ALTERED TOPOISOMERASE-I ACTIVITY AND RECOMBINATION ACTIVATING GENE-EXPRESSION IN A HUMAN LEUKEMIA-CELL LINE RESISTANT TO DOXORUBICIN

We examined the expression of the genes encoding topoisomerases I and II and those associated with V(D)J [variable(diversity)joining] recomb ination in two human T-cell acute lymphoblastic leukemia (T.ALL) cell lines, CEM and CEM/DOX. In CEM/DOX cells, which are resistant to doxor ubicin, the topoisomerase I gene was found to be 4-fold overexpressed and nuclear topoisomerase I relaxation activity was 2-fold greater in CEM/DOX than in CEM cells. Furthermore, the cleavable complex reaction induced by camptothecin, a specific topoisomerase I inhibitor, was fo und to be 2.5-increased in the presence of topoisomerase I extracted f rom CEM/DOX, in comparison to that in CEM cells. Conversely, the topoi somerase II mRNA levels, nuclear decatenation activities and (mAMSA) 9 -acridinylamino)methanesulfon-m-anisidide-induced cleavable complex fo rmation in CEM/DOX were similar to those of the doxorubicin-sensitive cells. The results indicate that topoisomerase I activity is elevated in CEM/DOX cells. Nevertheless, CEM/DOX cells were 11-fold more resist ant to camptothecin than were CEM cells, and cross-resistance to campt othecin was not reversed by verapamil. Furthermore, using an intact ce ll assay for DNA-protein complexes, we found that camptothecin-stimula ted cleavable complexes formed in CEM/DOX cells were increased in corr elation with the elevated topoisomerase I activity. These results sugg est that camptothecin resistance in CEM/DOX cells is due to different mechanism(s) than topoisomerase- or P-glycoprotein-associated multidru g resistance. The recombination activating gene, RAG1, which is one of the components of the site-specific V(D)J recombination complex, was 20-fold overexpressed in CEM/DOX cells. In contrast, RAG2 and T160 gen e transcripts, other components of the V(D)J complex, were at best poo rly detected in both sensitive and resistant cells. No specific V(D)J recombinase activity was found in CEM or CEM/DOX cells when the pJH201 transfection assay was used. The results indicate that CEM/DOX cells failed to generate V(D)J recombination although RAG1 gene is overexpre ssed. The mechanism of the RAG1 gene activation was not gene amplifica tion, and no rearrangement was detected in the RAG1 gene locus. RAG1 p resents homology with the yeast gene HPR1, itself homologous to yeast topoisomerase I and responsible for the control of recombination in so matic cells. Since DNA topoisomerases are themselves involved in the c ontrol of DNA topology, recombination and DNA repair, the possible coa ctivation of RAG1 and topoisomerase I genes in CEM/DOX cells is discus sed.