Treatment of a human papillomavirus type 31b-positive cell line with benzo[a]pyrene increases viral titer through activation of the Erk1/2 signaling pathway

Abstract

Numerous epidemiological studies have implicated cigarette smoking as a cofactor in the progression to cervical cancer. Tobacco-associated hydrocarbons have been found in cervical mucus, suggesting a possible interaction with human papillomavirus (HPV)-infected cells. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) is a major component of cigarette smoke condensate that has received significant attention due to its ability to induce carcinogenesis. We have previously demonstrated by conventional methods for determining viral titer that high concentrations of BaP increase HPV31b titers within the context of organotypic raft cultures compared with the level for vehicle controls. However, a definitive mechanism for explaining this increase in viral titer was lacking. Here, we show that BaP treatment activates the Ras-Raf-Mek1/2-Erk1/2 signaling pathway. The importance of Erk1/2 pathway activation to the BaP-mediated increase in viral titer was determined by Erk pathway inhibition with multiple Erk1/2 pathway inhibitors. Finally, BaP treatment activated p90RSK and its downstream target CDK1. These data indicate that the Erk1/2 signaling pathway plays an important role in mediating the response to BaP treatment that ultimately leads to increased viral titers.

Fig. 1.

(A) Quantitative RT-PCR infectivity assay. CIN-612 9E organotypic raft cultures were grown for 12 days and were treated with 1.0 μM BaP or an equivalent concentration of DMSO, and virus titers were measured by a quantitative RT-PCR infectivity assay. Data are presented as fold increases in E1^E4 level compared with the level for the DMSO control. (B) HPV31b gene expression. The virus lysates from panel A were used in Western blot analysis with antibodies specific for HPV L1, HPV E7, and actin.

Fig. 2.

Erk1/2 pathway activity. CIN-612 9E organotypic raft cultures were grown for 12 days and were treated with 1.0 μM BaP, an equivalent concentration of DMSO, or medium only. Cell lysates were generated and used in Western blot analysis with antibodies specific for total Ras, total Raf, total Mek1/2, phospho-Mek1/2, total Erk1/2, phospho-Erk1/2, or actin.

Fig. 3.

Effects of Erk1/2 pathway inhibition on viral titers. (A) Histochemical analysis. Shown are hematoxylin and eosin-stained CIN-612 9E raft culture tissue sections treated with 1.0 μM BaP, 1.0 μM BaP plus 20 μM U0126, or an equivalent concentration of DMSO. U0126 treatments were performed on days 7 and 8, 9 and 10, or 11 and 12. (B) Inhibition of Erk1/2 activation. Western blot analysis was performed on lysates from rafts treated with 1.0 μM BaP with or without 20 μM U0126. U0126 treatments were performed on days 7 and 8, 9 and 10, or 11 and 12. Cell lysates were prepared on day 8, 10, or 12, respectively. Western blot analyses were performed using a phospho-specific Erk1/2 antibody (see Materials and Methods) or GAPDH. (C) Quantitative RT-PCR infectivity assay of U0126-treated rafts. CIN-612 9E organotypic raft cultures were treated with 1.0 μM BaP with or without 20 μM U0126. U0126 treatments were performed as described for panel A. Virus titers were measured by a quantitative RT-PCR infectivity assay. Data are presented as fold increases in activation of E1^E4 transcript abundance compared with the level for DMSO treatment alone. Separately, CIN-612 9E organotypic raft cultures were grown without 1.0 μM BaP with or without 20 μM U0126. U0126 treatments were performed as described for panel A. Virus titers were measured as before and are presented as fold increases in activation of E1^E4 transcript abundance compared with the level for DMSO treatment alone. (D) Quantitative RT-PCR infectivity assay of PD98059-treated rafts. CIN-612 9E organotypic raft cultures were treated with 1.0 μM BaP with or without 50 μM PD98059. Virus titers were measured and reported as described for panel C.

Fig. 4.

(A) HPV31b early/late-promoter activity. CIN-612 9E cells were transfected with pGL2-Basic, pGL2B-630, or pGL2B-1706 and were grown in 1.6% methylcellulose with 1.0 μM BaP or an equivalent concentration of DMSO for 48 h. Cells were harvested, and promoter activity was determined by a luciferase assay. Data are represented as fold increases in activation over the level for the pGL2-Basic empty-vector control. (B and C) HPV31b late-transcript stability. CIN-612 9E organotypic raft cultures were grown for 12 days with 1.0 μM BaP or an equivalent concentration of DMSO. Total RNA was isolated and used in quantitative RT-PCR analysis with primers specific for HPV31b L1 (B) or L2 (C) transcripts. Data are represented as fold increases in activation over the level for the DMSO controls.

Fig. 5.

p90RSK activation. CIN-612 9E organotypic raft cultures were grown for 12 days with 1.0 μM BaP, BaP with 20 μM U0126, or an equivalent concentration of DMSO. U0126 treatments were performed on days 11 and 12. Cell lysates were generated and used in Western blot analysis with an antibody specific for total p90RSK and a phosphospecific p90RSK antibody that recognizes p90RSK phosphorylated at Thr573, indicative of activation.

Fig. 6.

U0126 inhibition of CDK1 kinase activity. Kinase assays were performed with cell lysates from rafts treated with 1.0 μM BaP with or without 20 μM U0126 or an equivalent concentration of DMSO as a control. U0126 treatments were performed on days 7 and 8, 9 and 10, or 11 and 12. Histone H1 was used as a CDK1 substrate.