Smokeless tobacco increases aneuploidy in oral HPV16 E6/E7-transformed keratinocytes in vitro.
BACKGROUND
The scope of this work was to study synergism between human papillomavirus (HPV) infection and tobacco in vitro, both known to be independent risk factors for oral cancer.


METHODS
HPV-positive and HPV-negative oral keratinocytes and oral HPV-negative fibroblasts were exposed to smokeless tobacco extract (STE) prepared from the Scandinavian (STE1) and US-type (STE2) snuff. Cell cycle profiles were determined with flow cytometry, and HPV E6/E7 mRNA expression in HPV-positive cells was assayed using RT-qPCR.


RESULTS
The exposure of HPV-positive keratinocytes with STE2 increased the number of aneuploid cells from 27% to 80% of which 44% were in S-phase, while none of the diploid cells were in S-phase. The changes after STE1 exposure were less than seen after STE2: from 27% to 31% of which 34% were in S-phase. STE had no effect on HPV16 E6/E7 expression in HPV-positive keratinocytes. In oral spontaneously transformed, HPV-negative keratinocytes, the number of aneuploid cells at G2-M stage increased after STE1 and STE2 exposure from 3% to 9% and 7%, respectively. In HPV-negative oral fibroblasts, the number of cells at G2-M phase increased from 11% to 21% after STE1 and 29% after STE2 exposure.


CONCLUSIONS
The effect of STE varied in the cell lines studied. STE2 increased significantly the proportion of aneuploid cells in HPV-positive oral keratinocytes, but not HPV16 E6/E7 expression. This indicates that tobacco products may enhance the effects of HPV 16 and the risk of DNA aneuploidy increasing risk to malignant transformation.
Department of Oral Pathology, Institute of Dentistry, Faculty of Medicine and MediCity Research Laboratory, University of Turku, Turku, Finland.
Marina
M
Merne
Jaana
J
Rautava
Merja
M
Ruutu
Stina
S
Syrjänen
D024422:Aneugens / Q000009:adverse effects*; D000782:Aneuploidy; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000502:physiology*; D004171:Diploidy; D005347:Fibroblasts / Q000187:drug effects / Q000821:virology; D016195:G2 Phase / Q000187:drug effects; D005881:Gingiva / Q000166:cytology / Q000821:virology; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000821:virology; D009061:Mouth Mucosa / Q000166:cytology / Q000187:drug effects* / Q000821:virology; D009538:Nicotine / Q000009:adverse effects; D009856:Oncogene Proteins, Viral / Q000032:analysis / Q000187:drug effects; D050725:Papillomavirus E7 Proteins / Q000032:analysis / Q000187:drug effects; D010936:Plant Extracts / Q000009:adverse effects*; D012097:Repressor Proteins / Q000032:analysis / Q000187:drug effects; D016196:S Phase / Q000187:drug effects; D012867:Skin / Q000166:cytology / Q000821:virology; D057891:Tetraploidy; D014030:Tobacco, Smokeless / Q000009:adverse effects*
D024422:Aneugens; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010936:Plant Extracts; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D009538:Nicotine
HPV; aneuploidy; oral cancer; oral mucosa; smokeless tobacco