Expression Of Telomerase And PinX1 Gene In Esophageal Carcinoma And The Biological Significance

Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. Telomeres are repetitive DNA sequences at the ends of linear chromosomes that are essential for chromosome protection and genomic stability, which play the key role in human cell replication and aging. Shortening of telomeres is associated with each round of cell division and eventually results in the cells apoptosis or death. Once telomeres are short, cells enter an irreversible growth arrest state called replicative senescence. So maintenance of telomere length is important in preventing the consumption of telomere DNA during cell division. Telomerase, a cellular reverse transcriptase, helps stabilize telomere length in human stem, reproductive and cancer cells by compensating this progressive telomere attrition through de novo addition of TTAGGG repeats to the chromosome ends. Among telomerase, the human telomerase reverse transcriptase (hTERT) is one of core components and plays a key role in the activation of telomerase. Though telomerase is considered to be necessary for the indefinite proliferation of human cells, several lines of studies show high level expressions of telomerase are found in tumor, which implicate that activation of telomerase plays a key role in the malignant transformation process. Since most cancers express telomerase, this indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering to malignant proliferation and involve in cancer development. Inhibition of telomerase results in gradual erosion of telomeres followed by cessation of proliferation or apoptosis, and thus may be a promising target for cancer therapy.As the endogenous suppressor genes of telomerase, PinX1 is regarded as one of potential tumor suppressor genes and involves in variety of tumor development. Recent reports showed that expression of PinX1 was obviously decreased and telomerase activity was enhanced in gastric carcinoma, colorectal cancer and leukemia. But it was also reported that there was no significant difference in the expression of PinX1 in the hepatumo tissues compared to that in normal control tissues, while high telomerase activity was found in the hepatumo tissues. Those contradictious researches showed the exact role of PinX1 genes in regulation of telomerase activity in tumor cells is still unclear. So it is also unclear whether PinX1 inhibits telomerase activity and/or suppresses tumor cell proliferation. It is achieved to set up an esophageal cancer cells model to get insight into the functions of PinX1 gene in regulating the telomerase activity. The development of telomerase inhibitors as therapeutic agents has become new method for cancer therapy and the study of PinX1 in esophageal cancer cells will contribute to the advanced therapeutic schemes.In order to explore the role of telomere and telomerase in carcinogenesis of esophageal epithelium, the telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC. Meanwhile, telomerase activity of esophageal carcinoma and atypical hyperplasia tissues was further detected by TRAP- argentation. Relationship between telomere length and telomerase activation and esophageal carcinoma clinical pathological features was analyzed to explore the involvement of telomere and telomerase in carcinogenesis of esophageal epithelium.In order to explore the role of PinX1 in carcinogenesis of esophageal epithelium and in inhibiting telomerase activation, the expression of PinX1 in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC and the proliferation and apoptosis of Eca 109 cells transfected with Exogenous PinX1 were assessed by RT-PCR, FCM and Western blot. Meanwhile, telomerase activity of Eca 109 cells transfected with PinX1 was further detected by TRAP- argentation.Methods:1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significanceesophageal carcinoma tissues from the patients without chemotherapy and radiotherapy from esophageal carcinoma high-risk area of China were collected in this study. All the specimens were verified by pathologic diagnosis, 44 cases of esophageal squamous cell carcinoma (ESCC), 50 dysplasia of esophageal squamous epithelium and 36 normal esophageal mucosa. 44 ESCC tissues included well (n=20) and moderately differentiated ESCC (n=11), poorly differentiated ESCC (n=13), fibrous membrane invasion (n=25), fibrous membrane untouched (n=19), lymph node metastasis positive (n=20), lymph node metastasis negative (n=24). 50 dysplasia of esophageal squamous epithelium included 22 cases of mild dysplasia and 28 cases of severe dysplasia. The telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by flow cytometry (FCM) and immunohistochemistry (IHC). Relationship between their expressions and clinical pathological features was analyzed.2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significanceEsophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 50 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 130 specimens. 80 ESCC tissues included well and moderately differentiated ESCC (n=39), poorly differentiated ESCC(n=11) ; fibrous membrane invasion(n=34), fibrous membrane untouched(n=16); lymph node metastasis positive(n=17), lymph node metastasis negative(n=33). PinX1 gene and protein expression in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and flow cytometry(FCM). Telomerase activity was detected by transcription-polymerase chain reaction- argentation (TRAP- argentation). Relationship between their expressions and clinical pathological features was analyzed.3 Effects of PinX1 gene on the proliferation and apoptosis and telomerase activity of esophageal cancer cellWe clone the whole length of PinX1 gene from gene bank and ligate the whole length of PinX1 gene to the pCDNA3.1 vector to construct the custom-crafted plasmid pCDNA3.1- PinX1. The positive clones pCDNA3.1- PinX1 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with pCDNA3.1 as control group. Eca109 cell transfected with pCDNA3.1- PinX1 and pCDNA3.1 was named Eca109/ PinX1 and Eca109/pCDNA3.1 cell respectively.Transfected rate was investigated by laser confocalmicroscopy and the effect of PinX1 on the growth of Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells was detected by MTS. The expression of PinX1 mRNA and protein in transfection Eca 109 cells were investigated