Human papillomavirus (HPV) has been classified as one of the causing factors of head and neck squamous cell carcinoma (HNSCC). However, little is known about HPV-related carcinogenesis in HNSCC. The purpose o f this s tudy i s to characterize immortalized human oral keratinocyte (IHOK) transfected by HPV16 E6/E7, IHOK/hcdk4 (IHOK transfected by pLXRN-hcdk4) and IHOK/hcdk4/hTERT (IHOK transfected by pLPC-hTERT-hcdk4) to reconstitute HNSCC in vitro. Conclusively, we established a new immortalized cell lines, IHOK/hcdk4 and IHOK/hcdk4/hTERT, to understand multistep carcinogenic process of oncogenic HPV16 E6/E7 in HNSCC.

Cellular imrnortali zation is thought to be an ear ly event during tumorigenesis. Telomerase reacLi vaLion by ecLopic hTERT expression is widely used for cellular imrnortali zation. This study was a imed Lo a na lyze establi sh immortalized human ora l epithelial cells(IOEC) and to reconstruct oral precancerous lesion by Lhree dimens iona l cultures. Telomerase activity was analyzed by Telomerase assays and Telomere longLh W:lS dcLccLcd by Termina l restri ction I"ragment analysis. bTERT gene was assayecl by tbe RT-PCR. p16lNK4 a‘ pHb. CDK2. P21CIP1. p27 and p53 were examined by western blotting. Three dimensiona l cu1ture using air - liquicl inLe rl"ace was pe rl"ormed. As results. IOEC was establi shed by ectopic ex pression of catalytic subunit• of telomerase‘ h1'EH1'. which is con tinuously maintained for more tban 120 population doublings(PDs) . IOEC showecl the expression 01" h1'ER1' and h1'H mHNA‘ elongated telomere length and higher telomerase activity. These cel ls showed no ex pression of p16lNK4a with retention 01" pRb and CDK2. Expression of p21CIP1. p27 a nd p53 may have no relation to immorta li ze oraJ epithelial cell s. Three dimensional culture of IOEC showed dysplastic strat ilïed epithelia l cell s. These results may serve as a useful moclel system for the study of oral carcinogenesis.