomerase -expressing cells. Indeed, when TANK1 protein overexpressed, telomere length increased in telomerase-positive tumor cells. According to the protein-counting model, very short telomeres would not bind sufficient amounts of TRF1. Long telomere would recruit a large amount of TRF1 protein, blocking telomerase-mediated telomeric elongation.Based on the current datas, the emerging view is that long telomeres recruit a larger amount of TRF1 and TANK1 mediated ADP-ribosylation inhibits binding of TRF1 into telomeric repeats. This indicates that TRF1 and TANK1 may serve as a potential target for cancer therapy. On the basis of the results from the studies above, we used immunohistochemical techniques to measure expression of telomerasereverse transcriptase (TERT) in 50 patients and detect the expression of TRF1 with RT-PCR and Western blot in 36 patients of telomerase positive. The cellular localization of TRF1 in NSCLC was observed by immunohistochemistry and the sequence of cellular nuclear localization signal was measured by DNA sequencing techniques. As the molecular mechanism of the TRF1 in NSCLC has not yet been clarified, this study could help to provide clues to the role of TRF1 in the telomere regulation.Part I The expression of TRF1 mRNA and TANK1 mRNA in Non-Small Cell Lung Cancer of telomerase positiveObjective To investigate the expression of TRF1 mRNA and TANK1 mRNA in NSCLC of telomerase positive and analyze the relationship between the TRF1 mRNA and TANK1 mRNA levels.Methods (1) From 2004 Nov to 2005 Nov, 50 patients with NSCLCs had undergone radical operations in The First Affiliated Hospital,College of Medicine,Zhejiang University. The diagnosis of lung cancer was obtained according to the pathohistologic examination in all cases. (2) The expressions of TERT were detected by immunohistochemistry. (3) The expressions of TRF1 mRNA and TANK1 mRNA in NSCLC with telomerase positive were investigated by RT-PCR.Results (1) Immunohistochemistry: TERT expression in NSCLC was found at the cell nuclear and cytoplasm. In 50 lung cancers specimens studied with the immunohistochemical method, 36(36/50, 72.0%) were showed telomerase positive. (2) Clinical information of telomerase positive patients: 36 patients with telomerase positive patients included 22 males and 14 females. There were 23 samples of adenocarcinoma and 13 samples of squamous cell carcinoma. (3) Expression of TRF1 mRNA in cancer tissues and paried noncancerous tissues: The mean±s ofTRF1 mRNA in cancer tissues and paired noncancerous tissues was ±0.119 and 2.015±2.337, respectively . It showed that TRF1mRNA levels were higher in paired noncancerous tissues than in cancer tissues (p=0.02). (4) There were no significant differences in TRF1 mRNA expression between adenocarcinoma and squamous cell carcinoma (P=0.773) . (5) The mean± s of TRF1 mRNA in lymph node metastasis and no lymph node metastasis was 0.893±0.619 and 1.334±0.769, respectively. No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.070) . (6) One-Way ANOVA was employed to evaluate the TRF1 mRNA expression in clinical stages. There were no significant differences in TRF1 mRNA expression among different clinical stages (P=0.364). (7) Expression of TRF1mRNA in variable grade of differentiation : The mean±s of TRF1mRNA in moderate differentiated and in poorly differentiated was 1.309±0.784 and 0.811±0.510, respectively. The results showed that the expression of TRF1 mRNA in poorly differentiated tumor was significantly down-regulated when compared with the moderated differentiated tumor (P=0.040) . (8) TANK1 mRNA was significantly up-regulated in cancer tissue when compared with the paired noncancerous tissue (P=0.04). (9) No significant difference of TANK1 mRNA level was found among sexes, different clinical stages, pathological subtypes and lymph node metastasis (p>0.05). (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TANK1 mRNA. No correlation was observed between TRF1 mRNA and TANK1 mRNA levels (r=0.098, P=0.605).Conclusions (1) Down-regulation of TRF1 mRNA expression was found in lung cancer tissues. (2) The expression of TRF1 mRNA was significantly associated with grade of tumor