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

Abstract

bEpidemiological studies identified 12 high-risk HPV (hrHPV) types and 8 probable/possible hrHPV types that display different cancer risks. Functional studies on transforming properties of hrHPV are mainly limited to HPV16 and -18, which induce immortalization of human foreskin keratinocytes (HFKs) by successive bypass of two proliferative life span barriers, senescence and crisis. Here, we systematically compared the in vitro immortalization capacities, as well as influences on p53, pRb, hTERT, growth behavior, and differentiation capacity, of nine hrHPV types (HPV16, -18, -31, -33, -35, -45, -51, -52, and -59), and two probable hrHPV types (HPV66 and -70). By retroviral transduction, the respective E6/E7 coding sequences were expressed in HFKs from two or three independent donors. Reduced p53 levels and low-level hTERT expression in early-passage cells, as seen in HPV16-, -31-, -33-, and -35-, and to a lesser extent HPV18-transduced HFKs, was associated with continuous growth and an increased immortalization capacity. Less frequent immortalization by HPV45 and -51 and immortalization by HPV66 and -70 was preceded by an intervening period of strongly reduced growth (crisis) without prior increase in hTERT expression. Immortalization by HPV59 was also preceded by a period crisis, despite the onset of low hTERT expression at early passage. HPV52 triggered an extended life span but failed to induce immortality. Variations in p53 and pRb levels were not correlated with differences in alternative E6/E7 mRNA splicing in all hrHPV-transduced HFKs. On collagen rafts, transductants showed disturbed differentiation reminiscent of precancerous lesions. In conclusion, in vitro oncogenic capacities differ between the established hrHPV types, and both some established and probable hrHPV types display weak or moderate immortalization potential. C ervical cancer, the third most common cancer among women worldwide, is caused by a persistent infection with certain types of human papillomavirus (HPV) (1, 2). Most HPV infections are transient and are cleared within 1 to 2 years. However, a fraction of infections eventually give rise to either squamous cell carcinoma (SCC) or adenocarcinoma (AdCA) of the cervix. SCCs develop from so-called cervical intraepithelial neoplasia (CIN) precursor lesions. Low-grade CIN lesions (CIN1) mostly reflect a productive infection in which viral replication and virion production are linked to the differentiation program of the epithelium. High-grade CIN lesions (CIN2/3) mainly represent transforming infections and are characterized by deregulated expression of the early viral E6 and E7 genes in the proliferating basal cells of the epithelium (3, 4).HPV types belonging to the alpha (␣) genus can infect the cervical mucosa (5) and are classified into low-risk (lrHPV) and high-risk (hrHPV) HPV based on their association with malignancy (6). Infections with lrHPV types, e.g., HPV type 6 (HPV6) and HPV11, are associated with benign warts or low-grade lesions, whereas infections with hrHPV can give rise ...

Immortalization capacity of HPV types is inversely related to chromosomal instability

“…Detectable hTERT mRNA expression, as determined in our previous study [14], coincided with occurrence of the 5p copy number gain in these cells. Conversely, in cells without crisis hTERT expression was detectable at earlier passages and these cells did not acquire the 5p gain (see Supplementary Table 1).…”

“…Chromothripsis was neither evident in pre-immortal cells (passage 15 cells; arrayCGH results), nor in cells at passage 20 and passage 30 (low-coverage sequencing results), but was retained in late immortal cells (passage 100; array CGH results) (Figure 5C). To the best of our knowledge, this is the first cell line with chromothripsis that is established in vitro and that exhibits an immortal, but non-transformed phenotype (unable to grow anchorage independent/data not shown) and represents a precancerous lesion when cultured on organotypic rafts [14]. …”

“…To determine whether in vitro immortalization capacities and associated host cell aberrations differ amongst the various hrHPV types, we have recently established a large series of HPV-type specific in vitro models. Primary human foreskin keratinocytes (HFKs) isolated from two to three independent donors were transduced with the viral oncogenes E6 and E7 of HPV16, 18, 31, 33, 35, 45, 51, 52, 59, 66 and 70 [14]. When studying the immortalization capacities of the different HPV-types, two main groups were distinguished based on their growth behavior in culture.…”

“…early passages representing pre-immortal cells in their extended lifespan and later passages representing immortal cell lines. Immortalization was previously determined by detection of telomerase activity and upregulated hTERT expression [14]. In case of HPV45-, 51-, 59-, 66- and 70-transduced cells these are the cells that escaped from crisis.…”

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

“…1). Thus, using engineered human epithelium resembling in vivo conditions based on near-diploid immortalized keratinocytes (NIKS [32]) we recently elucidated the phenotypic characteristics of both E6 gene variants in the context of the full HPV16 genome [31], building upon previous work on the effects of transduction with the E6 or E6/E7 genes only [27, 28, 33]. Using the organotypic model we observed that the AAE6 genome drives tumourigenesis by increasing epithelial proliferation, disrupting routine differentiation and apoptosis, evading the innate immune system and promoting immortalization [31].…”