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Journal of Virology, June 2008, p. 6084-6086, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.00103-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

How Does Tobacco Smoke Contribute to Cervical Carcinogenesis?

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Tobacco smoke is a well-established human papillomavirus (HPV) cofactor for the development of cervical precancer and cancer (3, 6), but the molecular mechanisms by which smoking increases the risk of cervical precancer and cancer remain unknown. There are several plausible explanations (2). One is that smoking inhibits the immune response to HPV. A second is that carcinogenic HPV-infected cells are exposed to smoking carcinogens that cause DNA damage while HPV oncoproteins block apoptosis and cell cycle arrest. Alam et al. (1) reported a molecular interaction between benzo[a]pyrene (BaP), a carcinogen found in cigarette smoke, and HPV synthesis, suggestive of yet another possible mechanism. They found evidence that "high concentrations of BaP resulted in a ten-fold increase in HPV31 viral titers, whereas treatment with low concentrations of BaP resulted in increased HPV genome copies, but not virion morphogenesis".

While Alam et al. (1) found that high concentrations induced high viral titers in their model system, it is notable that there was no evidence of a dose-response relationship between BaP concentration and HPV viral load that would lend biological plausibility. There is also no evidence that smokers have 1 µM BaP concentrations in their cervical tissue, which is more relevant than concentrations in cervical mucus.

Moreover, epidemiological studies have failed to consistently demonstrate an association of high HPV viral load, except for perhaps HPV type 16 (HPV16) (5), and incident cervical precancer or cancer. Nor has smoking been shown to elevate HPV viral load in humans. To examine the relationship of smoking and HPV viral load, data from atypical squamous cells of undetermined significance and a low-grade squamous-lesion triage study (ALTS)(8), a study in which cigarette smoking was shown to increase the risk of cervical precancer in carcinogenic HPV-positive women (6), were used. Using Hybrid Capture 2 signal strength as a semiquantitative measure of HPV viral load (4) among those women who were identified to have a single HPV genotype by PCR, no association of smoking status and semiquantitative HPV viral load was found for all women singly infected by any carcinogenic HPV genotype, by HPV31, or by HPV16 (Table 1). However, we note as a limitation that no method of viral load measurement that uses aliquots of exfoliated cervical cells can distinguish between two scenarios, lower viral load in many cells and higher viral load in fewer cells. It is plausible that smoking is associated with the latter condition and may be indicative of a higher risk of cervical precancer and cancer.

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TABLE 1. Comparison of the median Hybrid Capture 2 signal strengths, a semiquantitative measure of viral load (4), by smoking status and infection with a single carcinogenic HPV genotype in women participating in a low-grade squamous lesion triage studya

The HPV genome amplification due to exposure of HPV31b-infected raft cultures to low levels of BaP is an intriguing finding for the very reasons outlined by the authors (1), possible increased levels of the primary oncoproteins E6 and E7. Again, however, the authors did not find evidence of a dose-response effect, and only a twofold-higher effect than that for controls was observed. Unfortunately, there is a general lack of data on E6 and E7 expression and HPV natural history for reference. New assays that quantitatively measure HPV E6/E7 mRNA in human cervical specimens may be used to assess the relationship of smoking and E6/E7 expression.

To date, the molecular mechanism(s) by which smoking exerts an oncogenic effect on carcinogenic HPV-infected cells remains elusive. One recent study failed to find any difference in polycyclic aromatic hydrocarbon-DNA adduct formation between carcinogenic HPV-positive smokers and nonsmokers and between cases and controls (7). It is even uncertain at which stage(s) (HPV persistence, progression of persisting HPV infection to precancer, and/or invasion) smoking influences cervical carcinogenesis. Perhaps new tools, such as transgenic-mouse expression of HPV16 oncoproteins (9) or susceptible cell lines (10), may be useful for modeling the effects of smoking and smoking constituents on HPV biology. More laboratory and epidemiological research is needed to elucidate the etiologic role of smoking in cervical carcinogenesis.

