Dissecting NF-κB signaling induced by genotoxic agents via genetic complementation of NEMO-deficient 1.3E2 cells.
The transcription factor NF-κB regulates expression of a diverse set of genes to modulate multiple biological and pathological processes. Among these, NF-κB activation in response to genotoxic agents has received considerable attention due to its role in regulating cancer cell resistance to chemo- and radiation therapy. Furthermore, induction of this pathway by endogenous damage is further implicated in normal developmental processes, such as B cell development, and premature aging, among others. This pathway also serves as a signaling model in which nuclear initiated signals (DNA damage) are communicated to a cytoplasmic target (IκB kinase and NF-κB). Several of the critical molecular events of this nuclear to cytoplasmic NF-κB signaling cascade were discovered, in part, by genetic complementation analyses of the NEMO-deficient 1.3E2 mouse pre-B cell line. This chapter describes methods used to generate and analyze such reconstitution cell systems and certain caveats that are critical for proper interpretation of NEMO mutant defects.
Medical Scientist Training Program, Cellular and Molecular Biology Program, McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI, 53705, USA.
Shawn S
SS
Jackson
Shigeki
S
Miyamoto
D000818:Animals; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D004249:DNA Damage; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005816:Genetic Complementation Test; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency*; D051379:Mice; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
D047908:Intracellular Signaling Peptides and Proteins; D009153:Mutagens; C514981:NEMO protein, mouse; D016328:NF-kappa B