Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells.

Abstract

The human papillomaviruses (HPVs) are associated with specific benign and malignant lesions of the skin and mucosal epithelia. Cloned viral DNAs from HPV types 6b, 16, and 18 associated with different pathological manifestations of genital neoplasia in vivo were introduced into primary human cervical epithelial cells by electroporation. Cells transfected with HPV16 or HPV18 DNA acquired indefinite lifespans, distinct morphological alterations, and anchorageindependent growth (HPV18), and contain integrated transcriptionally active viral genomes. HPV6b or plasmid electroporated cells senesced at low passage. The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result in cervical carcinoma in vivo.The human papillomaviruses (HPVs) offer a unique approach to the studies of human epithelial cell carcinogenesis. Clinical and epidemiological studies combined with molecular analysis of the >50 viruses identified to date (1-3) have revealed an apparent anatomical restriction of HPV types with specific benign and malignant lesions of the skin and mucosa (4). Approximately 20% of the HPVs that have been identified were isolated from anogenital tissues (4-9); however, HPV6 and HPV11 predominate in benign exophytic and endophytic condylomas, whereas HPV16 and HPV18 predominate in premalignant and malignant genital tissues. Ninety-five percent of cervical cancers and cervical intraepithelial neoplasias occur within the transformation zone of the uterine cervix (10) and reportedly contain HPV16, HPV18, or related types of HPV. To investigate the oncogenic functions by which these viruses induce these specific types of premalignant and malignant lesions in vivo, we have introduced cloned viral DNAs representing HPV6b, -16, and -18 into primary cervical epithelial cells by electroporation and present a characterization of the altered biological properties of the cells, which we attribute to the presence of the different HPV genomes. Parallel cell cultures were also electroporated with cloned simian virus 40 (SV40) DNA, which we and others have shown to partially transform primary human keratinocytes derived from neonatal foreskins (11,12). MATERIALS AND METHODS Establishment of Primary Cervical Epithelial Cultures.Normal cervical epithelial cells were derived from explant cultures of the portio surface and transformation zone of cervix uteri obtained from 35-to 40-year-old women who had undergone hysterectomies for benign uterine fibroids. Primary cultures were initiated and maintained as described (13) Electroporation and Subculturing of Cervical Cells. Twenty-one days postinitiation of the primary cultures, when the cells were actively growing and at 75-90% confluency, explants were removed by aspiration and the cell monolayers were disaggregated by exposure to 0.25% trypsin/0.02% EDTA (GIBCO) for 30 s, and incubation at 37°C for 5-10 min. Cells were washed and resuspended (4-6 x 106 cells per ml) in Ca2l-, Mg2+-free Dulbecco's phosphate-buffered saline...

Viral integration sites in human papilloma virus-33-immortalized cervical keratinocyte cell lines

“…This was further strengthened by our previous observation [12] that cell populations that have integrated the viral DNA at different sites could not be stably established in culture. This interpretation of our results is in agreement with many data obtained by Southern blotting describing a single integration site of HPV-16 or -18 in cervical cancer cell lines or in in vitro transfected lines [5,7,15,16,[24][25][26][27][28]. Moreover, some studies reporting HPV integration at a single site in high grade lesions emphasize the monoclonal origin of cervical cancers and support our results [19].…”

“…These data are in agreement with many reports describing HPV-16 or -18 integration in cancer cells of the cervix [5,6,[15][16][17]. Moreover, in contrast with nononcogenic HPVs, which are mostly in an episomal state, HPV-16 or -18 are preferentially found integrated in high grade lesions [18][19][20][21][22][23].…”

The Human Papillomavirus E6 protein and its contribution to malignant progression

“…Numerous studies have shown that high-risk E6 and E7 are together su cient to induce immortalization of primary human keratinocytes (MuÈ nger et al, 1989;Hawley-Nelson et al, 1989), while the low-risk HPV proteins are completely inactive in this assay Pecoraro et al, 1989). It is also interesting to note that these keratinocytes, although immortalized, will not form tumours in nude mice.…”

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.

“…Studies of human cell trans formation have shown that HPV-6 does not efficiently immortalize primary, human epithelial cells (Schlegel et al 1988;Pecoraro et al 1989;Halbert et al 1992). There is no evidence for cellular degradation of p53 by HPV-6 E6 (Scheffner et al 1990;Werness et al 1990;Crook et al 1991), and the affinity of HPV-6 E7 protein for cellular retinoblastoma protein (pRb) is ~ 10-fold lower than the affinity of HPV-16 E7 for pRb (Munger et al 1989b;Barbosa et al 1990;Gage et al 1990; data not shown).…”