Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways.
A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high-risk human papillomavirus-type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53- and pRb-pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature-sensitive SV40 large T-antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read-out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb-pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53- and pRb-pathways.
Smeets
Serge J
SJ
Department of Otolaryngology/Head-Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands.
van der Plas
Marlon
M
Schaaij-Visser
Tieneke B M
TB
van Veen
Eva A M
EA
van Meerloo
Johan
J
Braakhuis
Boudewijn J M
BJ
Steenbergen
Renske D M
RD
Brakenhoff
Ruud H
RH
D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000096:biosynthesis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D027383:Papillomaviridae / Q000378:metabolism; D010641:Phenotype; D012097:Repressor Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D002118:Calcium