A bombesin receptor subtype-3 peptide increases nuclear oncogene expression in a MEK-1 dependent manner in human lung cancer cells

A synthetic peptide, (D-Phe(6), beta -Ala(11) Phe(13), Nle(14))bombesin-(6- 14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transf ected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta -Ala(11), Ph e(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta -Ala(11), Phe(13), Nle(14))bomb esin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein ki nase phosphorylation caused by (D-Phe(6), beta -Ala(11), Phe(13), Nle(14))b ombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mi togen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta -Ala(11), Phe(13), Nle(14))bombes in-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 ac tivation caused by (D-Phe(6), beta -Ala(11), Phe(13), Nle(14))bombesin-(6-1 4) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6) , P-Ala(11), Phe(13), Nle(14))bombesin-(6- 14) caused increased c-fos as we ll as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold incr ease in c-fos mRNA caused by 100 nM (D-Phe(6), beta -Ala(11), Phe(13), Nle( 14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), pro(26), pS i(26,27),Phe(27))GRP(20-27) (BW2258U89), a GRP receptor antagonist. These r esults indicate that (D-Phe(6), beta -Ala(11), Phe(13), Nle(14))bombesin-(6 -14) caused increased nuclear oncogene expression and upstream events inclu de mitogen activated protein kinase phosphorylation and Elk-1 activation. ( C) 2001 Published by Elsevier Science B.V.