Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates

Abstract

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

FIG. 1.

(A) Sequences of the TFOs and of the duplex containing the 16-bp (underlined) triplex target site used in this study. The experimental construction for cell experiments is shown: an insert of 54 bp, containing the original triplex target (plasmid pWT), was cloned within the 5′ transcribed but untranslated region of the firefly luciferase gene (luc), under the control of the SV40 promoter, between the HindIII and NcoI sites of vector pGL3Pr. The 54-bp region around the triplex site is shown. The region on the 3′ side of the triple helix, i.e., the 3′ side of the oligopurine strand of the duplex, contains two Topo I-mediated cleavage sites, b and c, observed in the presence of free CPT. All Topo I cleavage sites observed in vitro are indicated by letters. M, 5-methyl-2′-deoxycytidine; P, 5-propynyl-2′-deoxyuridine. The 16-nt sequence of the HIV-CPT(10) conjugate used as a control is shown. (B) Structures of the CPT conjugate derivatives used in this study.

FIG. 2.

Cellular activities of TFO-CPT conjugates. HeLa cell lysates were assayed for firefly and Renilla luciferase activities after transfection by Superfect. The firefly luciferase construct was introduced with the control pTK-RL vector in the absence (black bars) or presence of TFO-NPh2, TFO-CPT(10), or HIV-CPT(10) at 0.05 μM (hatched bars), 0.1 μM (white bars), or 0.5 μM (dotted bars). The results presented are the mean firefly/Renilla luciferase luminescence intensity ratios of triplicates (relative luciferase activity) and are normalized to the value obtained in the absence of the oligonucleotides (none). (A) pWT construct at 24 h, 48 h, and 72 h after transfection. (B) pGL3Pr control vector at 24 h, 48 h, and 72 h after transfection. Conjugates TFO-CPT(10) and TFO-CPT(7) gave equivalent results.

FIG. 3.

Stability of TFO-P and conjugate TFO-CPT(10) in cell medium. The 5′-end-radiolabeled oligonucleotides (left panel) were incubated in cell culture medium (DMEM containing 10% fetal bovine serum) at 37°C. Aliquots were removed after 24 h (central panel) or 72 h (right panel) and analyzed on a 20% denaturing gel.

FIG. 4.

Immunoblot analysis of Topo I-DNA covalent complexes stabilized by Topo I inhibitor in HeLa cell nuclei. HeLa cell nuclei (5 × 10⁶) were incubated for 3 h at 37°C in the presence or absence of compounds (5 μM). The lysates obtained were applied to a CsCl gradient and centrifuged overnight. Twelve fractions were collected and analyzed by slot blot assay. Fractions 1 to 4 contained free Topo I. Topo I/DNA covalent complexes were detected in fractions 8 to 11. Results are representative of three independent experiments.

FIG. 5.

Modified duplexes are derived from duplex WT by mutations (in bold) at the triplex site (MUT) or at cleavage sites b and c, replaced by either no cleavage site (B2) or by a known Topo I cleavage site, B (TID).

FIG. 6.

Sequence analysis of the Topo I-mediated cleavage products on the original 324-bp target duplexes (WT), the 324-bp duplex mutated at the triplex site (MUT), and two 324-bp duplexes mutated at sites b and c (B2 and TID). (A) WT: target duplex (lane 1) and duplex incubated with Topo I (lane 2) in the presence of 5 μM cCPT (lane 3), 0.5 μM TFO-CPT(10) (lane 4), or 0.5 μM TFO-CPT(7) (lane 5). (B) MUT: duplex mutated at the triplex site (lane 1) and duplex incubated with Topo I (lane 2) in the presence of 5 μM cCPT (lane 3), 0.5 μM TFO-CPT(10) (lane 4), 0.5 μM TFO-CPT(7) (lane 5), or 1 μM MGB-CPT(10) (lane 6). (C) B2: duplex lacking cleavage sites b and c (lane 3), duplex incubated with DNase I in the absence (lane 1) or presence of 0.5 μM TFO (lane 2), and duplex incubated with Topo I (lane 4) in the presence of 5 μM cCPT (lane 5), 0.5 μM TFO-CPT(10) (lanes 6), or 0.5 μM TFO-CPT(7) (lane 7). (D) TID: duplex containing a known CPT cleavage site, B (lane 1), and duplex incubated with Topo I (lane 2) in the presence of 5 μM cCPT (lane 3) or 7CPT (lane 4), 0.5 μM TFO-CPT(10) (lanes 5), or 0.5 μM TFO-CPT(7) (lane 6). Adenine/guanine-specific Maxam-Gilbert chemical cleavage reactions were used as markers. The positions of the cleavage sites are indicated (sites a to f). The region corresponding to the triplex site is indicated (TH), as is its orientation. nt, nucleotides.

FIG. 7.

Quantification of the intensity of cleavage for each target in the presence of CPT (gray bars) at 5 μM, conjugate TFO-CPT(10) (hatched bars) at 0.5 μM, and MGB-CPT(10) (black bars) at 1 μM as described in Materials and Methods. Four independent experiments were analyzed. TFO-CPT(7) behaves the same as TFO-CPT(10), and its data were omitted for simplicity. a.u., arbitrary units.

FIG. 8.

Cellular activity of the conjugates on pB2 at 24 h after transfection. The firefly luciferase construct was introduced with the control pTK-RL vector in the absence (black bars) or presence of TFO-NPh2, TFO-CPT(10), or HIV-CPT(10) at 0.05 μM (hatched bars), 0.1 μM (white bars), or 0.5 μM (dotted bars). The relative luciferase activity reported is normalized to that of cells in the absence of oligonucleotides (none). The experiments were run in triplicate and repeated 3 to 10 times, and the error bars show standard error calculations. Conditions were the same as in Fig. 2.