Fig 1.
Structure and histology of the cervical TZ.
A. Schematic representation of the cervix showing the TZ between ectocervix and endocervix. B. Histology of the cervical TZ showing the stratified squamous epithelium and underlying Nabothian cysts. C. Schematic showing the surface features of ectocervix, endocervix and TZ that aid in tissue dissection. The ectocervix is easily identified because the surface is smooth, white, and shiny with no mucous. The endocervix surface is rough, red in color, and covered with mucous. The TZ contains Nabothian cysts (swollen glands due to occlusion of ducts by squamous metaplasia). These large cysts are easily visible and diagnostic for the TZ. D. Photograph of a typical cervical specimen showing each region.
Fig 2.
Cell morphology and keratin expression of monolayer cervical cell cultures.
A. Phase contrast microscopy of primary human cervical cells from each region showing different cell morphology. B. K14 and K18 immunostaining showing different expression in cell culture in vitro. C. K17 and MMP7 immunostaining of cells cultured from each region of cervix. Irrelevant primary antibodies were used as the negative control for immunostaining.
Fig 3.
Quantitative analysis of cell marker expression by epithelial cells from each cervical region.
Freshly isolated cells from each cervical region were stained with antibodies against K14, K18, K17, p63 or MMP7. Bars represent the mean ± standard error of three experiments using samples from different donors. The asterisks indicate statistical differences [** P < 0.01;*** P < 0.001].
Fig 4.
Transfection efficiency and immortalization efficiency after transfection with HPV16, HPV18 or SV40 DNA.
A. Transfection efficiency of beta-galactosidase reporter gene in early passage cells from ectocervix, TZ and endocervix. The bars represent the mean ± standard error of 13 experiments using samples from different donors. B. Immortalization efficiency of cervical cells from each region after normalization for differences in transfection rate. Each experiment had negative controls consisting of cultures transfected with only the neomycin gene, and all negative control cultures became senescent after 2 to 3 passages. The bars represent the mean ± standard error of experiments from 24 donors (HPV16) or 12 donors (SV40). C. Immortalization efficiency of cervical cells by HPV18 after normalization for differences in transfection rate. The bars represent the mean ± standard error of experiments from five donors (HPV18). D. Immortalization efficiency of cervical cells by HPV16 when cells were maintained on collagen-coated cell culture plates. The asterisks indicate statistical differences [** P < 0.01; *** P < 0.001].
Fig 5.
Infection and immortalization by retroviruses encoding HPV16 E6/E7.
A. Infection efficiency of cells from ectocervix, TZ and endocervix. The bars represent the mean ± standard error of eight experiments using samples from different donors. B. Immortalization efficiency of cervical cells from each region that was normalized for differences in infection rate. Bars represent the mean ± standard error of eight experiments using samples from different donors. The asterisks show statistical differences [** P < 0.01; *** P < 0.001].
Fig 6.
Immortalization by transfection with the complete HPV16 genome using co-cultures of epithelial and stromal cells.
A. The bars represent the mean ± standard error of five experiments using samples from different donors. No significant difference was observed using ANOVA and Tukey Post-hoc test (SPSS), p > 0.05.
Fig 7.
Keratin expression in HPV-immortalized cell lines.
A. Immunofluorescence staining for K14 and K18 in TZ, ectocervix and endocervix-derived immortalized CX16-RV2 cell lines. B. Quantitative analysis of K14 and K18 staining intensity in 12 HPV16 immortalized cervical cell lines. Bars represent the mean ± standard error of four experiments using samples from different donors. The asterisks show statistical differences [*** P < 0.001].
Table 1.
Properties of HPV16-immortalized cell lines.