FIGURE 3.

H1650 human lung adenocarcinoma cells are PTEN-deficient and express high leptin and leptin receptor mRNA. Comparison of different lung cancer cell lines reveals H1650 cells are PTEN-deficient (A) and express the highest levels of LEP and its receptor LEPR mRNA (B). C, leptin and leptin receptor protein levels as a result of PTEN deletion as assessed in H1650 cells, suggesting the likely presence of a functional LEP signaling pathway in lung epithelial cells. D, dose-dependent increase in H1650 cell proliferation (∼3-fold) was observed when cells were treated for 48 h with increasing concentrations (50, 100, and 200 ng/ml; lane 2–4) of human recombinant LEP. E, continuous impedance sensing measurements identify greater wound closure efficiency in cells treated with 100 ng/ml leptin prior to wounding as compared with control cells without leptin. F, identification of the LEP gene core promoter region in H1650 lung cancer cell line revealed a proximal enhancer containing NRF-1 and δ-binding sites. Subconfluent cultures were transiently transfected with various promoter-reporter deletion constructs derived from 5′-upstream regulatory sequence of the LEP gene. Luciferase activity was expressed relative to the base-line luciferase activity of a promoter-less luciferase reporter construct (pGL3-Basic) set to unity. Data are represented from four independent experiments performed in triplicate (± S.E.; *, p value ≤ 0.05). G, diagrammatic representation of the most active promoter region −149 to +21 bp of the LEP gene (Luc-150), including proximal enhancers depicting the positions of NRF-1 and C/EBP sites.