Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The E-ring lactone is the Achilles' heel of camptothecin derivatives: although it is considered necessary for the inhibition of the enzyme topoisomerase I (topo1), the opening of the lactone into a carboxylate abolishes the generation of topo1-mediated DNA breaks. S38809 is a novel camptothecin analog with a stable 5-membered E-ring ketone; therefore, it lacks the lactone function. DNA relaxation and cleavage assays revealed that S38809 functions as a typical topo1 poison by stimulating DNA cleavage at T downward arrow G sites. The activity was strongly dependent on the stereochemistry of the C-7 carbon atom that bears the hydroxy group. S38809 proved to be a potent cytotoxic agent, with a mean IC50 of 5.4 nM versus 11.6 nM for topotecan and 3.3 nM for SN38 (the active metabolite of irinotecan) on a panel of 31 human tumor cell lines. The cytotoxicity of S38809 and its ability to stabilize cleavable complexes was considerably reduced in camptothecin-resistant cells that express a mutated topo1, confirming that topo1 is its primary target. Cell death induced by topo1 poisoning requires the conversion of DNA single-strand breaks into double-strand breaks that can be detected by the formation of phosphorylated histone H2AX. In HCT116 cells, topotecan, SN38, and S38809 induced histone H2AX phosphorylation in S phase of the cell cycle, with S38809 being as potent as SN38 and 5-fold more potent than topotecan. In vivo, S38809 showed a marked antitumor activity against HCT116 xenografts. These findings open a new route for improving the pharmacological properties of camptothecin derivatives.
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PMID:Novel stable camptothecin derivatives replacing the E-ring lactone by a ketone function are potent inhibitors of topoisomerase I and promising antitumor drugs. 1749 37

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G(2) cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G(2) checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G(2) population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G(2) checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIalpha to relax supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIalpha, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.
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PMID:Inhibition of topoisomerase IIalpha and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials. 1751 99

To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone gamma-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.
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PMID:The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses. 1751 3

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 approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-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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PMID:Nonclassic functions of human topoisomerase I: genome-wide and pharmacologic analyses. 1787 16

Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those drug-induced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G(2)-M and a dependence on functional p53 for their cytotoxicity. Dose-dependent gamma-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These gamma-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0).
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PMID:Novel indenoisoquinolines NSC 725776 and NSC 724998 produce persistent topoisomerase I cleavage complexes and overcome multidrug resistance. 1797 83

In order to enhance the cytotoxicity of radiation, camptothecin (CPT), an inhibitor of DNA topoisomerase I, was added to the cultured glioma cell lines before irradiation (IR). Radiation responses of five glioblastoma cell lines (U87-MG, U373-MG, GHE, GaMG and SNB-19) treated with CPT were analyzed in terms of cell and colony counts, cell cycle progression, expression of histone gamma H2AX, DNA repair protein Rad50, survivin, cleaved caspase 3, p53 and of topoisomerase I. CPT enhanced the radiotoxicity in U87-MG and SNB-19 cell lines if cell and colony counts were used as the end-points. In contrast, pre-treatment with CPT of U373-MG, GHE and GaMG cell lines did not enhance cytotoxicity of IR in terms of cell and colony counts but accelerated DNA damage repair assessed by Rad50 foci. CPT treated glioma cells revealed at least two subpopulations with respect to the expression of histone gamma H2AX, a marker of DNA double-strand breaks. The cell lines tested also differed in the expression of survivin, cleaved caspase 3, p53 and of topoisomerase I. The failure of CPT to enhance the radiotoxicity of glioma U373-MG, GHE and GaMG cell lines in terms of cell and colony counts was found to correlate with accelerated DNA damage repair, and with low expression of topoisomerase I, a target of CPT.
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PMID:Differential response of human glioblastoma cell lines to combined camptothecin and ionizing radiation treatment. 1861 57

Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their alpha-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone gamma-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone gamma-H2AX could be used as a biomarker for the upcoming clinical trials of S39625.
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PMID:Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters. 1808 16

