Human telomerase reverse transcriptase activated by E6 oncoprotein is required for human papillomavirus-16/18-infected lung tumorigenesis.
OBJECTIVE
Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development.


METHODS
Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively.


RESULTS
hTERT mRNA levels in E6-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage I) were greater than levels in advanced tumors (stages II and III). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi.


CONCLUSIONS
Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.
Cheng
Ya-Wen
YW
Institutes of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China.
Wu
Tzu-Chin
TC
Chen
Chih-Yi
CY
Chou
Ming-Chih
MC
Ko
Jiunn-Liang
JL
Lee
Huei
H
D000368:Aged; D005260:Female; D005786:Gene Expression Regulation; D052162:Human papillomavirus 16; D052161:Human papillomavirus 18; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D017403:In Situ Hybridization; D008175:Lung Neoplasms / Q000201:enzymology* / Q000821:virology; D008297:Male; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D030361:Papillomavirus Infections / Q000201:enzymology*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor; D019098:Telomerase / Q000096:biosynthesis*; D046888:Tissue Array Analysis; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C068651:E6 protein, Human papillomavirus type 16; C489427:MYC protein, human; D009856:Oncogene Proteins, Viral; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D016159:Tumor Suppressor Protein p53; C509186:TERT protein, human; D019098:Telomerase