Human papillomaviruses (HPVs) are small DNA tumor viruses that infect, persist in, and cause proliferative lesions in the epithelial cells of the skin, ectoderm-derived mucosae, and their adnexa. Mucosal HPV types are associated with most if not all carcinomas of the uterine cervix, many anogenital cancers, and ~25% of head and neck cancers (HNCs) (reviewed in reference 55
). HNCs arising in the oropharynx or tonsils are HPV positive in over 50% of the cases. Although the same oncogenic or “high-risk” (HR) HPV types are found in the cervix, in the anogenital area, and in HNC, their prevalence is strikingly different: in cervical and anogenital cancers, HPV type 16 (HPV-16) is found in ~50% of the tumors, followed by HPV-18 (~20%), while the remaining cases harbor over 15 other HR HPV types, including HPV-31 and other well-characterized as well as additional, novel HPV types. In contrast, over 90 to 95% of HPV-positive HNCs contain HPV-16, whereas HPV-18, HPV-31, or other HR HPVs are infrequent (10
). Furthermore, HR HPVs appear to vary in oncogenic potential: HPV-18 is associated with more advanced cervical disease that progresses more rapidly and typically has a worse prognosis than that caused by other HR HPV types (7
). In contrast, other mucosal, “low-risk” (LR) HPV types (HPV-6 or -11) are not commonly associated with malignant progression even though they also give rise to clinically significant disease, such as genital warts and recurrent respiratory papillomatosis in the larynx and trachea that require repeated therapeutic interventions.
In vivo, HPV persists in the nuclei of epithelial cells in the form of unintegrated, supercoiled circular plasmid DNA. Spontaneous immortalization of human keratinocytes in culture occurs rarely, yet keratinocyte lines from primary skin (HaCaT) (5
) and the cervix (NIKS) (1
) have been described. Early experiments demonstrated that HR HPV genomes can expand the life span of, or “immortalize,” primary human foreskin keratinocytes in culture (2
). However, the culture conditions precluded the establishment and maintenance of persistently replicating, extrachromosomal HPV plasmids in these previous studies. In contrast to persistently infected cells, most HPV-associated cervical, anogenital, and head and neck carcinomas contain disrupted, integrated HR HPV DNA (19
). The integrated HR HPV genomes invariably express two viral early proteins, E6 and E7, that bind to and disrupt the functions of the tumor suppressor gene products p53 and pRB, respectively, and interact with a number of additional cellular targets (70
). The E6 and E7 gene products of both LR and HR mucosal HPV types are required for persistence, yet only those of HR HPV strains effectively immortalize epithelial cells in culture when ectopically expressed under the control of heterologous or retroviral promoters (55
As initially demonstrated by Laimins and coworkers (27
), recircularized HR HPV genomes can establish persistent replication in human primary foreskin keratinocytes. Under optimal conditions, such cells harbor extrachromosomal plasmid HPV genomes, exhibit an extended life span in vitro, undergo dysplastic differentiation in organotypic “raft” cultures, and have been used to dissect early and late events of the viral life cycle, including viral gene expression, vegetative viral DNA replication, and virion synthesis (16
). Similar analyses have been extended to cervical keratinocytes (3
) and airway epithelial cells (39
). However, previous studies that exploited HPV persistence in primary human epithelial cells primarily investigated the viral life cycle and have not examined individual virus genome-cell interactions.
In this study, we directly compared the capacity of HR HPV-16, -18, and -31 as well as the LR HPV-11 to establish persistence and lead to growth alterations in primary human epithelial cells derived from the three main mucosal sites where HPV-associated cancers arise: the cervix, the tonsillar fossa, and foreskin. In addition to assessing early gene promoter activity and initial genome amplification (23
), we utilized a quantitative colony formation assay followed by subculturing multiple individual cell clones stemming from individual virus genome-cell interactions (36
). In contrast to previous studies, this approach allowed us to quantify the viral genome capacity to extend the cellular life span (“immortalize”) in culture and to alter their growth properties and genomic stability in relation to the viral HPV genome status and viral gene transcription, all parameters which could otherwise be masked in heterogeneic mass cultures.
We found that, in culture, tonsillar epithelial cells did not reflect the apparent preference for HPV-16 observed in patients. The efficiency of extended-growth clone formation was solely a function of HR HPV type: HPV-18 was strikingly more effective than HPV-16 or -31 in establishing growing cell clones harboring extrachromosomal plasmid HPV genomes in all keratinocytes tested regardless of their site of origin. Furthermore, in contrast to HPV-16, which exhibits considerable functional differences among its geographic variant genomes harboring altered control sequences (26
), we found that the similarly diverse HPV-18 upstream regulatory region sequences (URRs) did not confer the same variant-dependent activity.
Despite shortened population doubling (PD) times and dysplastic differentiation of the cells in organotypic culture, HPV persistence did not lead to chromosomal instability. Because of their apparent long-term stability in culture, these HR HPV-containing keratinocyte clones can serve as a representative model to study cellular and viral changes in the progression from early persistent HR HPV infection to cancer.