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

Abstract

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

FIG. 1.

(A) Model of the physical status of the HPV genome within the context of viral persistence, alteration of growth phenotypes, and host cell immortalization. (B and C) Organization of the HPV-16 genome (B) or detection of HPV-16 genomes in HNC tumor biopsy specimens by Southern blotting of total DNA (C). (D) Physical status of viral genomes in clonal HPV-immortalized tonsillar epithelial cells (HPV-16/HTE) as determined by Southern blotting of total DNA digested with a “no-cutter” BglII or “single-cutter” BamHI enzyme. HPV DNA forms are as follows: I, integrated; C, concatameric; S, supercoiled; L, linearized. Faster-migrating HPV fragments are derived from digestion of integrated HPV DNAs.

FIG. 2.

Analysis of HPV-16 integration patterns. (A) Schematic of the distinctive structure of viral transcripts derived from intact HPV-16 plasmid genomes. (B) Structures of viral transcripts (type A or B) derived from either type I or type II integrated HPV DNA. (C) Example of 3′RACE (APOT) analysis of resolved amplicons derived from total RNA extracted from SiHa, HPV-16 W12E, HPV-16/HFK, primary (HPV-negative) HFK cells, and HNC tumor biopsy specimens T1 and T2.

FIG. 3.

c-myc expression is altered in some HPV-16-immortalized integrated clonal human keratinocytes (HK). (A) c-myc protein levels were quantified by Western blot analysis of 30 μg of total protein extracted from clonal HPV⁺/HK cell lines (defined in Table 2) with integration at chromosome 10 (lane 3, clone HFK-14) and in chromosome 8 (c-myc) (lanes 4 to 6) compared to c-myc levels in primary HFK cells. Murine fibroblast (MF) cell extracts were used to demonstrate human c-myc antiserum specificity. (B) c-myc RNA levels quantified by Northern blot analysis of total RNA isolated from clonal HPV⁺/HFK cell lines and control cultures. The blot was stripped and rehybridized with a probe specific for cellular involucrin RNA. HeLa cells were used as a control for elevated c-myc levels compared to those of HFK (primary foreskin keratinocytes). Murine fibroblasts were used as a negative c-myc control.

FIG. 4.

Clonal HPV-immortalized keratinocytes do not necessarily acquire altered viral transcription phenotypes after extended culturing. Total RNA extracted from a set of six clonal HPV-positive keratinocytes prior to and following extended culturing was analyzed by RNase protection assays. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.