Poly(ADP-Ribose) Polymerase-1 Could Facilitate the Religation of Topoisomerase I-linked DNA Inhibited by Camptothecin

Background: PARP inhibitors and topoisomerase I poisons (Top1p) synergize by an unknown mechanism. Results: Although Parp1 deletion fails to increase Top1p sensitivity, transfection with catalytically inactive PARP1 or its isolated DNA binding domain does sensitize. Conclusion: PARP inhibitors poison PARP1 to diminish repair of topoisomerase I-triggered DNA damage. Significance: These results predict that tumors with elevated PARP1 will be particularly sensitive to Top1p/PARP inhibitor combinations.

Section: Discussionmentioning

confidence: 96%

“…3E, third panel) (33)(34)(35)(36)(37). This model would predict that increased Topo1cc covalent complexes would be formed in the presence of PARP inhibitors.…”

Section: Parp Inhibition Does Not Directly Alter the Cell Cycle Topomentioning

confidence: 99%

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Enhanced Killing of Cancer Cells by Poly(ADP-ribose) Polymerase Inhibitors and Topoisomerase I Inhibitors Reflects Poisoning of Both Enzymes

Patel

Flatten

Schneider

et al. 2012

“…9C) compared with interphase cells. Although the ability of phosphorylation in the N-terminal domain to affect events at the topo I active site might seem counterintuitive, previous studies have demonstrated that removal of the N-terminal domain modestly decreases topo I enzymatic activity and CPT sensitivity (47,48). Crystallization of topo I with an intact N terminus, a feat that has not been reported to date, appears to be required to determine whether phosphorylation of Ser 21 enhances activity and CPT sensitivity through an interaction with portions of the N-terminal domain previously implicated in topo I activation or through interaction with different regions of the polypeptide.…”

mentioning

confidence: 99%

Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Hackbarth¹,

Gálvez-Peralta

Dai

et al. 2008

Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. and Ser 394 can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser 21 phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells. Human topo I2 is a type IB topoisomerase that relieves positive and negative DNA supercoiling caused by transcription, replication, and chromosome condensation (1). The 91-kDa, 765-amino acid polypeptide contains four domains: a poorly conserved lysine-rich N-terminal domain that contains nuclear and nucleolar localization signals, a linker region, and the core and C-terminal domains that contain the residues important for DNA interaction and relaxation of supercoils (2). A transesterification reaction at the active site of topo I ligates Tyr 723 of the enzyme to the 3Ј phosphate of the DNA, thereby creating a nick in the DNA backbone (3). This nick allows controlled rotation of the DNA to relieve supercoils. Mutation of Tyr 723 prevents the transesterification reaction and abolishes all relaxation activity (4). The anticancer drug CPT and its derivatives slow topo I-mediated DNA relaxation (5, 6) and inhibit the religation reaction step of the enzyme (7, 8), trapping topo I on DNA (9, 10) and causing cell death (11,12).A number of observations have raised the possibility that phosphorylation can modulate the activity and CPT sensitivity of topo I. Treatment with calf intestine alkaline phosphatase decreases topo I enzymatic activity in vitro (13-15). Conversely subsequent treatment with PKC or CKII, two kinases that copurify with topo I and phosphorylate it in vitro (16 -18), stimulates topo I activity 2-3-fold (14,15,19) and enhances the ability of CPT to trap covalent topo I-DNA cleavage complexes (20), suggesting that phosphorylation by these kinases might make topo I more sensitive to CPT.Despite its potential importance, many aspects of topo I phosphorylation remain poorly understood. The number of phosphorylation sites, for example, remains unclear because the number of phosphopeptides detected after metabolic labeling and immunopreci...

“…Top1 ubiquitination and subsequent degradation by the 26 S proteasome (48) have been suggested as an early step in the repair of Top1cc prior to tyrosyl-DNA phosphodiesterase (Tdp1) action (49). PARP-1 also binds to the N-terminal of Top1 and facilitates the religation of Top1cc (50). It is therefore plausible that the caspase-3-dependent cleavage of Top1 serves to prevent the repair of Top1cc during apoptosis.…”

Section: Caspase-3-cleaved Top1 Is Preferentially Involved In the Apomentioning

confidence: 99%

Topoisomerase I Requirement for Death Receptor-induced Apoptotic Nuclear Fission

Sordet

Goldman

Redon

et al. 2008

Topoisomerase I (Top1) is known to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. However, it can be trapped on DNA as cleavage complexes (Top1cc) by oxidative and carcinogenic DNA lesions, base damage, and camptothecin treatment. We show here that Top1 is also functionally involved in death receptor-induced programmed cell death. In cells exposed to TRAIL or Fas ligand, Top1cc form at the onset of apoptosis. Those apoptotic Top1cc are prevented by caspase inhibition and Bax inactivation, indicating that both caspases and the mitochondrial death pathway are required for their formation. Accordingly, direct activation of the mitochondrial pathway by BH3 mimetic molecules induces apoptotic Top1cc. We also show that TRAIL-induced apoptotic Top1cc are preferentially formed by caspase-3-cleaved Top1 at sites of oxidative DNA lesions with an average of one apoptotic Top1cc/100 kbp. Examination of Top1 knockdown cells treated with TRAIL revealed similar DNA fragmentation but a marked decrease in apoptotic nuclear fission with reduced formation of nuclear bodies. Thus, we propose that Top1 contributes to the full apoptotic responses induced by TRAIL.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 2 is a promising therapeutic agent because it induces apoptosis in a wide variety of cancer cells without affecting normal tissues (1, 2). TRAIL belongs to the TNF family of cytokines, including TNF␣ and Fas ligand, which induce apoptosis by binding to their cognate plasma membrane receptors (3). The binding of TRAIL to the DR4 or DR5 receptors and the binding of Fas ligand to Fas receptor cause the intracellular death domains of those receptors to trimerize, which leads to the recruitment of FADD and the activation of caspase-8. Caspase-8 then cleaves and thereby activates caspase-3 either directly (type I cells) or/and indirectly (type II cells) (4) by activating the mitochondrial death pathway through the cleavage of Bid (5). Cleaved Bid binds to and activates the pro-apoptotic Bcl-2 relatives Bax and Bak proteins, causing the release of mitochondrial cytochrome c and the activation of caspase-9 and caspase-3 (6). Activated caspase-3 (and other downstream caspases) cleaves a broad array of intracellular targets including DNA topoisomerase I (Top1) (7). It is also required to induce the controlled rearrangement and degradation of nuclear structures with chromatin condensation, DNA fragmentation, nuclear fission, and release of apoptotic nuclear bodies in the extracellular space (8). TRAIL-induced apoptosis also involves an accumulation of intracellular reactive oxygen species (ROS) (9).Top1 removes DNA superhelical tensions generated during transcription, replication, and chromatin remodeling (10) and is essential in higher eukaryotes (11). It relaxes DNA by forming transient DNA single-strand breaks that are produced as Top1 forms a covalent bond between its active site tyrosine (Tyr 723 ) and a 3Ј-DNA phosphate. These Top1 cleavage complexes (Top1cc) allow con...