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1. Alam, S., M. J. Conway, H. S. Chen, and C. Meyers. 2008. The cigarette smoke carcinogen benzo[a]pyrene enhances human papillomavirus synthesis. J. Virol. 82:1053-1058.[Abstract/Free Full Text]
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2. Castellsague, X., and N. Munoz. 2003. Chapter 3: cofactors in human papillomavirus carcinogenesis—role of parity, oral contraceptives, and tobacco smoking. J. Natl. Cancer Inst. Monogr. 2003:20-28.[Abstract/Free Full Text]
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3. Castle, P. E., S. Wacholder, A. T. Lorincz, D. R. Scott, M. E. Sherman, A. G. Glass, B. B. Rush, J. E. Schussler, and M. Schiffman. 2002. A prospective study of high-grade cervical neoplasia risk among human papillomavirus-infected women. J. Natl. Cancer Inst. 94:1406-1414.[Abstract/Free Full Text]
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4. Gravitt, P. E., R. D. Burk, A. Lorincz, R. Herrero, A. Hildesheim, M. E. Sherman, M. C. Bratti, A. C. Rodriguez, K. J. Helzlsouer, and M. Schiffman. 2003. A comparison between real-time polymerase chain reaction and hybrid capture 2 for human papillomavirus DNA quantitation. Cancer Epidemiol. Biomarkers Prev. 12:477-484.[Abstract/Free Full Text]
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5. Gravitt, P. E., M. B. Kovacic, R. Herrero, M. Schiffman, C. Bratti, A. Hildesheim, J. Morales, M. Alfaro, M. E. Sherman, S. Wacholder, A. C. Rodriguez, and R. D. Burk. 2007. High load for most high risk human papillomavirus genotypes is associated with prevalent cervical cancer precursors but only HPV16 load predicts the development of incident disease. Int. J. Cancer. 121:2787-2793.[CrossRef][Medline]
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6. McIntyre-Seltman, K., P. E. Castle, R. Guido, M. Schiffman, and C. M. Wheeler. 2005. Smoking is a risk factor for cervical intraepithelial neoplasia grade 3 among oncogenic human papillomavirus DNA-positive women with equivocal or mildly abnormal cytology. Cancer Epidemiol. Biomarkers Prev. 14:1165-1170.[Abstract/Free Full Text]
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7. Pratt, M. M., P. Sirajuddin, M. C. Poirier, M. Schiffman, A. G. Glass, D. R. Scott, B. B. Rush, O. A. Olivero, and P. E. Castle. 2007. Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: an immunohistochemistry study. Mutat. Res. 624:114-123.[Medline]
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8. Schiffman, M., and M. E. Adrianza. 2000. ASCUS-LSIL Triage Study. Design, methods and characteristics of trial participants. Acta Cytol. 44:726-742.[Medline]
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9. Song, S., A. Liem, J. A. Miller, and P. F. Lambert. 2000. Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis. Virology 267:141-150.[CrossRef][Medline]
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10. Spardy, N., A. Duensing, D. Charles, N. Haines, T. Nakahara, P. F. Lambert, and S. Duensing. 2007. The human papillomavirus type 16 E7 oncoprotein activates the Fanconi anemia (FA) pathway and causes accelerated chromosomal instability in FA cells. J. Virol. 81:13265-13270.[Abstract/Free Full Text]

						Philip E. Castle Division of Cancer Epidemiology and Genetics National Cancer Institute 6120 Executive Blvd., Room 5004, MSC 7234 Bethesda, Maryland 20892-7234 Phone: (301) 435-3976 Fax: (301) 402-0916 E-mail: castlep{at}mail.nih.gov

Author's Reply

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Since most HPV infections are spontaneously cleared (4), viral persistence and viral load are thought to be necessary for cancer progression (12, 16). Multiple epidemiological studies have suggested a correlation between cigarette smoking among HPV-infected women as a cofactor for the development of cervical cancer (8, 13, 15). Using organotypic "raft" cultures, we studied the effect of BaP, a major carcinogenic component of cigarette smoke, on the productive life cycle of three high-risk HPV types (1). To date, raft cultures are the best physiologically relevant in vitro model system available, one which closely mimics the natural replication of the virus as it occurs in vivo (7). We showed that treatment with 1 µM BaP resulted in a 10-fold increase in viral titers while treatment with 0.001 µM BaP resulted in a 2-fold increase in the number of genome copies (1). Thus, our studies suggest that exposure to cigarette smoke carcinogens such as BaP could lead to manipulation of host cell- and/or HPV-specific functions resulting in enhancement of the "total viral load," with respect to both increased virion synthesis and viral genome amplification. Increased viral titers may be important for infection of secondary sites around the primary lesion, and an increase in the number of genome copies may result in a concomitant increase in the number of templates from which the E6 and E7 oncogenes may be transcribed (5). Upregulation of genome amplification may also increase the probability of viral DNA integration into the host genome, a milestone in the development of cervical cancer. Based on our studies, we present a novel finding that BaP-regulated enhancement of both virion synthesis and amplification of genome copies may potentially result in increased persistence of the virus in HPV-infected women who smoke.

