CHARACTERIZATION OF GENES WHICH EXHIBIT REDUCED EXPRESSION DURING THERETINOIC ACID-INDUCED DIFFERENTIATION OF F9 TERATOCARCINOMA CELLS - INVOLVEMENT OF CYCLIN D3 IN RA-MEDIATED GROWTH ARREST

In the presence of retinoic acid (RA), F9 murine teratocarcinoma cells differentiate into cells resembling the extra-embryonic endoderm of t he early mouse embryo. Using differential hybridization, we have clone d and characterized six cDNAs corresponding to mRNAs that exhibit redu ced expression in F9 cells following RA treatment. Two of these cDNAs encode novel genes (REX-2 and REX-3). The other isolated cDNAs encode genes that have been previously described in other contexts: 1-4 (cycl in D3); 2-10 (pyruvate kinase); 2-12 (glutathione S-transferase); and 2-17 (GLUT 3). The mRNA levels of these genes are reduced by RA or RA plus theophylline and cAMP (RACT) only after 48 h of treatment, and co ntinue to decrease at 96 h. The half-lives of these mRNAs are not chan ged by RA treatment, indicating that these mRNAs may be regulated thro ugh a transcriptional mechanism. In isoleucine-deprived cells, which a re growth arrested but do not differentiate, the steady state mRNA lev els of genes Rex 2, Rex 3, pyruvate kinase and GLUT 3 are not reduced, in contrast to cyclin D3 and glutathione S-transferase. The expressio n of the REX-2, REX-3, pyruvate kinase, glutathione S-transferase and GLUT 3 genes is reduced by RACT to the same extent in F9 RAR gamma-/- and RAR alpha-/- lines as in F9-Wt. In contrast, cyclin D3 exhibits lo wer mRNA expression in F9 RAR gamma-/- and RAR alpha-/- stem cells, an d this mRNA is not decreased by RACT treatment. Overexpression of cycl in D3 blocks the RA-induced growth arrest of F9 cells, indicating that the downregulation of this gene following RA treatment may constitute a necessary step in the cascade of events leading to growth inhibitio n by RA. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.