Our long-term goal is to elucidate the principles by which cAMP controls cell development, proliferation and differentiation. cAMP regulates a striking number of physiologic processes, including intermediary metabolism, cellular proliferation, and neuronal signaling by altering basic patterns of gene expression via activation of cAMP response element (CRE)-directed transcription. The mechanism of the CRE-directed transcription in cell proliferation, however, is largely unexplored. Studies of the CRE-transcription in tumor cells compared to normal cells should contribute to the knowledge base for pharmaceutical design. To elucidate the role of the cAMP enhancer (CRE) in the control of cell proliferation, we used transcription factor-decoy oligonucleotide approach. Studies have shown that synthetic double- stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into target cells as decoy cis-elements to bind the factor and alter gene transcription. Because the CRE cis-element TGACGTCA is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self- hybridizes to form a duplex/hairpin. We investigated if synthetic single-stranded oligonucleotide of the CRE-palindromic and hairpin- forming oligonucleotides could penetrate cells, bind sequence-specific DNA-binding proteins, and interfere with the CRE-directed transcription in vivo. We found that a 24-mer CRE palindrome oligonucleotide (trioctamer of TGACGTCA) effectively competed with the cis-element for binding transcription factors and inhibited the gene transcription in a wide variety of cell types. Our finding that the CRE oligonucleotide inhibits the basal expression of RIa and Ca genes strongly indicates that the CRE oligonucleotide can indeed compete with the cis-CRE element in binding CREB. Because the CRE oligonucleotide can interfere with CREB binding to the CRE enhancer, it is expected that the oligonucleotide could produce a more profound effect on the mRNA reduction under cAMP-induced conditions. Thus, the CRE oligonucleotide can interfere with both the basal and cAMP-induced expression of the endogenous CRE-containing genes. CREB is known to associate with (e.g., heterodimerize) a variety of other transcription factors (e.g., members of the Jun/Fos family) and c-fos is cAMP-inducible. These findings demonstrate the cross-talk between CRE and AP-1. We found that the CRE- palindromic-oligonucleotide specifically interferes with both the CRE- and AP-1 directed transcription in vivo. Surprigingly, the CRE-decoy oligonucleotide restrained tumor cell proliferation without affecting the growth of non-cancerous cells. The dual blockage of two important signal transduction pathways, CRE-PKA and AP-1-PKC, could be causally related, at least in part, to the cancer cell specific growth inhibitory effect of the CRE decoy oligonucleotide.10268-03 LTIB is incorporated into this project. - Transcription, cyclic AMP response element, Transcription factor-decoy oligonucleotide, CRE and AP-1 Cross talk, cAMP and EGF Cross talk, Inhibition of tumor growth in vivo,
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