ALTERATIONS IN THE CYCLIC-AMP SIGNAL-TRANSDUCTION PATHWAY REGULATING RIBONUCLEOTIDE REDUCTASE GENE-EXPRESSION IN MALIGNANT H-RAS TRANSFORMED-CELL LINES

Ribonucleotide reductase is a highly regulated activity responsible fo r reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways impo rtant in controlling the expression of the two genes that code for rib onucleotide reductase, R1 and R2. A series of radiation and H-ras tran sformed mouse 10T1/2 cell lines with increasing malignant potential we re exposed to stimulators of cAMP synthesis (forskolin and cholera tox in), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) an d a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elev ations in the expression of the R1 and R2 genes at the message and pro tein levels were observed in malignant metastatic populations, which w ere not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase g ene expression occurred without any detectable modifications in the ra tes of DNA synthesis, showing that they were regulated by a novel mech anism independent of the S phase of the cell cycle. Furthermore, studi es with forskolin (a stimulator of the protein kinase A signal pathway ) and the tumor promoter 12-0-tetradecanoylphorbol-1 3-acetate (a stim ulator of the protein kinase C signal pathway), alone or in combinatio n, indicated that their effects on R1 and R2 gene expression in a high ly malignant cell line were greater than when they were tested individ ually, suggesting that the two pathways modulating R1 and R2 gene expr ession can cooperate to regulate ribonucleotide reduction, and interes tingly this can occur in a synergistic fashion. Also, a direct relatio nship between H-ras expression and ribonucleotide reductase gene expre ssion was observed; analysis of forskolin mediated elevations in R1 an d R2 message levels closely correlated with the levels of H-ras expres sion in the various cell lines. In total, these studies demonstrate th at ribonucleotide reductase expression is controlled by a complex proc ess, and malignant ras transformed cells contain alterations in the re gulation of signal transduction pathways that lead to novel modificati ons in ribonucleotide reductase gene expression. This signal mechanism , which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase e xpression in a S phase independent manner during periods of DNA repair . (C) 1994 Wiley-Liss, Inc.