The Jun kinase 2 isoform is preferentially required for epidermal growth factor-induced transformation of human A549 lung carcinoma cells

We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathw ay in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Best, R McKay, N. Dean, and D. Merc ola, J. Biol. Chem. 272:33422-33429, 1997). Several isoforms families of JN K, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative sp licing of three different genes and have distinct substrate binding propert ies. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been scree ned, and two high-affinity and specific candidates have been identified. An tisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 mu M but did not alter JNK2 mR NA or protein levels. Conversely, antisense JNK2 specifically eliminated JN K2 steady-state mRNA and protein expression with an EC50 of 0.1 mu M. Antis ense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-ind uced total JNK activity, whereas sense and scrambled-sequence control oligo nucleotides had no effect. The elimination of mRNA, protein, and JNK activi ties lasted 48 and 72 h following a single Lipofectin treatment with antise nse JNK1 and JNK2, respectively, indicating sufficient duration for examini ng the impact of specific elimination on the phenotype. Direct proliferatio n assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did . EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment bu t not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but net JNK1 is utilized for mediating the effects of EGF. This study represents the fir st demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.