THE EFFECTS OF RADIATION ON THE EXPRESSION OF A NEWLY CLONED AND CHARACTERIZED RAT CYCLIN-B MESSENGER-RNA

Purpose: To evaluate the hypothesis that the radiation induced G2 dela y in the cell cycle is associated with radiation induced effects on cy clin B expression in a rodent cell system. Methods and Materials: Two rodent, rat and Chinese hamster, cyclin B cDNAs were cloned and charac terized. The two rodent species were 85% and 86% identical, respective ly, when compared to the human cyclin B, indicating that they are the rodent homologues of cyclin B. 3.7 cells (rat embryo cells transformed by H-ras and v-myc) were synchronized and then irradiated. Flow cytom etry and Northern blots were performed to evaluate the effects of radi ation on cyclin expression in relation to phase of the cell cycle. Res ults: Examination of the rodent cyclin B sequences revealed only two r egions with significant divergence to the human sequence, one in the l ysine rich region adjacent to the cyclin destruction box, which is the putative site for ubiquitination, and one at the C terminal end. Alth ough many of the amino acids diverged in the lysine rich region, the p ositions of the lysines themselves were virtually invariant suggesting their potential importance in ubiquitination. Both rodent species wer e also noted to have a PEST-like sequence which occurs in the human, b ut not in nonmammalian cyclins cloned to date and could also potential ly contribute to rapid destruction. The rat and Chinese hamster mRNAs contain much longer 3' untranslated regions than the published human s equence with multiple AUUUA and AUUU motifs which are seen in other mR NAs with rapid turnover times. This feature has not been previously fo und in cyclin mRNAs. In addition we have found that in the 3' region o f the rodent cDNAs we find two potential polyadenylation sites suggest ing that this gene may have several transcripts. Our studies suggest t hat multiple mechanisms of control of mammalian cyclin B destruction e xist, both at the mRNA and protein level. Evidence is also provided th at the levels of rat cyclin B mRNA peaks during G2/M. Irradiation is s hown to induce a G2 delay in synchronized 3.7 cells, compared to unirr adiated controls, and the delay is temporally related to decreased lev els of cyclin B mRNA expression. Since the G2 delay induced by ionizin g radiation may contribute to the ability of cells to survive irradiat ion, cyclin B expression may be a key component in the determination o f sensitivity or resistance to radiation therapy. Conclusion: The isol ation and characterization of two rodent cyclin B's confirm that multi ple mechanisms of control of mammalian cyclin B destruction exist. Our studies show that rat cyclin B expression is influenced by radiation and is temporally related to the delay in the G2 phase induced by radi ation.