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, 8 (1), 15402

HPV16-Immortalized Cells From Human Transformation Zone and Endocervix Are More Dysplastic Than Ectocervical Cells in Organotypic Culture

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HPV16-Immortalized Cells From Human Transformation Zone and Endocervix Are More Dysplastic Than Ectocervical Cells in Organotypic Culture

Han Deng et al. Sci Rep.

Abstract

A major risk factor for cervical cancer is persistent infection with high-risk human papillomaviruses (HPV) which can cause cervical intraepithelial neoplasia. Greater than 90% of cervical cancers develop in the transformation zone (TZ), a small region of metaplastic squamous epithelium at the squamocolumnar junction between endocervix and ectocervix. However, it is unclear why this region is highly susceptible to malignant progression. We hypothesized that cells from TZ were more susceptible to dysplastic differentiation, a precursor to cervical cancer. We used three-dimensional organotypic culture to compare differentiation of HPV16-immortalized epithelial cell lines derived from ectocervix, TZ, and endocervix. We show that immortal cells from TZ or endocervix form epithelia that are more dysplastic than immortal cells from ectocervix. A higher percentage of immortal cells from TZ and endocervix express the proliferation marker Ki-67 and are positive for phospho-Akt. Immortal cells from TZ and endocervix invade collagen rafts and express increased levels of matrix metalloproteinase-1. Inhibition of MMP-1 or Akt activity blocks invasion. We conclude that HPV16-immortalized cells cultured from TZ or endocervix are more susceptible to dysplastic differentiation, and this might enhance their susceptibility to cervical cancer.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic of the cervical transformation zone. (Top) View of cervix as seen through gynecologist’s speculum showing ectocervix, TZ with Nabothian cysts, and endocervix. (Bottom) Cross section of transformation zone showing columnar epithelium of endocervix and stratified squamous epithelium of TZ and ectocervix. Nabothian cysts form when mucous ducts of endocervix become occluded by overgrowth of stratified squamous epithelium from newly formed TZ. Brown shading illustrates cells derived from endocervical reserve cells.
Figure 2
Figure 2
Primary cervical epithelial cells form differentiated stratified squamous epithelia when grown in organotypic culture. (Top row) Normal primary cells were cultured from ectocervix, TZ, or endocervix and maintained for 10 days on collagen rafts containing human cervical stromal cells. Dotted lines indicate position of basement membranes. (Bottom row) Ectocervical, TZ and endocervical cells simultaneously stained for K14 (red), K18 (green) and DAPI (blue).
Figure 3
Figure 3
HPV16-immortalized cells from TZ and endocervix are more dysplastic. (a) HPV16-immortalized cells derived from ectocervix, TZ, or endocervix were maintained for 10 days on rafts with cervical stromal cells. All cell lines were tested at approximately 80 to 95 population doublings. Dotted lines indicate position of basement membrane and the upper extent of epithelial cells. Upper row of figures were stained with H&E and lower row were simultaneously stained for K14, K18 and DAPI. (b) Mean dysplastic index (degree of dysplasia) ± standard error of 24 different HPV16-immortalized cell lines (8 from ectocervix, TZ and endocervix) maintained in organotypic culture on rafts formed with 3T3-J2 mouse cells or human stromal cells. Bars above graph show values that are statistically different (3 asterisks = p < 0.001).
Figure 4
Figure 4
HPV16-immortalized cells from TZ and endocervix have more Ki-67 positive cells. (a) HPV16-immortalized cell lines derived from ectocervix, TZ, or endocervix were maintained for 10 days on collagen rafts with cervical stromal cells and then stained with DAPI to visualize cell nuclei and Ki-67 antibody to show proliferating cells. Cervical cancer cells served as a positive control for cell proliferation. Dotted lines indicate position of basement membrane and upper extent of epithelial cells. (b) Mean percentage of Ki-67 positive cells ± standard error of 12 different HPV16-immortalized cell lines (4 from ectocervix, TZ and endocervix) maintained in organotypic culture on rafts formed with cervical stromal cells. Bars above graph show values that are statistically different (3 asterisks = p < 0.001).
Figure 5
Figure 5
HPV16-immortalized cells from TZ and endocervix express more phospho-Akt. (a) HPV16-immortalized cell lines from ectocervix, TZ, or endocervix were maintained for 10 days on collagen rafts with cervical stromal cells and then stained with fluorescent antibody to phospho-Akt. Control indicates cells that were stained with an irrelevant antibody. Dotted lines indicate the position of the basement membrane and the upper extent of epithelial cells. (b) Mean percentage of phospho-Akt positive cells ± standard error of nine different HPV16-immortalized cell lines (3 from ectocervix, TZ and endocervix). Bars show values that are statistically different (3 asterisks = p < 0.001).
Figure 6
Figure 6
HPV16-immortalized cells from TZ and endocervix are more invasive. (a) HPV16-immortalized cells derived from ectocervix, TZ, or endocervix (all 85 population doublings) were grown for 10 days on rafts with either 3T3-J2 mouse cells or human stromal cells. Dotted lines indicate position of basement membranes. (b) Mean invasion index (degree of invasion into collagen raft) ± standard error of 24 different HPV16-immortalized cell lines (8 from ectocervix, TZ and endocervix) maintained in organotypic culture on rafts formed with 3T3-J2 cells or human stromal cells. Bars show values that are statistically different (3 asterisks = p < 0.001).
Figure 7
Figure 7
HPV16-immortalized cells from TZ and endocervix express higher levels of MMP-1. (a) HPV16-immortalized cells from ectocervix, TZ, or endocervix were maintained for 10 days on collagen rafts with cervical stromal cells, and then stained with fluorescent antibody to MMP-1. Control indicates cells stained with an irrelevant antibody. Dotted lines indicate the position of the basement membrane and the upper extent of epithelial cells. (b) Mean percentage of MMP-1 cells ± standard error of nine different HPV16-immortalized cell lines (3 from ectocervix, TZ and endocervix). Bars show values that are statistically different (3 asterisks = p < 0.001).
Figure 8
Figure 8
Inhibition of Akt or MMP-1 blocks invasion of HPV16-immortalized cells. (a) HPV16-immortalized TZ cells were seeded on collagen rafts with cervical stromal cells and then treated for 10 days with the Akt inhibitor SC 66 (2 µg/ml) or the MMP inhibitor GM6001 (10 μM) in DMSO. Untreated cultures received only DMSO. Dotted lines indicate position of basement membranes. (b) Mean invasion index ± standard error of three organotypic cultures. Bars show values that are statistically different (3 asterisks = p < 0.001).

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