Study On The Mechanism Of Telomere Length Maintenance Of Human Gastric Cancer Cell Line SGC-7901 By Tankyrase

Background: Telomeres are large nucleoprotein complexes that protect the ends of chromosomes against degradation and fusion. It contain repeated TTAGGG sequences.Telomeric repeats are synthesized by the enzyme telomerase.Telomere length maintenance is important for cancer cells to keep immortality.Many results have suggested that telomerase play a key role both in the gastric carcinogenesis and in the process of progress.It is generally believed that the telomere length be maintenanced by telomerase at gastric cancer.In recent years many factors have been found to be associated with telomere control besides telomerase. Telomere-binding proteins TRF1 and TRF2 play pivotal roles in telomere protection and maintenance in mammalian cells. TRF1 can negatively regulate telomere length by inhibiting the interaction between telomerase and the telomeres. Several proteins have been shown to associate with TRF1 and TRF2.Tankyrase,originally identified as a TRF1 binding protein,is a member of the growing family of poly(ADP-ribose) polymerase (PARPs). Tankyrase mediated ADP-ribosylation inhibits binding of TRF1 to telomeric repeats in vitro. This indicates tankyrase is a positive regulator of telomere length and may serve as a potential target for cancer therapy. In order to investigate if tankyrase play a role in the telomere length maintenance and its mechanism at SGC-7901, we constructed antisense tankyrase eukaryotic expressing vector and then tranfected it into human gastric cancer cell line SGC-7901 as well as the cells which had been transfected with antisense hTR(7901-ahTR) and hTRT(7901-ahTRT) with DOTAP liposome firstly. Secondly, the activity of telomerase and the length of telomere were detected by PCR-ELISA or Southern Blot respectively .In order to explore the possibility of tankyrase to act as a new target for cancer gene therapy, the effect of antisense tankyrase on the biological behaviour of SGC-7901 was observed lastly. Objectives: 1. To construct and identify an antisense tankyrase gene eukaryotic expressing vector by recombinant technology. 2. To investigate the role and mechanism of tankyrase in the telomere maintenance of human gastric cancer cell line SGC-7901. 3. To observe the effect of antisense gene of tankyrase on the biological behaviour of gastric cancer cell line SGC-7901. 4. To explore the potential value of in the gene therapy of gastric cancer. Methods and Results: 1. The construction and identification of antisense tankyrase gene eukaryotic expressing vector. Firstly, a 3.5kb fragment of tankyrase cDNA was obtained from the plasmid via EcoRⅠdigestion, and inserted into the multiclone site EcoRⅠof eukaryotic expressing vector pcDNA3.1/Zeocin(-) plasmid by recombinant technique.The resulting antisense recombinant was identified by endonuclease cutting with BamH Ⅰand DNA sequencing ,which was named pcDNA-aTNKS. 2.Gene transfection and identification of tansfectants The transfection of antisense tankyrase recombinant and plasmid pcDNA3.1/Zeo(-) was mediated by liposome named DOTAP. The transfectants were screened by Zeocin or Zeocin plus G418 respectively and then idendified by detecting Zeocin resistant gene(ZeocinR) using PCR technique.By this way,we obtained stable transfectants termed 7901-pcDNA, 7901-neo-pcDNA, 7901-aTNKS, 7901-ahTR-aTNKS and 7901-ahTRT-aTNKS. We also used Western Blot technique to determine if the expression of tankyrase was inhibited by antisense tankyrase gene in these stable transfectants.The results suggested that endogenous tankyrase expression could be efficiently down-regulated by transfection of antisense tankyrase gene in cultured SGC-7901 cells. 3. The effect of antisense tankyrase on the expression of telomerase subunits The mRNA of telomerase subunits TP1,hTR or hTRT was detected by RT-PCR.No significant difference of these subunits expression was found after antisense tankyrase transfection.These results indicate that tankyrase do not regulate the telomerase expression. 4. The effect of antisense tankyrase on the expression of TRF1 and TRF2 The two telomeric proteins,TRF1 and TRF2 were detected by immunochemistry andWestern Blot.All antisense genes observed in this experiment including tankyrase, hTR and hTRT did not have effect on the expression of TRF1 and TRF2 compared to the control group. 