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Differences between Drug-sensitive and -resistant Human Leukemic CEM Cells in c-jun Expression, AP-1 DNA-binding Activity, and Formation of Jun/Fos Family Dimers, and Their Association with Internucleosomal DNA Ladders after Treatment with VM-26¹

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [R. K., W. T. B.], and Department of Pharmacology, University of Tennessee, Memphis, Tennessee 38163 [W. T. B.]

Although the DNA topoisomerases are critical intracellular targets of a number of clinically important anticancer drugs, the mechanism(s) by which inhibition of these enzymes causes cell death are poorly understood. We found that treatment of human leukemic lymphoblasts (CCRF-CEM) with teniposide (VM-26), under conditions that stabilize DNA-topoisomerase II complexes, caused the formation of internucleosomal DNA ladders. However, it appeared unlikely that the VM-26-stabilized DNA-topoisomerase II-cleavable complexes directly produce these internucleosomal DNA ladders, since similar nucleosomal DNA ladders were observed following either continuous or a short (1 h) exposure of cells to VM-26. Under continuous exposure to VM-26, the internucleosomal DNA ladders were associated with the transient induction of c-jun mRNA in a dose-dependent fashion, reaching maximum expression at 6 h after treatment with VM-26 and being down-regulated to basal levels by 12 h. The induction of c-jun mRNA by VM-26 apparently preceded DNA ladder formation. However, in CEM sublines selected for resistance to VM-26 (CEEM/VM-1 and CEM/VM-1-5; 50- and 140-fold resistant, respectively) and which display the phenotype of multidrug resistance associated with altered DNA topoisomerase II (at-MDR), we found that the induction of c-jun mRNA by VM-26 and subsequent DNA ladder formation were progressively attenuated in proportion to the resistance of the cells, apparently due in part to decreased stabilization of DNA-topoisomerase II-cleavable complexes. Further, the attenuated induction of c-jun in the at-MDR cells was found to be associated with a decreased rate of c-jun transcription and an increase in the instability of its mRNA following VM-26 treatment. The attenuation of c-jun mRNA induction was also reflected in decreased production of c-Jun protein in the at-MDR cells. Of interest was the fact that no significant induction of c-fos mRNA by VM-26 was observed in either CEM or at-MDR cells. Furthermore, the induction of c-jun was related to the activation of AP-1 DNA-binding activity in a time- and dose-dependent manner in CEM cells, whereas the activation of AP-1 binding was attenuated in at-MDR cells in proportion to their resistance to VM-26. Using Jun and Fos family member antibody inhibition experiments in gel-mobility shift assays, we found that AP-1-binding activity appeared to be preferentially mediated by c-Jun/Fra-1 heterodimers in both CEM and at-MDR cells. However, unlike CEM cells, the Fra-2 protein was also involved in the increased AP-1 complexes formed with toxic doses of VM-26 in the at-MDR cells. Collectively, these results suggest that attenuated DNA-protein complex formation after VM-26 administration is associated with attenuation of c-jun gene induction and AP-1-binding activity in the at-MDR cells, and reflects the decreased susceptibility of these cells for cell death in response to DNA damage by VM-26. Our results further suggest that VM-26-mediated signal transduction pathways in the at-MDR cells may differ from those of CEM cells in the activation of different target genes through AP-1 sites, and these genes may play a role in the expression of resistance to VM-26 in the at-MDR cells.

¹ This work was supported in part by National Cancer Institute Research Grant CA 40570, Program Grant CA 23099, and Cancer Center Support Grant CA 21765, and in part by American Lebanese Syrian Associated Charities.

² To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, Saint Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38101-0318.

Received 4/29/94. Accepted 7/20/94.

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