Activation of the NF-kappaB p50/p65 Complex in Human Lung Cancer Cell Lines

Choi, Hyung Seok; Yoo, Chul Gyu; Lee, Choon Taek; Kim, Young Whan; Han, Sung Koo; Shim, Young Soo

Abstract

BACKGROUND: NF-kappaB is a characteristic transcriptional factor whose functional activity is determined by post- translational modification of protein and subsequent change of subcellular localization. The involvement of the NF- kappaB family of the transcription factors in the control of such vital cellular functions as immune response, acute phase reaction, replication of certain viruses and development and differentiation of cells has been clearly documented in many previous studies. Several recent observations have suggested that the NF-kappaB might also be involved in the carcinogenesis of some hematological and solid tumors. Investigating the possibility that members of the NF- kappaB family participate in the molecular control of malignant cell transformation could provide invaluable information on both molecular pathogenesis and cancer-related gene therapy. METHOD: To determine the expression patterns and functional roles of NF-kappaB family transcription factors in human lung cancer cell lines NCI -H792, NCI-H709, NCI-H226 and NCI-H157 were analys ed by western blot, using their respective antibodies. The nuclear and the cytoplasmic fraction of protein extract of these cell lines were subsequently obtained and NF- kappaB expression in each fraction was again determined by western blot analysis. The type of NF-kappaB complex present in the cells was determined by immunoprecipitation. To detect the binding ability of cell- line nuclear extracts to the kappaB consensus oligonucleotide, electrophoretic mobility shift assay(EMSA) was performed.
RESULTS
In the cultured human lung cancer cell lines tested, transcription factors of the NF- kappaB family, namely the p50 and p65 subunit were expressed and localized in the nuclear fraction of the cellular extract by western blot analysis and immunocytochemistry. Immunoprecipitation assay showed that in the cell, the p50 and p65 subunits made NF- kappaB complex. Finally it was shown by Electrophoretic Mobility Shift Assay(EMSA) that nuclear extracts of lung cancer cell lines are able to bind to NF-kappaB consensus DNA sequences.
CONCLUSION
These data suggest that in human lung cancer cell lines the NF-kappaB p50/p65 complex might be activated, and strengthen the hypothesis that NF-kappaB family transcription factors might be involved in the carcinogenesis of human lung cancer.