AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS:...AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.展开更多
OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer in...OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer invasiveness, and lymphatic metastasis. METHODS: Telomerase activity in esophageal SCC tissues, adjacent dysplasia tissues and normal epithelia from the surgical edge were assessed by microdissection-TRAP (telomeric repeat amplification protocol)-silver staining assay. RESULTS: Telomerase activity was detected in 37 (82.2%) of 45 esophageal tumors, 23 (79.3%) of 29 dysplasias, and 2 (5%) of 40 normal epithelia. There was a significant difference in activity between dysplasia and normal epithelium, as well as between tumor and normal epithelium. Twenty-six (92.9%) of 28 tumors with lymphatic metastasis had detectable telomerase activity compared to 11 (64.7%) of 17 non-lymphatic metastasis tumors. These relationships were statistically significant (P展开更多
基金the National Natural Science Foundation of Chines,No.39830380
文摘AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.
文摘OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer invasiveness, and lymphatic metastasis. METHODS: Telomerase activity in esophageal SCC tissues, adjacent dysplasia tissues and normal epithelia from the surgical edge were assessed by microdissection-TRAP (telomeric repeat amplification protocol)-silver staining assay. RESULTS: Telomerase activity was detected in 37 (82.2%) of 45 esophageal tumors, 23 (79.3%) of 29 dysplasias, and 2 (5%) of 40 normal epithelia. There was a significant difference in activity between dysplasia and normal epithelium, as well as between tumor and normal epithelium. Twenty-six (92.9%) of 28 tumors with lymphatic metastasis had detectable telomerase activity compared to 11 (64.7%) of 17 non-lymphatic metastasis tumors. These relationships were statistically significant (P
