The cigarette smoke carcinogen benzo[a]pyrene enhances human papillomavirus synthesis

Abstract

Epidemiological studies suggest that cigarette smoke carcinogens are cofactors which synergize with human papillomavirus (HPV) to increase the risk of cervical cancer progression. Benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, is detected in the cervical mucus and may interact with HPV. Exposure of cervical cells to high concentrations of BaP resulted in a 10-fold increase in HPV type 31 (HPV31) viral titers, whereas treatment with low concentrations of BaP resulted in an increased number of HPV genome copies but not an increase in virion morphogenesis. BaP exposure also increased HPV16 and HPV18 viral titers. Overall, BaP modulation of the HPV life cycle could potentially enhance viral persistence, host tissue carcinogenesis, and permissiveness for cancer progression.

FIG. 1.

Histochemical analysis. Shown are hematoxylin and eosin-stained CIN-612 9E raft culture tissue sections treated with a range of BaP concentrations as indicated.

FIG. 2.

Southern blotting and densitometric analysis were performed to determine the effect of BaP treatment on HPV31b genome copies. (A) A representative Southern blot showing the F1 (episomal), FII (nicked), and FIII (HindIII linearized) forms of the HPV31b genome. Five micrograms of total cellular DNA was digested with HindIII, which linearizes the HPV31b genome at nucleotide 2455. Blots were detected with a ³²P-labeled total HPV31-specific DNA probe generated by random primer extension, followed by autoradiography. (B) Comparative densitometric analysis of the FIII bands from Southern blots of BaP treatments compared with those of DMSO treatments and medium-only controls. The medium-only control was set to 1. Results indicate the averages of three independent experiments ± standard error of the means (*, P < 0.005 relative to that of medium-only control samples; †, P < 0.005 relative to that of DMSO-treated control samples). The paired Student t test was used to calculate P values.

FIG. 3.

HPV infectivity assay and L1 expression. (A) Infectious titer of the HPV31b control raft tissues and tissues treated with BaP in the presence of C8:0 (left panel) and in the absence of C8:0 (right panel). Shown is a 2% agarose gel of nested reverse transcription (RT)-PCR-amplified HPV31 E1̂E4 spliced transcript and β-actin in reactions with increasing dilutions of the virus stocks shown on the left. (B) The effect of BaP on L1 capsid protein expression as determined from Western blots. (C) Neutralization of HPV31b virus particles from BaP-treated and control raft cultures, using monoclonal H31.Ab, followed by detection of the E1̂E4 spliced transcript in the infectivity assay is shown. The H31.Ab was used at a dilution of 1:20 in HaCat medium. Virus stocks were used at the dilutions shown on the left. (D) Infectious titers of HPV16 and HPV18 control raft tissues and tissues treated with BaP. HPV16- and HPV18-positive cell lines HPV16(114/b)wt:3 and HPV18wt:4, respectively, were used to generate raft cultures for these assays. Shown are 2% agarose gels of nested RT-PCR-amplified HPV16 and HPV18 E1̂E4 spliced transcript and β-actin bands. Reactions were performed with increasing dilutions of the virus stocks shown on the left of each panel.