5. The expression of c-myc and bcl-2 We used RT-PCR technique to detect the changes of c-myc and bcl-2 on transcription level and Western Blot technique to detect that on translation level.The results showed the expression of oncogene c-myc was significantly down-regulated in 7901-aTNKS, 7901-ahTR, 7901-ahTRT,7901-ahTR-aTNKS and 7901-ahTRT-aTNKS cells. And the apoptosis-associated gene bcl-2 expression was also down-regulated remarkably in these cells except 7901-aTNKS. 6. The effect of antisense tankyrase on the telomerase activity The telomerase activity was detected using PCR-ELISA. The data showed there was no obvious difference between the telomerase activity of 7901-aTNKS and SGC-7901cells, while the telomerase activity was decreased significantly when treated with antisense hTR or antisense hTRT. This suggested that antisense tankyrase could not inhibit the ability of adding telomeric fragments to TX Primer by telomerase, unlike antisense hTR or hTRT. 7. The effect of antisense tankyrase on the telomere length The hybridization of telomere DNA fragments with the telomere probe was performed using Southern Blot. The intensity of the signal of scanned images of autoradiograph was determined using ImageQuant TL software.The mean telomere length was SGC-7901>7901 -pcDNA>7901-neo-pcDNA>7901-ahTR>7901-ahTRT>7901-aTNKS>7901-ahTR-aTNKS>7901-ahTRT-aTNKS.This data suggested that down-regulation of tankyrase expression could shorten telomere length more efficiently than that of hTR or hTRT. 8. The effect of antisense tankyrase on the biological behaviour of SGC-7901 Flow cytometric analysis displayed an accumulation of cells in the G0/G1 phase of cell cycle and a decrease in proliferative index after antisense tankyrase transfection. Growth curve by MTT method also showed the proliferation was suppressed in 7901-aTNKS.The appearance of 7901-aTNKS under light microscope was homologous compared with its parent cells.We found 7901-aTNKS cells were usually large with an abundant cytoplasm and decreased nuclear division,the ratio of nuclei to cytoplasm and atypia using HE staining,while the appearance of SGC-7901 and 7901-pcDNA was heterogenous.Nuclei inthese cells were large and hyperchromatic.Nuclear division, the ratio of nuclei to cytoplasm and atypia were increased.Multinucleate giant cells and giant nucleate cells were usually present. Ultrastructure under transmission electronic microscope revealed that organelles in 7901-aTNKS were abundant compared with its parent cells.And many secreting vacuoles with microvilli in its inner emerged in cytoplasma in these cells. On the contrary, organelles in 7901 and 7901-pcDNA were decreased with enlarged nuclei, irregular nuclear membranes, high ratio of nucleus to cytoplasm and prominent nucleoli. No tumorigenicity was found in nude mice even if 4×106 7901-aTNKS cells were injected subcutaneously. Conclusions: 1. The antisense eukaryotic expressing vector of tankyrase was successfully constructed by recombinant technology. 2. Tankyrase plays an important role in the telomere maintenance of gastric cancer cell line SGC-7901.Tankyrase can allow access of telomerase to telomeres in the mechanism for telomere maintenance by telomerase. 3. Tankyrase attaches itself to telomere maintenance by ADP-ribosylation of TRF1. However, antisense tankyrase has no evident impact on the expression of telomere binding protein TRF1 and TRF2.This indicates tankyrase may be an acceptor in the signal of telomere length and do not regulate the expression of TRF1 or TRF2. 4. Although telomere elongation by tankyrase correlates with telomerase, antisense tankyrase has no remarkable effect on the expression of telomerase subunits in the telomerase-positive cells. 5. A combined transfection of antisense tankyrase and antisense hTR or hTRT can further shorten the telomere length. This indicates that there is a synergic action of antisense tankyrase with antisense hTR or hTRT for telomere length shortening. We presume antisense tankyrase can increase the sensitivity to telomerase inhibitors. 6. Antisense tankyrase can down-regulate the expression of oncogene c-myc at its transcription and translation levels, which is related to the changes of biological behaviour of SGC-7901 cells. 7.Antisense gene therapy of tankyrase can partially reverse malignant phenotypes of SGC-7901 cells and induce differentiation. 8. Inhibition of tankyrase expression by antisense strategy may provide a newapproach to gene therapy for human gastric cancer.