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Yves Pommier – One of the best experts on this subject based on the ideXlab platform.
the intra s phase checkpoint affects both dna replication initiation and elongation single cell and dna fiber analysesMolecular and Cellular Biology, 2007Co-Authors: Jennifer A. Seiler, Mirit I Aladjem, Chiara Conti, Ali Syed, Yves PommierAbstract:
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 γ-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.
The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analysesCancer Research, 2007Co-Authors: Yves Pommier, Jennifer A. Seiler, Chiara Conti, Mirit I AladjemAbstract:
5227 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 DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. Following a short, non-toxic 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 the late-S-, compared to early-S-phase cells, and was due to both an inhibition of initiation and elongation. DNA replication was actively inhibited by checkpoint activation since 7–Hydroxystaurosporine (UCN-01) or a specific Chk1 inhibitor restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced CPT-induced DNA damage at replication foci sites where histone gamma-H2AX colocalized with replication foci. Together, our study demonstrates that the intra S-phase checkpoint is exerted not only upon replication initiation but also upon DNA elongation.
p21cdkn1a allows the repair of replication mediated dna double strand breaks induced by topoisomerase i and is inactivated by the checkpoint kinase inhibitor 7 HydroxystaurosporineOncogene, 2006Co-Authors: Takahisa Furuta, R L Hayward, Ling Hua Meng, Haruyuki Takemura, Gregory J Aune, William M Bonner, Mirit I Aladjem, Kurt W Kohn, Yves PommierAbstract:
This study provides evidence for the importance of p21 CDKN1A for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21 CDKN1A and p53 enhance camptothecin-induced histone H2AX phosphorylation (yH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, yH2AX reverses after camptothecin removal. By contrast, yH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21 CDKN1A (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced yH2AX formation in p21-/- cells, we conclude that enhanced yH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chkl/Chk2 inhibitor 7–Hydroxystaurosporine (UCN-01) also increases camptothecin-induced yH2AX formation and inhibits camptothecin-induced p21 CDKN1A upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that yH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21 CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.
William Plunkett – One of the best experts on this subject based on the ideXlab platform.
Inhibition of topoisomerase IIα and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trialsMolecular cancer therapeutics, 2007Co-Authors: Lei Guo, Xiaojun Liu, Kiyohiro Nishikawa, William PlunkettAbstract:
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.
Pharmacodynamics of cytarabine alone and in combination with 7–Hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial.Blood, 2005Co-Authors: Deepa Sampath, Zheng Shi, Jorge E. Cortes, Zeev Estrov, Michael Andreeff, Varsha Gandhi, William PlunkettAbstract:
Chk1 and Akt signaling facilitate survival of cells treated with nucleoside analogues. Activation of Chk1 in response to cytarabine (ara-C) induced an S-phase checkpoint characterized by the inhibition of Cdk2, cell cycle arrest, no change in constitutively active Akt, or low-stress kinase signaling in ML-1 cells. However, inhibition of Chk1 by UCN-01 in S-phase-arrested cells resulted in an abrogation of the checkpoint, inhibition of Akt, activation of JNK, and a rapid induction of apoptosis. Similarly, primary acute myelogenous leukemia (AML) blasts exposed to ara-C and UCN-01 demonstrated a selective loss in cloning potential when compared with normal progenitors. Therefore, we evaluated a pilot clinical trial of ara-C in combination with UCN-01 in patients with relapsed AML. Blasts from some patients demonstrated a previously activated Chk1-Cdk2 DNA damage response pathway that decreased during therapy. Constitutively phosphorylated Akt kinase declined on addition of UCN-01 to the ara-C infusion, an action accompanied by an activation of JNK and reduction in absolute AML blast counts. Thus, use of UCN-01 in combination with ara-C decreases Chk1 phosphorylation, inhibits the Akt survival pathway, and activates JNK during the course of therapy, offering a rationale for the cytotoxic action of this combination during AML treatment. (Blood. 2006;107:2517-2524)
Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7–Hydroxystaurosporine (UCN-01).Oncology reports, 2003Co-Authors: Li Ying Yang, Hong Jiang, Kelly M. Rangel, William PlunkettAbstract:
Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7–Hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.
Takashi Tsuruo – One of the best experts on this subject based on the ideXlab platform.
Survival-signaling pathway as a promising target for cancer chemotherapyCancer Chemotherapy and Pharmacology, 2003Co-Authors: Naoya Fujita, Takashi TsuruoAbstract:
The serine/threonine kinase AKT, also known as PKB or RAC-PK, is a key molecule for protecting cells from undergoing apoptosis. Several studies have suggested that the AKT-mediated survival-signaling pathway is an attractive target for cancer chemotherapy: (1) the AKT pathway is relatively inactive in resting cells; (2) amplification of the AKT gene occurs in some tumors; (3) loss of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) is common in tumors and its loss constitutively activates AKT; (4) AKT is activated at the cancer invasion front. To clarify which drugs exhibit their cytotoxicity by inhibiting the AKT pathway, we screened anticancer drugs that could downregulate phospho-AKT levels and AKT kinase activity. We found that UCN-01 (7–Hydroxystaurosporine), heat-shock protein 90 (HSP90) inhibitors, and topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) possessed the ability to interfere with the AKT pathway. UCN-01 directly suppressed upstream AKT kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) (IC_50
Interference with PDK1-Akt survival signaling pathway by UCN-01 (7–Hydroxystaurosporine)Oncogene, 2002Co-Authors: Saori Sato, Naoya Fujita, Takashi TsuruoAbstract:
3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the AGC subfamily of protein kinases. In particular, PDK1 plays an important role in the regulation of Akt/PKB survival pathway by phosphorylating Akt on Thr308. Here we show that UCN-01 (7–Hydroxystaurosporine), a drug now in clinical trials and with a unique fingerprint pattern, induced dephosphorylation and inactivation of Akt, resulting in the turn-off of the survival signals and the induction of apoptosis. Further analysis revealed that UCN-01-mediated Akt inactivation was caused by inhibiting upstream Akt kinase PDK1 (IC50=33 nM) both in vitro and from cells, but not by suppressing Akt itself or phosphatidylinositide-3-OH kinase. UCN-01-induced PDK1 inhibition was also observed in in vivo murine and human tumor xenografts. Overexpression of active form of Akt diminished the cytotoxic effects of UCN-01, suggesting that UCN-01 may in part exert its cytotoxicity by inhibiting PDK1-Akt survival pathway. Because UCN-01 has already proved to have potent anti-tumor activity in vivo, PDK1-Akt survival pathway is a new, attractive target for cancer chemotherapy.