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective cytotoxin that has shown significant activity in advanced clinical trials in combination with radiotherapy and cisplatin. The current study aimed to advance our understanding of tirapazamine-induced lesions and the pathways involved in their repair. We show that homologous recombination plays a critical role in repair of tirapazamine-induced damage because cells defective in homologous recombination proteins XRCC2, XRCC3, Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with the involvement of homologous recombination repair, we observed extensive sister chromatid exchanges after treatment with tirapazamine. We also show that the nonhomologous end-joining pathway, which predominantly deals with frank double-strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In addition, we show that tirapazamine preferentially kills mutants both with defects in XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable DNA-topoisomerase I cleavable complexes. We further show that gamma H2AX, an indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle. These observations lead us to an overall model of tirapazamine damage in which DNA single-strand breaks, base damage, and DNA-protein cross-links (including topoisomerase I and II cleavable complexes) produce stalling and collapse of replication forks, the resolution of which results in DSB intermediates, requiring homologous recombination and XPF/ERCC1 for their repair.
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PMID:Homologous recombination is the principal pathway for the repair of DNA damage induced by tirapazamine in mammalian cells. 1817 18

The results of a phase I clinical trial of the topoisomerase I (Topo I) poison CPT-11 followed by the cyclin-dependent kinase inhibitor flavopiridol in patients with advanced solid tumors indicate that patients whose tumors were wild-type, but not mutant, for p53 obtained the most clinical benefit from this combination therapy. We elected to elucidate the mechanistic basis for this effect in isogenic-paired HCT116 colon cancer cells that were either wild-type (+/+) or null (-/-) for p53. With the combination therapy of SN-38 (the active metabolite of CPT-11) followed by flavopiridol, the induction of apoptosis was 5-fold greater in the p53+/+ cells compared with the p53-/- cells. This sequential treatment induced phosphorylation of p53 at Ser(15), which interacted with Rad51, a DNA repair protein involved in homologous recombination. Rad51 bound to p53-Ser(15) within the first 5 hours of combination therapy, and then was transcriptionally suppressed at 24 hours by flavopiridol only in p53+/+ cells. Microarray analysis also revealed suppression of Rad51 in a p53-dependent manner. Depletion of Rad51 by small interfering RNA (siRNA) sensitized both p53+/+ and p53-/- cells to SN-38-induced apoptosis with increase of gamma H2AX, a marker of DNA damage. Conversely, overexpression of Rad51 rescued p53+/+ cells from SN-->F-induced apoptosis. Because flavopiridol inhibits Cdk9, we found that inhibition of Cdk9 by DRB or by siRNA could recapitulate the flavopiridol effects, with suppression of Rad51 and induction of apoptosis only in p53+/+ cells. In conclusion, after DNA damage by Topo I poisons, flavopiridol targets homologous recombination through a p53-dependent down-regulation of Rad51, resulting in enhancement of apoptosis.
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PMID:The cyclin-dependent kinase inhibitor flavopiridol potentiates the effects of topoisomerase I poisons by suppressing Rad51 expression in a p53-dependent manner. 1838 38

ST1968, a novel hydrophilic camptothecin analogue of the 7-oxyiminomethyl series, is characterised by the formation of stable DNA-topoisomerase I cleavable complex and by a promising profile of antitumour activity. The present study was designed to extend preclinical evaluation of the novel camptothecin in human squamous cell carcinoma (SCC) models. ST1968 exhibited an impressive activity with a high cure rate in SCC models. ST1968 produced 100% of complete response without evidence of regrowth in tumours characterised by susceptibility to drug-induced apoptosis (FaDu, A431 and A2780). In contrast to irinotecan, ST1968 still showed an excellent, persisting activity in models less susceptible to apoptosis induction (KB, Caski and SiHa), in which drug treatment elicited a persistent DNA damage response, as documented by phosphorylation of p53, RPA-2 and histone H2AX, resulting in delayed apoptosis and senescence. This behaviour was associated with a marked cellular/tumour drug accumulation. In conclusion, ST1968 exhibited an outstanding antitumour activity superior to that of irinotecan against SCC. A high intracellular accumulation, resulting in fast apoptosis or DNA damage persistence, appeared to be a critical determinant of SCC sensitivity to ST1968.
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PMID:Intracellular accumulation and DNA damage persistence as determinants of human squamous cell carcinoma hypersensitivity to the novel camptothecin ST1968. 1844 21


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