Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation.
Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.
Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Jinjin
J
Guo
Michelle
M
Chu
Tetteh
T
Abbeyquaye
Chang-Yan
CY
Chen
D000362:Agar / Q000737:chemistry; D000818:Animals; D015139:Blotting, Southern; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D004249:DNA Damage; D004351:Drug Resistance; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D016193:G1 Phase; D005731:Ganglionic Stimulants / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D008168:Lung / Q000166:cytology* / Q000187:drug effects; D008727:Methotrexate / Q000494:pharmacology; D009538:Nicotine / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D013936:Thymidine / Q000737:chemistry; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D005731:Ganglionic Stimulants; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D009538:Nicotine; D000362:Agar; D006861:Hydrogen Peroxide; D013762:Tetrahydrofolate Dehydrogenase; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D018631:ras Proteins; D013936:Thymidine; D008727:Methotrexate