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Northern analysis of the apoptosis-associated genes Gadd34 and Gadd45 verified their higher expression in JNK2AS-treated cells (Fig. 4C)Citation , and the mRNA levels of Gadd153, another gene whose expression is often up-regulated by conditions that induce Gadd34 and Gadd45, were also elevated. The induction of the Gadd genes suggested that JNK2AS-treated cells were under stress and prompted us to examine the expression of two other stress-regulated genes: (a) Grp78, a molecular chaperone involved in protein folding; and (b) the cyclin-dependent kinase inhibitor p21Cip1/Waf1, which we had found previously to be up-regulated in other JNK2AS-treated cells (17 , 18) . Both genes were markedly up-regulated in JNK2AS-treated cells (Fig. 4C)Citation , suggesting that depletion of JNK2 in PC3 cells facilitates the induction of several independent stress-signaling pathways. Activation of the JNK pathway is important for the phosphorylation and activation of protein components of AP-1 transcription factor complexes (1, 2, 3, 4 , 5, 6, 7 , 28) , suggesting that genes regulated by AP-1-dependent transcription could be negatively affected by JNKAS treatment. Indeed, we detected reduced expression of several AP-1-regulated genes, including lamin A-C (29) , and integrin b-4 (Ref. 30 ; Fig. 4DCitation ). Among other genes whose lower expression after JNK2AS treatment was verified by Northern blotting were HMG-I(Y), a protein highly expressed in prostate cancer (31) and recently identified as a myc-regulated oncogene (32) , Lsm5 (33) , transcobalamin, and DSS1 (Ref. 34 ; Fig. 4Citation D, and data not shown). Importantly, although most JNK2AS-altered genes appeared to be specific for treatment with this oligonucleotide, some gene expression changes were also seen in the JNK1AS- and JNKScr-treated groups, indicating that a more global response to the uptake of oligonucleotides (Fig. 4E)Citation was elicited. Within this group were two IFN-inducible genes, ISGF-3 and ISG15, suggesting that application of phosphorothioate oligonucleotides could induce IFN-dependent responses.
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