Topoisomerase I (Top1) catalyzes two transesterification reactions: single-strand DNA cleavage and religation that are normally coupled for the relaxation of DNA supercoiling in transcribing and replicating chromatin. A variety of endogenous DNA modifications, potent anticancer drugs and carcinogens uncouple these two reactions, resulting in the accumulation of Top1 cleavage complexes. Top1 cleavage complexes damage DNA and kill cells by generating replication-mediated DNA double-strand breaks (DSBs) and by stalling transcription complexes. The repair of Top1-mediated DNA lesions involves integrated pathways that are conserved from yeasts to humans. Top1-mediated DNA damage and cell cycle checkpoint responses can be studied biochemically and genetically in yeast and human cells with known genetic defects. Defects in these repair/checkpoint pathways, which promote tumor development, explain, at least in part, the selectivity of camptothecins and other Top1 inhibitors for cancer cells.
A series of novel thiosemicarbazone derivatives bearing condensed heterocyclic carboxaldehyde moieties were designed and synthesized. Among them, TSC24 exhibited broad antiproliferative activity in a panel of human tumor cells and suppressed tumor growth in mice. The mechanism research revealed that TSC24 was not only an iron chelator but also a topoisomerase IIalpha catalytic inhibitor. Its inhibition on topoisomerase IIalpha was due to direct interaction with the ATPase domain of topoisomerase IIalpha which led to the block of ATP hydrolysis. Molecular docking predicted that TSC24 might bind at the ATP binding site, which was confirmed by the competitive inhibition assay. These results about the mechanisms involved in the anticancer activities of thiosemicarbazones will aid in the rational design of novel topoisomerase II-targeted drugs and will provide insights into the discovery and development of novel cancer therapeutics based on the dual activity to chelate iron and to inhibit the catalytic activity of topoisomerase IIalpha.
The Mre11⅐Rad50⅐Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRNdeficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G 2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.
Tetrandrine is an antitumor alkaloid isolated from the root of Stephania tetrandra. We find that micromolar concentrations of tetrandrine irreversibly inhibit the proliferation of human colon carcinoma cells in MTT and clonogenic assays by arresting cells in G 1 . Tetrandrine induces G 1 arrest before the restriction point in nocodazole-and serum-starved synchronized HT29 cells, without affecting the G 1 -S transition in aphidicolin-synchronized cells. Tetrandrine-induced G 1 arrest is followed by apoptosis as shown by fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated nick end labeling, and annexin V staining assays. Tetrandrine-induced early G 1 arrest is mediated by at least three different mechanisms. First, tetrandrine inhibits purified cyclin-dependent kinase 2 (CDK2)/cyclin E and CDK4 without affecting significantly CDK2/cyclin A, CDK1/cyclin B, and CDK6. Second, tetrandrine induces the proteasome-dependent degradation of CDK4, CDK6, cyclin D1, and E2F1. Third, tetrandrine increases the expression of p53 and p21Cip1 in wild-type p53 HCT116 cells. Collectively, these results show that tetrandrine arrests cells in G 1 by convergent mechanisms, including down-regulation of E2F1 and up-regulation of p53/p21Cip1 .
DNA topoisomerase I (Top1) is essential for removing DNA supercoiling generated in transcribing and replicating chromatin (11,67). Top1 relaxes positively and negatively supercoiled DNA by introducing reversible DNA single-strand breaks associated with covalent Top1-DNA complexes. Camptothecin, a natural alkaloid, selectively targets the Top1-DNA complex by stabilizing the covalent Top1-DNA cleavage intermediate (33,47,65). Camptothecin and its derivatives, irinotecan and topotecan, are potent anticancer drugs currently being used successfully in the treatment of colon and ovarian cancer (4,46,64). The cytotoxic action of camptothecin is manifested when a replication fork encounters the drug-stabilized cleavage complex (31, 34). At these sites, extension of the replicating strand up to the end of the Top1-mediated break in the template strand generates a replication double-strand break ("replication runoff") as demonstrated by ligation-mediated PCR (62) and the induction of ␥-H2AX (23) (http://discover.nci.nih.gov/ pommier/pommier.htm). Camptothecin is, therefore, a wellcharacterized pharmacological tool for studying the molecular mechanisms involved in cellular responses to replicative stress (23,48,59,62). Top1 cleavage complexes and, therefore, replication double-strand breaks can form in response to common DNA lesions including abasic sites, mismatches, oxidative base lesions, base adducts, and strand breaks (49, 51).Histone H2AX phosphorylated on serine 139, termed ␥-H2AX, is one of the earliest known markers of camptothecin-induced replication-associated damage (23). More generally, ␥-H2AX is a marker of DNA double-strand breaks (45, 54). ␥-H2AX has been proposed to anchor the broken chromosome ends together and recruit DNA repair elements (5,20,23,45,53). We have shown previously that ␥-H2AX is critical for the recruitment of the Mre11-Rad50-Nbs1 (MRN) complex in camptothecin-treated cells and that H2AX deficiency renders cells hypersensitive to camptothecin (23,53). Using aphidicolin, we also showed that blocking replicative polymerases abrogates ␥-H2AX formation (23), indicating that ␥-H2AX forms in response to replication-associated doublestrand breaks induced by camptothecin.The causative gene of the cancer-predisposing genetic disease Bloom's syndrome, BLM, is a member of the RecQ family of DNA helicases (28). BLM is considered a caretaker of the genome (28, 39) and a key component in DNA damage response signaling (22,52). Evolutionarily conserved and essential for the maintenance of genomic stability, BLM promotes branch migration of Holliday junctions in vitro in an ATPdriven fashion (36,38,40,66,70). BLM functions in association with topoisomerase III␣ (Top3␣) (68), a type I class of topoisomerases (11,37,67,69). The BLM-Top3␣ complex can resolve recombination intermediates and prevent the collapse of replication forks and consequent DNA double-strand
The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced f5-fold and Top2A compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone ;-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.
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