by RT-PCR, FCM as well as Western-blot. The changes of telomerase activity in Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells were measured by TRAP- argentation. Results:1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significance Value of Q-FISH was detected by FCM in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group. Value of Q-FISH in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was a negative correlation betwen telomere length and cytologic grade(r=-0.79, P<0.01).FI value of hTERT in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group was 0.87±0.18, 1.13±0.19, 1.39±0.24 and 1.84±0.21. FI value of hTERT in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). FI value of hTERT was positive related with cytologic grade(r=0.84, P<0.01); According to the standard: value of FI>1.0 was positive, expreesing rate of hTERT portain was 68.18%,92.86% and 95.45% in groups of mild dysplasia, severe dyaplsia and carcinoma. expressing rate in cancinoma group and severe dysplasia group were higher than that in mild dysplasia group (P<0.01).From normal group to cancinoma group value of DI (DNA Index) was 1.03±0.34, 1.06±0.28, 1.17±0.25 and 1.54±0.37. DI value in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was positive correlation betwen value of DI and cytologic grade (r=0.62, P<0.01). With the cytologic grade progressing value of PI (proliferation index) increased, value of PI (28.79±5.47) in cancinoma group was higher than that (18.97±5.27) in severe dysplasia group. Value of PI was positive related with cytologic grade (r=0.64, P<0.01).There was show significant positive correlation betwen value of DI and PI(r=0.76, P<0.01), and negative correlation between Q-FISH value of telomere and FI value of hTERT(r=-7.49, P<0.01), as well as Q-FISH value of telomere vs. value of DI(r=-0.41, P<0.01).The expression of hTERT located in nucleus of esophageal epithelial cells and cancerous cells by Immunohistochemistry staining. The positive rate of hTERT in incisal margin normal tissue was 11.1%(4/36), and those in para-tumorous dysplasia tissues and cancinoma were 44.0%(22/50) and 86.37%(38/44), there was instinct difference of positive expression among them (P<0.01). Expression of hTERT had no relationgship observed with age, gender, differentation grade, lymph node matastasis and invasive degree of patients (P>0.05).2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significanceThe positive rate of telomerase activity were 84%,62% and 6% respectively in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal. Telomerase activity in esophageal cancer tissues and dysplasia of esophageal squamous epithelium were significantly higher than that in normal tissues (P<0.05).The A value of telomerase activity were 1.457±0.838, 0.429±0.346 and 0.073±0.039 in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal (P<0.05). The A value of telomerase activity was related to the tumor tissue grade and lymph no demetastasis (P<0.05, P<0.01), but not to other clinicopathological features in ESCCS (P>0.05).The expression of PinX1 mRNA and protein in esophageal cancer tissues were significantly lower than that in normal tissues (P<0.01). In esophageal cancer tissues, there were no difference in the expression of PinX1 mRNA and protein between age and gender (P>0.05), but the expression of PinX1 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly lower than that in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative tissues (P<0.05).There was significant negative correlation between telomerase activity expression and PinX1 protein expression (rs =-0.883,P=0.000).3 Transfection of PinX1 gene, its influence to esophageal cancer cell and telomerase activityEca109/PinX1 and Eca109/pCDNA3.1 cell were established successfully. PinX1-transfected Eca109 cells grew more slowly than the cells without transfection or transfected with void vectors (P<0.05). The growth of PinX1-transfected cells was retarded and blocked into G0/G1 stage. The apoptosis rate was higher in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only by FCM.The expression of PinX1 mRNA and protein in Eca109/PinX1 cells was significantly higher than that in Eca109/pCDNA3.1 and Eca109 cells (P<0.05). The expression of telomerase activity was lower in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only (P<0.05). There was significant positive correlation between telomerase activity expression and PI expression (rs=0.451,P=0.000).Conclusions:1 Shortening of telomeres and increased hTERT expression, DNA content and PI value were found in ESC, which showed telomeres and telomerase are associated to carcinogenesis of esophageal epithelium. The difference of telomere length and hTERT expression in tumor of initial period of carcinogenesis might be a useful cancer biomarker for early diagnosis of ESC.2 Telomerase activity was increased in atypical hyperplasia tissues than that in normal tissues of the esophagus. Upon to the malignant degrade of pathological changes in ESC, the telomerase activity increased more in malignant tumor. The results suggested that telomerase is involved in tumor progression and serves as an early event in ESC, and may contribute to early diagnosis of ESC and supervising the development of ESC. The A value of telomerase activity was related to the tissue grade and lymph node metastasis in ESC, but not to other clinicopathologieal features. It suggested that the A value of telomerase activity may be used as a sign to estimate the curative effect and to appraise the prognosis of ESC.The down-expression of PinX1 was founded in ESC. The down grade is depended for the clinical stage and pathological grade in ESC. PinX1 gene may be used as one of the molecular markers to determine the malignant degree, metastasis potency and predicting progression of ESC. Telomerase activity expression was significant negative correlation to PinX1 expression, suggested that PinX1 can decrease telomerase activation.3 Within the successfully established Eca109/PinX1esophageal cancer cell model, the esophageal cancer Eca109 cells growths were inhibited by PinX1 transfenction evidenced by increased apoptosis and an arrest of G0/G1 phase and telomerase activity of Eca 109 cells was decreased by PinX1 gene. The results show PinX1 serves as an inhibitors of telomerase activity to suppress tumor development.