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...

Murine natural killer (NK) and natural cytotoxic (NC) cells showed different patterns of augmentation of lytic activity after preincubation for 24 h with either poly-IC, interleukin 2 (IL-2), or interleukin 3 (IL-3): (a) Poly-IC augmented only NK cells, with no effect on NC activity, as we have previously observed (4); (b) IL-2 augmented both NK and NC activity; and (c) IL-3 augmented only NC lysis, without affecting NK activity. In addition, both precursor and the augmented effector cells showed differences in expression of the Qa-5 surface marker: NK precursors and effectors are Qa 5+, whereas NC precursors and effector cells are Qa-5-.

Natural cell-mediated cytotoxicity (NCMC), measured against a variety of tumors, is mediated by at least two sub-populations of effector cells: natural cytotoxic (NC) and natural killer (NK). The studies described in this report show that target lysis by NC cells requires a prolonged (18- to 24 h) assay period, whereas NC-susceptible targets can be lysed in short-term (4h) [3H]-proline assays using allo-sensitized CTL or mitogen-activated cytotoxic populations. NC cells are not "activated" during the long-term assay as indicated by: (1) demonstrating that lysis of NC-susceptible targets in long-term (20 h) 51Cr assays is still the function of a Qa-5- effector cell (NC) and (2) the fact that preincubation of the NC effector cell with susceptible targets for 18 h did not result in the activation of an NK-like population (kinetics of target lysis were comparable to those noted with fresh NC cell preparations). We show that NC cell activity is preserved in both the beige mutant and the PL/J mouse strains, both of which exhibit low NK cell activity, even in long-term assays. These combined studies support the view that NC and NK cell activities are the function of distinct cell types and not the property of a single cell class under different states of activation.