Induction of topoisomerase I cleavage complexes by 1-beta -D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells

Abstract

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4- to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the +1 position (immediately 3') relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5'-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388/CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.

Figure 1

Enhancement of top1 cleavage complexes by ara-C incorporation at the +1 position of a top1 cleavage site. (A) Structures of deoxycytidine (C) and ara-C. (B) Modified Tetrahymena hexadecameric rDNA sequence with a strong top1 cleavage site (10, 31) indicated by the arrowhead was labeled with [³²P]cordycepin (*A) at the 3′ terminus of the scissile (upper) strand. Top1 cleavage yields a 23-mer product. Oligonucleotides were synthesized with either C or ara-C at the indicated positions. (C) Enhancement of top1 cleavage by ara-C at the +1 but not at the +2 position of the nonscissile strand. Oligonucleotides with the indicated base pair at the +1 or +2 position relative to the top1 cleavage site are shown above lanes. (D) Enhancement of top1 cleavage by ara-C at the +1 position of the scissile strand. For each oligonucleotide used in C and D: Lanes A, DNA alone; lanes B and C, + top1; lanes D and E, + top1 + 10 μM CPT. Reactions were performed at 25°C for 15 min and stopped either immediately with 0.5% SDS (lanes B and D) or were first incubated with 0.5 M NaCl (final concentration) for an additional 30 min at 25°C before addition of 0.5% SDS (lanes C and E).

Figure 2

Ara-C incorporation at the +1 position of a top1 cleavage site inhibits the religation of top1 cleavage complexes. (A) Ara-C inhibits the religation of top1 cleavage complexes. Acceptors were generated from substrates shown in C (see text for details). The unlabeled acceptors were then incubated with 10-fold excess of 3′-labeled, 23-mer donor strand for 5 s, 10 s, 0.5 min, 1 min, 3 min, 5 min, 10 min, or 30 min (lanes 1–8, respectively), and reactions were stopped with 0.5% SDS. (B) Quantitation of the gel shown in A. (C) Kinetics of top1-induced DNA cleavage. Oligonucleotides containing either C or ara-C at the +1 position were incubated with top1 for 5 s, 10 s, 0.5 min, 1 min, 3 min, 5 min, 10 min, or 30 min (lanes 1–8, respectively), and reactions were stopped with 0.5% SDS. (D) Effects of Ara-C incorporation on noncovalent top1 binding to DNA. Oligonucleotides with C or ara-C at the +1 position of the nonscissile strand (see Fig. 1B) were incubated with 0, 25, 50, 100, or 250 ng of purified human top1Y727Fp for 5 min at 25°C (lanes 1–5, respectively), and electromobility shift assay was performed. (A and C) Lanes a represent the DNA alone.

Figure 3

Top1 cleavage complexes in human leukemia CEM cells treated with ara-C. Approximately 10⁶ cells were treated with 10 μM ara-C for 15 h or with 1 μM CPT or 100 μM VP-16 for 1 h at 37°C. Cells were then lysed with 1% sarkosyl, and subjected to the ICE bioassay (see text for details). Cesium chloride fractions are indicated by numbers 1–20. (A) Top1 immunoblotting. (B) DNA staining of fractions 6–12 with ethidium bromide after electrophoresis. (C) Top2 immunoblotting. Presence of covalent topoisomerase cleavage complexes is indicated by the brackets.

Figure 4

Ara-C incorporation produces top1 cleavage complexes in human leukemia CEM cells. Detection of top1 cleavage complexes was performed by using the ICE assay as described in Fig. 3, and top1 immunoblotting of the DNA-containing fractions (numbers at the bottom of each panel) are presented. Dose-response and kinetics of top1 trapping by ara-C in CEM cells are shown in A and B, respectively. (C) Aphidicolin cotreatment inhibits the production of top1-DNA complexes by ara-C. Cells were treated with 1 μM aphidicolin alone (Aph), 10 μM ara-C alone (Ara-C) for 7 h, or with a combination of ara-C and aphidicolin (Ara-C + Aph) (1 h pretreatment with aphidicolin followed by aphidicolin and ara-C for 6 h) at 37°C. (D) Persistence of ara-C-induced top1-DNA complexes. Cells were treated with Ara-C (1 μM, 15 h), and top1-DNA complexes were measured either immediately (Ara-C) or after removal of the drug and incubation in drug-free medium for 1 or 2 h (Rev. 1 h and Rev. 2 h, respectively).

Figure 5

Top1-deficient P388/CPT45 cells are resistant to ara-C. (A) Top1 content of P388 and P388/CPT45 cells were analyzed by Western blotting using the C21 human top1 mAb. Lanes 1, 10⁵ cells; lanes 2, 2 × 10⁵ cells. Numbers on the right indicate the migration position of molecular mass markers. (B) Growth inhibition in P388 (○) and P388/CPT45 cells (●) was measured by MTT assay after continuous treatment with ara-C for 3 and 5 days, respectively.