Human Papillomavirus (HPV) Type 18 Induces Extended Growth in Primary Human Cervical, Tonsillar, or Foreskin Keratinocytes More Effectively than Other High-Risk Mucosal HPVs

Abstract

Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in ϳ25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of fulllength mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.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 ϳ25% 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 ϳ50% of the tumors, followed by HPV-18 (ϳ20%), 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,

Differential In Vitro Immortalization Capacity of Eleven, Probable High-Risk Human Papillomavirus Types

“…However, the potential existence of type-dependent mechanisms of immune escape and the role of the tumor microenvironment are still largely unexplored. Furthermore, we cannot rule out the possibility that the oncogenicity of HPV45, -51, -52, and -59 is epithelium dependent, although no such cell type dependency was demonstrated for HPV11, -16, -18, and -31 (55).…”

“…Although this observation was surprising given its relatively high prevalence in cervical cancer worldwide (7), one may speculate that HPV45 is more effective in the induction of subsequent transformation events, such as anchorage-independent growth and colony formation, as was shown for HPV18 compared to HPV16 (55).…”

Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis

“…In vitro studies with HPV-E6/E7 immortalized keratinocytes and raft cultures have shown that a persistent HPV infection is not sufficient for malignant transformation. [14][15][16] This transformation can be achieved by treating them for example with tobacco mutagens. 17 However, patients with HPV-positive carcinomas are significantly less likely found in smokers and drinkers.…”

Human Papillomavirus Type 16 (HPV-16) Genomes Integrated in Head and Neck Cancers and in HPV-16-Immortalized Human Keratinocyte Clones Express Chimeric Virus-Cell mRNAs Similar to Those Found in Cervical Cancers

“…Advances in keratinocyte culture techniques have permitted the study of the life cycle of HR HPV types that replicate efficiently and persist in primary human keratinocytes, such as HPV-16, HPV-31 (32,34,35), and to a more limited extent HPV-18 (28,36). However, relatively little is known about the progression of the infected cell toward a malignant phenotype in vivo or in culture.…”

“…In this study, we compared the physical state of HPV DNAs, mRNA sequences, and spliced structures in nine HPV-16-containing HNC tumors to those found in sets of isogenic clones of primary human keratinocytes derived from tonsillar or foreskin epithelia immortalized by the introduction of plasmid HPV-16 genomes (28,49). These culture reagents and assays provide the necessary tools to answer questions regarding viral integration and oncogenic progression that otherwise might not be effectively addressed with clinical material alone and offer an effective model system for examining HR HPV integration and its role in HPV-associated carcinogenesis.…”