Modulation of human telomerase reverse transcriptase in hepatocellular carcinoma.
OBJECTIVE
Most cancer cells acquire immortal capability by telomerase activation. The human telomerase reverse transcriptase gene (hTERT) is considered to be the major determinant of the enzymatic activity of human telomerase, and the hTERT promoter contains several c-Myc binding sites that mediate hTERT transcriptional activation. Few studies have examined the role of hTERT in hepatocarcinogenesis, and the relationship between c-Myc and telomerase in human hepatocellular carcinoma tissue is unknown.


METHODS
We measured hTERT mRNA levels and c-Myc oncoprotein expression in 57 patients with hepatocellular carcinoma using in situ hybridization and immunohistochemistry, respectively. The transcription regulation of hTERT was evaluated by transient transfection of pGL3-1375 into the human hepatocellular carcinoma cell line J5. To determine the relationship between c-Myc and the hTERT promoter, a 1375-bp DNA fragment encompassing the promoter was placed upstream of the luciferase reporter gene and transiently transfected into the cell line. Two additional hTERT promoter constructs (-776 and -100 bp region) and an hTERT promoter-LUC construct containing 2 c-Myc mutations (pGL3-181 MycMT) were also used for luciferase assays.


RESULTS
In 30 of 57 cases (52%), hTERT mRNA expression was associated with c-Myc protein expression. However, 16 of 57 cases (28%) showed strong hTERT mRNA detection without c-Myc protein expression, and 11 cases (19%) showed weak hTERT mRNA expression and strong c-Myc expression. Although luciferase activity was decreased between upstream 1 375 bp and 776 bp, there was no significant difference between upstream 776 bp and 100 bp. Finally, there was no significant decrease in activity after transfection of the hTERT promoter-LUC construct.


CONCLUSIONS
The results indicate that c-Myc does not play a major role in gene regulation of the catalytic subunit of telomerase (hTERT) in human hepatocellular carcinoma. Other regulatory elements or epigenetic phenomena should be further investigated to understand hTERT gene regulation in human hepatocellular carcinoma.
Division of General Surgery, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan, China.
Cheng-Jueng
CJ
Chen
Satoru
S
Kyo
Yao-Chi
YC
Liu
Yeung-Leung
YL
Cheng
Chung-Bao
CB
Hsieh
De-Chuan
DC
Chan
Jyh-Cherng
JC
Yu
Horng-Jyh
HJ
Harn
D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000503:physiopathology*; D002460:Cell Line; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000503:physiopathology*; D008156:Luciferases / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D008156:Luciferases; D019098:Telomerase