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

Abstract

1-␤-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 P388͞ CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.camptothecin ͉ DNA repair ͉ DNA damage ͉ nucleoside analog D NA topoisomerases I (top1) are essential and ubiquitous enzymes (1, 2). They are critical for DNA replication and transcription by regulating the topological state of DNA by means of reversible transesterification reactions (3-5). Eukaryotic top1 reversibly cleaves one strand of the DNA by binding covalently to the 3Ј end of the broken DNA (6). This intermediate is referred to as the top1 cleavage complex. Top1 also mediates religation of the DNA. Under normal conditions, the religation step of the equilibrium is favored and only a small fraction of the DNA is cleaved (4). Top1 inhibitors, such as camptothecin (CPT) and its derivatives, stabilize (trap) the cleavage complexes by inhibiting top1-mediated DNA religation (7,8). Trapping of cleavage complexes by CPT converts the top1 enzyme into a cellular poison, and top1-mediated DNA damage probably results from replication or transcription complex collisions with CPT-arrested top1-DNA covalent complexes (7,8).DNA damages, such as base mismatches, abasic sites, UV photo-lesions, and ethenoadenine adducts, can also stabilize top1 cleavage complexes by inhibiting top1-mediated DNA religation (9-11). Oxidized bases, and benzo[a]pyrene adducts, on the other hand, enhance top1 cleavage complexes by enhancing the cleavage step (forward rate) of the nicking-closing reaction (12, 13).

Mechanisms of apoptosis induction by nucleoside analogs

“…Although the exact nature of the damage caused by nucleoside analogs in stalling replication forks is poorly understood, it follows that cellular responses to these agents are likely to overlap those elicited by drugs such as hydroxyurea (HU) that also cause replication forks to stall by limiting dNTP production. Also, crystallographic studies have demonstrated that incorporation of ara-C into DNA causes localized alterations in the DNA duplex (Schweitzer et al, 1994) and stabilizes covalent topoisomerase I-DNA complexes, thus converting the enzyme into a cellular poison (Pourquier et al, 2000;Chrencik et al, 2003). This suggests that cellular responses to nucleoside analogs are also likely to overlap those elicited by the camptothecin family of topoisomerase I active agents.…”

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

“…So far, it could be shown that htopoI cleavage complex formation arises in response to various DNA damaging treatments in cultured cells (Subramanian et al, 1998;Pommier et al, 2000;Pourquier et al, 2000Pourquier et al, , 2001Pourquier et al, , 2002Daroui et al, 2004;Soe et al, 2004;Sordet et al, 2004a, b). Previously, it was discussed that htopoI molecules could be trapped due to a direct recognition of the DNA damage, followed by cleavage of the DNA strand.…”

“…Furthermore, the fact that numerous chemotherapeutic drugs seem to kill tumor cells, for example by inducing htopoI cleavage complexes (Pourquier et al, , 2002Sordet et al, 2004b), makes the htopoI damage response a highly relevant and important pathway to analyse in further detail, since the so obtained knowledge may help to improve or understand the action of chemotherapeutics.…”

“…Since then, the so-called 'htopoI damage response' has been under investigation. It could be shown that the htopoI response is triggered by various kinds of DNA damage, like oxidative lesions induced by hydrogen peroxide (Daroui et al, 2004), arsenic compounds (Sordet et al, 2004b), or staurosporine (Sordet et al, 2004a), bulky adducts caused by, for example, benzo[a]-pyrene , base methylations (Pourquier et al, 2001), UV lesions (Subramanian et al, 1998;Soe et al, 2004), or antimetabolite incorporation (Pourquier et al, , 2002. Although the purpose of cleavage complex formation is still unclear, current data suggest that there might be an involvement in DNA repair (Subramanian et al, 1998;Mao and Muller, 2003) and/or apoptosis (Soe et al, 2004;Sordet et al, 2004a, b).…”

Modulation of ara-CTP levels by fludarabine and hydroxyurea in leukemic cells

“…1,2 Besides, further investigation indicates inhibition of topoisomerase I as an additional mechanism for ara-C cytotoxicity. 3 In clinical studies, cellular accumulation and retention of ara-CTP have been correlated with manifestations of ara-Cmediated cytoxicity and clinical outcome. [4][5][6] Resistance against ara-C is the major cause of treatment failure.…”