4-(Methylnitrosamino)-1-(3-pyridy1)-1-butanone　(NNK)　is　an　important　tobacco-specific　nitrosamine　(TSNA)　that　induces　malignant　tumors　in　rodents.　High-risk　human　papillomavirus　(hr-HPV)　infection　is　an　important　cause　of　several　human　cancers.　Epidemiological　evidence　has　shown　that　HPV　cooperatively　induces　carcinogenesis　with　tobacco　smoke.　In　the　present　study,　the　synergistic　carcinogenesis　of　NNK　and　HPV18　was　investigated.　Immortalized　human　esophageal　epithelial　SHEE　cells　containing　the　HPV18　E6E7　gene　were　constructed　by　lentiviral　transfection.　SHEE-E6E7　cells　were　exposed　to　NNK　along　with　SHEE-V　cells　without　HPV18　E6E7　as　a　negative　control.　The　cooperation　of　NNK　and　HPV　was　examined　by　wound-healing,　transwell,　and　colony-forming　assays.　The　results　showed　that　NNK　exposure　promoted　the　migration,　invasion,　and　proliferation　abilities　of　both　SHEE-E6E7　and　SHEE-V　cells;　however,　the　changes　in　these　phenotypic　features　were　remarkably　stronger　in　SHEE-E6E7　cells　than　those　in　SHEE-V　cells.　Our　findings　indicate　that　NNK　promotes　malignant　transformation　of　human　esophageal　epithelial　cells　and　suggest　a　synergistic　carcinogenesis　with　the　HPV18　E6E7　oncogene.　As　reported　previously,　the　formation　of　pyridyloxybutylated　DNA　adducts　is　a　crucial　step　in　NNK-mediated　carcinogenesis.　In　order　to　clarify　the　influence　of　HPV　on　the　formation　of　NNK-induced　DNA　adducts,　the　amounts　of　4-hydroxy-1-(3-pyridyl)-1-butanone　(HPB)-releasing　DNA　adducts　were　determined　using　high-performance　liquid　chromatography-electrospray　ionization-tandem　mass　spectrometry.　We　observed　that　the　levels　of　HPB-releasing　adducts　in　SHEE-E6E7　cells　were　significantly　higher　(p　＜　0.01)　than　those　of　SHEE-V　cells,　which　was　in　line　with　results　of　the　phenotypic　assays.　In　conclusion,　this　study　provides　direct　evidence　that　NNK　and　HPV18　exhibit　a　synergistic　effect　on　formation　of　DNA　adducts,　resulting　in　malignant　transformation　of　esophageal　epithelial　cells.　Such　knowledge　on　the　interaction　between　infection　and　smoking　habits　in　the　development　of　cancers　informs　cancer-prevention　strategies.　Further　studies　to　delineate　the　molecular　mechanism　and　to　identify　specific　intervention　targets　are　worthwhile.