Long-term effect of interferon on keratinocytes that maintain human papillomavirus type 31

Abstract

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.

FIG. 1.

Growth of HPV-31-positive cells is severely retarded by IFN-β. Multiple dishes of keratinocytes were seeded at 4 × 10⁵ cells per 6-cm dish 1 day prior to IFN treatment. Beginning on the next day (day 0), cells were either left untreated or treated with IFN-β at 1,000 U/ml. One dish from control or IFN-treated cells was harvested on the indicated date, and viable cells were counted. (A) NHKs; (B) SCC-13 carcinoma cell line; (C) LKP31 cell line; (D) CIN 612 9E cell line.

FIG. 2.

The morphology, size, and shape of HPV-31-positive cells are dramatically changed upon IFN treatment. CIN 612 9E cells were either left untreated (A and D) or treated with IFN-β for 6 days (B, C, and E), and the morphology of cells was observed with a phase-contrast microscope (magnification, ×70) (A through C). The same samples were analyzed by flow cytometry, and the forward-scatter (FS) and side-scatter (SS) patterns of control and IFN-treated cells are shown (D and E).

FIG. 3.

Levels of episomal HPV-31 DNA gradually decrease upon prolonged IFN treatment. LKP31 cells were treated with IFN-β at passage 15. The IFN-resistant population was expanded, and the low-molecular-weight DNA was harvested at different passages. Ten micrograms of harvested DNA was examined by Southern blot analysis with an HPV-31-specific probe. The positions of episomal, relaxed-circle, and integrated viral DNAs are indicated at the left or right of each panel. Panels A and B represent two independent experiments. Levels of viral DNA from control untreated LKP31 cells at passage 27 (A) or passages 11 and 29 (B) are also shown.

FIG. 4.

IFN induces apoptosis in HPV-31-positive cells. LKP31 cells grown on coverslips were either left untreated (A) or treated with IFN-β at 1,000 U/ml for 6 days (B). Coverslips were processed for a TUNEL apoptosis assay (upper panels) and counterstained with DAPI (lower panels). Images of TUNEL and DAPI staining of CIN 612 9E cells treated with IFN-β under the same conditions are also shown (C).

FIG. 5.

IFN induces apoptosis in HPV-31-positive cells. NHK or LKP31 cells were either left untreated (A and C) or treated with IFN-β at 1,000 U/ml for 4 days (B and D). Cells were harvested and processed for annexin V-FITC-propidium iodide (PI) staining. The percentage of cells that were FITC positive or FITC-PI double positive is indicated in each quadrant.

FIG. 6.

Western blot analysis of the expression of apoptosis-related genes in cells treated with IFN. (A) NHKs, LKP31 cells, or CIN 612 9E cells were either left untreated or treated with IFN-β at 1,000 U/ml for 4 days. Cells were harvested, and protein lysates were separated by SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred onto a membrane and probed with a FasL-specific antibody. The same blot was stripped and probed with an anti-Bcl-X antibody. (B and C) NHKs or LKP31 cells were either left untreated or treated with IFN-β at 1,000 U/ml for various times. Protein lysates were harvested at the indicated times posttreatment and subjected to Western blot analysis for the expression of caspase 1 (B) and TRAIL (C).

FIG. 7.

IFN induces senescence in HPV-31-positive cells. (A and C) CIN 612 9E cells grown on coverslips were either left untreated (A) or treated with IFN-β at 1,000 U/ml for 6 days (C). The cells were then processed for β-Gal staining. (B) LKP31 cells treated under the same conditions were stained for β-Gal.

FIG. 8.

Growth rate assays with E6E7 (A) and HeLa (B) cells. Cells were seeded at 4 × 10⁵ (E6E7) or 1 × 10⁵ (HeLa) per 6-cm dish 1 day prior to IFN treatment. The next day (day 0), cells were either left untreated or treated with IFN-β at 1,000 U/ml. One dish each from control or IFN-treated cells was harvested, and the number of viable cells was counted at the indicated times.

FIG. 9.

(A through C) Growth rate assays with A431, D1, and F4 cells. Cells were seeded at 10⁵ per 6-cm dish 1 day prior to IFN treatment. From the next day (day 0) on, cells were either left untreated or treated with IFN-β at 1,000 U/ml. One dish from either control or IFN-treated cells was harvested, and the number of viable cells was counted, at the indicated times. (D) RT-PCR analysis of E2 expression in two independent E2 clones upon induction with doxycycline (Dox). (E) Luciferase assay of two E2-expressing clones transiently transfected with either the control reporter construct (pGL3Basic) or the luciferase reporter construct with multiple E2 binding sites (pGL3Basic/6XE2BS) upon induction with doxycycline. Control-luciferase, pGL3Basic; E2RE-luciferase, pGL3Basic/6XE2BS.