ABSTRACT
Telomeres of human chromosomes contain a G-rich 3'-overhang that adopts an intramolecular G-quadruplex structure in vitro which blocks the catalytic reaction of telomerase. Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalyzed by telomerase and can therefore act as antitumor agents. We have identified by Fluorescence Resonance Energy Transfer a new series of quinoline-based G-quadruplex ligands that also exhibit potent and specific anti-telomerase activity with IC50 in the nanomolar concentration range. Long term treatment of tumor cells at subapoptotic dosage induces a delayed growth arrest that depends on the initial telomere length. This growth arrest is associated with telomere erosion and the appearance of the senescent cell phenotype (large size and expression of beta-galactosidase activity). Our data show that a G-quadruplex interacting agent is able to impair telomerase function in a tumor cell thus providing a basis for the development of new anticancer agents.
Subject(s)
Apoptosis , DNA , Telomere/drug effects , Triazines/pharmacology , Cell Line, Transformed , Cellular Senescence , G-Quadruplexes , Humans , Ligands , Molecular Structure , Telomerase/metabolism , Triazines/chemistry , Tumor Cells, CulturedABSTRACT
The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC(50) values of 18-100 nM in a standard TRAP assay.
Subject(s)
DNA/chemistry , Ethidium/chemistry , Nucleic Acid Conformation , Telomerase/antagonists & inhibitors , DNA/genetics , Fluorescent Dyes/chemistry , Guanine/chemistry , Humans , Oligonucleotides/chemistry , Oligonucleotides/genetics , Spectrometry, Fluorescence , Telomerase/genetics , Telomerase/metabolism , Telomere/enzymology , Telomere/geneticsABSTRACT
We examined the expression of the genes encoding topoisomerases I and II and those associated with V(D)J [variable(diversity)joining] recombination in two human T-cell acute lymphoblastic leukemia (T.ALL) cell lines, CEM and CEM/DOX. In CEM/DOX cells, which are resistant to doxorubicin, 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 found to be 2.5-increased in the presence of topoisomerase I extracted from CEM/DOX, in comparison to that in CEM cells. Conversely, the topoisomerase II mRNA levels, nuclear decatenation activities and (mAMSA) 4'(9-acridinylamino)methanesulfon-m-anisidide-induced cleavable complex formation 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 resistant to camptothecin than were CEM cells, and cross-resistance to camptothecin was not reversed by verapamil. Furthermore, using an intact cell assay for DNA-protein complexes, we found that camptothecin-stimulated cleavable complexes formed in CEM/DOX cells were increased in correlation with the elevated topoisomerase I activity. These results suggest that camptothecin resistance in CEM/DOX cells is due to different mechanism(s) than topoisomerase- or P-glycoprotein-associated multidrug 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 gene transcripts, other components of the V(D)J complex, were at best poorly 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 overexpressed. The mechanism of the RAG1 gene activation was not gene amplification, and no rearrangement was detected in the RAG1 gene locus. RAG1 presents homology with the yeast gene HPR1, itself homologous to yeast topoisomerase I and responsible for the control of recombination in somatic cells. Since DNA topoisomerases are themselves involved in the control of DNA topology, recombination and DNA repair, the possible coactivation of RAG1 and topoisomerase I genes in CEM/DOX cells is discussed.