While BaP has been detected in cervical mucus, its concentration has yet to be determined (6). In contrast, other cigarette smoke components have been found in measurable concentrations within a variable range. Our rationale for choosing the range of BaP concentrations tested was based on published data on other cigarette smoke carcinogens which have been quantified in cervical mucus. For example, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a well-characterized cigarette carcinogen, has been detected at a concentration as high as 0.56 µM (9), whereas noncarcinogenic components such as nicotine and its metabolite cotinine have been detected at concentrations of 3 µM and 0.36 µM, respectively (11). Interestingly, both nicotine and cotinine were found to be strongly concentrated in the cervical mucus in comparison to their levels in serum (3). With respect to this issue raised by Dr. Castle, we would like to point out that no studies have been performed to actually measure BaP concentrations within cervical cells or, for that matter, any other cigarette carcinogen. It may be assumed that active or passive cellular processes concentrate BaP and its metabolites inside or outside the cell depending on a variety of conditions related to the dynamics of cellular homeostasis, which in turn may be dependent on the number of cigarettes smoked per day. In our opinion, intracellular concentrations of BaP and its concentration in the cervical mucus bathing the extracellular surfaces may in combination determine the effective BaP concentration and its ultimate effect on the viral life cycle.

Our published studies did not reflect a linear dose dependence of HPV genome amplification upon BaP treatment because we reported the results obtained at data points using serial 10-fold decreases in BaP final concentrations (1). However, our unpublished studies did show a bimodal BaP dosage response when a wider range of BaP concentrations was utilized (Fig. 1). Our studies showed that treatment with low concentrations of BaP increased genome amplification, and increased amplification has previously been shown to correlate with increased oncogene expression (5). In addition, high levels of E6/E7 transcript expression have been well correlated with the viral DNA load (10) as well as with poor prognosis in cervical cancer patients (2, 14). On the other side of the spectrum, it is a moot point to argue whether or not the dosage effect of BaP has a linear response on HPV genome amplification, especially since the end point of our assay measured the production of infectious virus, which is a definitive stage of the viral life cycle.

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FIG. 1. Southern blotting and densitometric analysis were performed to determine the effect of BaP treatment on HPV31b genome copies. Five micrograms of total cellular DNA was digested with HindIII, which linearizes the HPV31b genome at nucleotide 2455. Blots were detected with 32P-labeled total HPV31-specific DNA probe generated by random primer extension, followed by autoradiography. Densitometric analysis was then performed with the FIII bands from Southern blots of BaP treatments, and results were compared with those of no-BaP controls, which was set to 1. Results indicate analysis of one representative experiment.

While the Hybrid Capture 2 assays presented by Dr. Castle are informative, they are as yet inconclusive. We would like to respectfully point out that these assays are designed to measure a type of end point which is significantly different from those used in our studies and unsuitable for side-by-side comparisons. The differences are attributed to the types of tissues used in these instances. The Hybrid Capture 2 assays utilized exfoliated cells, and the limitations to using these types of patient samples have been pointed out by Dr. Castle. In comparison, we used raft tissues which were derived from a well-defined homogeneous system and which are amenable to reproducible results over multiple experiments (1).

Our initial studies were started based on the epidemiological studies which first proposed the connection between cigarette smoking and the increased risk of developing cervical cancer (15). Our findings have yielded some unexpected yet exciting data which suggest that cigarette smoke carcinogen exposure increases HPV viral load with respect to both virion synthesis and genome copies. On the other hand, much has been published regarding the current ideas on the positive relationship between persistent HPV infections and viral loads as a possible link in cancer progression (16). Thus, our studies provide the basis for a marriage of the two ideas, in turn giving rise to the novel possibility that exposure to cigarette carcinogens induces robust conditions (virus and/or host cell specific) which positively support persistence of the virus. Our studies provide a foundation for examining the molecular mechanisms by which carcinogenic constituents of cigarette smoke regulate host cellular factors as well as the viral life cycle which together may determine cervical carcinogenesis.

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						Craig Meyers* Samina Alam Michael J. Conway Department of Microbiology and Immunology 500 University Drive Pennsylvania State University College of Medicine Hershey, Pennsylvania 17033
						* Phone: (717) 531-6240. Fax: (717) 531-4600 E-mail: cmm10{at}psu.edu

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Journal of Virology, June 2008, p. 6084-6086, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.00103-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Castle, P. E. Articles by Conway, M. J. Search for Related Content PubMed PubMed Citation Articles by Castle, P. E. Articles by Conway, M. J.