Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell.
OBJECTIVE
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.


CONCLUSIONS
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.
Shen
Zhong-Ying
ZY
Department of Tumor Pathology, Medical College of Shantou University, 22 Xinling Road, Shantou 515031, Guandong Province, China. zhongyingshen@yahoo.com
Xu
Li-Yan
LY
Li
En-Min
EM
Cai
Wei-Jia
WJ
Chen
Min-Hua
MH
Shen
Jian
J
Zeng
Yi
Y
D017209:Apoptosis / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D048429:Cell Size; D002471:Cell Transformation, Neoplastic; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D004947:Esophagus / Q000166:cytology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*
D019098:Telomerase