Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein

Abstract

Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.

FIG. 1.

Induction of the E7 protein during growth in methylcellulose-containing medium. (A) Caski cells were grown in methylcellulose-containing medium and were harvested at the indicated times. Cell lysates (200 μg) were separated by SDS-PAGE and analyzed by Western blot assay using E7 antibody and p27 antibody. Cell lysates (25 μg) were probed with pan-cytokeratin antibody and tubulin antibody. (B) Exponentially growing Caski cells as adherent culture and Caski cells growing in methylcellulose-containing medium for 14 h were analyzed by flow cytometry. HOK-16B cells (C) and BaP-T cells (D) grown in methylcellulose-containing medium were harvested at the indicated time. Cell lysates—200 μg for E7, 100 μg for involucrin, and 25 μg for tubulin—were analyzed using an immunoblot assay. Caski cells (E) and BaP-T cells (F) cultured in medium containing 1.5 mM CaCl₂ were harvested at the indicated times. Cell lysates (200 μg for E7, 50 μg for tubulin, and 250 μg for involucrin) were analyzed using an immunoblot assay.

FIG. 2.

The half-life of E7 did not change significantly during differentiation. (A) The Caski cells grown as a monolayer or in methylcellulose-containing medium (14 h) were labeled with [³⁵S]methionine-cysteine for 2 h and chased with cold methionine for the indicated periods of time. The ³⁵S-labeled E7 protein was immunoprecipitated and analyzed by SDS-PAGE. The level of ³⁵S-E7 was quantified by a phosphorimager and plotted against the time of chase. The averages of two independent experiments are presented. (B) The Caski cells grown as a monolayer or in methylcellulose-containing medium for 6 h, 12 h, and 18 h were treated with cycloheximide (50 μg/ml) for 1 to 4 h. Cell lysates (200 μg) were separated by SDS-PAGE and analyzed by Western blot assay using E7 antibody.

FIG. 3.

The steady-state level of the E7 mRNA did not change significantly during growth in methylcellulose-containing medium. Total RNA was isolated from Caski cells or HOK-16B cells at the indicated times following growth in methylcellulose-containing medium. RNA (10 μg) was hybridized to ³²P-labeled antisense-E7 and antisense-cyclophilin probes and processed by an RNase protection assay as described in Materials and Methods. The ³²P-labeled E7 and cyclophilin probes are shown.

FIG. 4.

Increased polysome association of the E7 mRNA during differentiation. Caski cells (left) and BaP-T cells (right) were grown in a monolayer culture (A) or in methylcellulose-containing medium for 14 h (B). Cytosolic extracts were fractionated on a sucrose gradient (15 to 40%) as described in Materials and Methods. RNA isolated from each fraction (numbers indicated at the top) was analyzed for the presence of the E7 mRNA and Skp2 mRNA by an RNase protection assay. Staining for rRNA by ethidium bromide in gradient fractions is shown for each sample. Fractions 1 to 5 represent subpolysomal RNA, and fractions 6 to 9 represent polysome-associated RNA.

FIG. 5.

Induced and sustained phosphorylation of 4E-BP1 in differentiating HPV-containing Caski cells but not in normal human keratinocytes or non-HPV HaCaT cells. Cell lysates of normal human keratinocytes (150 μg), Caski cells (100 μg), and HaCaT cells (100 μg) grown in methylcellulose-containing medium for the indicated times were fractionated by SDS-PAGE and analyzed by Western blot assay using antibodies against phospho-4E-BP1 (Ser65), 4E-BP1, eIF4E, and phospho-eIF4E (Ser 209). Two hundred micrograms of Caski cell lysates was analyzed for E7 protein using a Western blot assay.

FIG. 6.

Changes in the levels of 4E-BP1 and eIF4E during growth in methylcellulose-containing medium. Lysates of primary oral keratinocytes (200 μg), HOK-16B cells (150 μg), BaP-T cells (100 μg), Caski cells (50 μg), SCC-25 cells (100 μg), and C33A cells (50 μg) grown in monolayer cultures or in methylcellulose-containing medium were analyzed by a Western blot assay using 4E-BP1 and eIF4E antibodies.

FIG. 7.

(A) Increased level of 4E-BP1 associates with the m⁷GTP resin in differentiating HaCaT cells but not in Caski cells. Lysates (500 μg) of Caski cells or HaCaT cells grown in methylcellulose-containing medium (for 14 h) were incubated with m⁷GTP-Sepharose beads as described in Materials and Methods. The proteins bound to the beads were analyzed for 4E-BP1 and eIF4E using a Western blot assay. (B) Rapamycin blocks expression of E7 protein in Caski cells. Caski cells grown as adherent culture or in methylcellulose-containing medium were treated with rapamycin (100 ng/ml) for 12 h. (Left) Lysates from attached (350 μg) or differentiated (200 μg) cells were analyzed for E7, tubulin (25 μg), and 4E-BP1 (100 μg) using a Western blot assay. (Right) Total RNA (10 μg) isolated from dimethyl sulfoxide (DMSO)- or rapamycin-treated cells was analyzed for E7 mRNA and cyclophilin mRNA using an RNase protection assay.