differentiation. (3) Up-regulation of TANK1 mRNA was found in lung cancer tissues.Part II The expression of TRF1 protein in Non-Small Cell Lung CancerObjective To detect the expression of TRF1 protein in NSCLC and analyze the relationship between the TRF1 and clinic factors.Methods The levels of TRF1 in cancer tissues and paired noncancerous tissues were measured by Western blot.Results (1) Expression of TRF1 in cancer tissues and paried noncancerous tissues: 36 lung cancers samples studied with Western blot method, however, the expression of TRF1 in cancer tissues and paried noncancerous tissues was 30 and 33 samples, respectively. (2) TRF1 expression of cancer tissues and paried noncancerous tissues in 30 cases: The mean±s of TRF1 in cancer tissues and in paired noncancerous tissues was 0.552±0.329 and 0.652±0.476, respectively. The results showed that TRF1 levels were higher in paired noncancerous tissues than in cancer tissues (p=0.028). (3) There were no significant differences in TRF1 expression between adenocarcinoma and squamous cell carcinoma (P=0.601) .(4) The mean±s of TRF1 in the positive lymph node and in the negative lymph node was 0.593±0.309 and 0.512±0.354, respectively . No significant difference was found between in lymph node metastasis and no lymph node metastasis (P=0.509) . (5) One-Way ANOVA was employed to evaluate the relationship between TRF1 expression and clinical stages. There was no significant difference in TRF1 expression levels among clinical stages (P=0.076). (6) Expression of TRF1 in variable grade of differentiation : The mean±s of TRF1 in tumor with moderate differentiation and in tumor with poor differentiation was 0.481±0.148and 0.634±0.451, respectively . The results showed that the expression of TRF1 was no significantly different between moderate differentiated tumor and poorly moderatedifferentiated (P=0.24) (10) Spearman test was employed to evaluate the correlation between TRF1 mRNA and TRF1 protein. No correlation was observed between TRF1 mRNA and TRF1 protein levels (r=0. 099, t=0.603, P>0.05).Conclusions (1)Down-regulation of TRF1 expression was found in lung cancer tissues.(2) No significant difference of TRF1 expression was found among sexes, different clinical stages, pathological subtypes and lymph node metastasisPart III Cellular localization of TRF1 protein in Non-Small Cell Lung Cancer by immunohistochemistryObjective To investigate the cellular localization of TRF1 in NSCLC.Methods Cellular localization of TRF1 in NSCLC was evaluated by immunohistochemistry.Results (1) The expression of TRF1 protein was evaluated by immunohistochemical staining and it mainly accumulated in the cytoplasm. In most cells, the signal was generally weak. TRF1 immunoreactivity was expressed at variable intensity and distribution. The positive rates of TRF1 were 16 of 36 cases (44.4%) in cancer tissues, while 18 of 36 cases (50.0 % ) in paired noncancerous tissues. There was no significant difference in the positive rates of TRF1 between the cancer tissues and paired noncancerous tissues (P>0.05).The positive rates of both nucleus and cytoplasm in cancer tissues were 22.22%, and 33.33% in paired noncancerous tissues. No significant difference was found between them, and also between adenocarcinoma and squamous cell carcinoma.Conclusions (1) Immunohistochemistry results showed that the positive stainingpercentages of TRF1 were 44.44% in cancer tissues and 50% in paired noncancerous tissues. (2) Immunohistochemistry results indicate that the expression of TRF1 protein mainly accumulated in the cytoplasm.Part IV DNA sequencing analysis of the nuclear localization signal of TRF1 in Non-Small Cell Lung CancerObjective To investigate the base mutation of nuclear localization signal DNA sequence of TRF1 in NSCLC.Methods The nuclear localization signal (NLS) of TRF1 was predicted by PSORT II, and then the NLS sequence was detected by sequencing.Results The results showed that there was no difference in the NLS sequence of TRF1 between cancer tissues and paired noncancerous tissues.Conclusions There was no base mutation in TRF1 NLS sequence of NSCLC.
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