Biomedical subjects

Kurt W Kohn

Publications and source records attributed to Kurt W Kohn.

22 records · Page 2Linked to original sources

PubMed2003

Apoptosis induced by topoisomerase inhibitors.

Topoisomerase inhibitors are among the most efficient inducers of apoptosis. The main pathways leading from topoisomerase-mediated DNA damage to cell death involve activation of caspases in the cytoplasm by proapoptotic molecules released from mitochondria. In some cells, apoptotic response also involves the death receptor Fas (APO-1/CD95). The engagement of these apoptotic effector pathways is tightly controlled by upstream regulatory pathways that respond to DNA lesions-induced by topoisomerase inhibitors in cells undergoing apoptosis. These include the proapoptotic Chk2, c-Abl and SAPK/JNK pathways, the survival PI(3)kinase-Akt-dependent pathway and the transcription factors p53 and NF-kappaB. Initiation of cellular responses to DNA lesions-induced by topoisomerase inhibitors is ensured by the protein kinases DNA-PK, ATM and ATR, which bind to DNA breaks. These kinases commonly called "DNA sensors" mediate their effects (DNA repair, cell cycle arrest and/or apoptosis) by phosphorylating a large number of substrates, including several downstream kinases such as c-Abl and the checkpoint protein Chk2. c-Abl induces apoptosis by activating cell death pathways (e.g., SAPK, p53 and p73) and inhibiting cell survival pathways [e.g., PI(3)kinase]. The DNA-damage regulating kinase Chk2, in addition to its role in cell cycle arrest and/or DNA repair, can induce apoptosis by phosphorylation/activation of the promyelocytic leukemia (PML) protein and p53. Finally, we will review the recent observations that support a role for topoisomerases in chromatin fragmentation during the execution phase of apoptosis.

Antineoplastic Agents↗

PubMed2002

UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1.

UCN-01 (7-hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. It has been shown previously that UCN-01 abrogates DNA-damage-induced G(2) checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site previously identified for Chk2 (or/and Chk1) kinase. We found that in human colon carcinoma HCT116 cells, IR treatment enhanced Chk2 kinase activity, whereas Chk1 activity remained unchanged, which suggested that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN-01 inhibits Chk2 immunoprecipitated from HCT116 cells (IC(50), approximately 10 nM). In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G(2) arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G(2) checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was no p21(Waf1/Cip1) induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells.

Alkaloids↗

PubMed2002

Transcriptional regulation of mitotic genes by camptothecin-induced DNA damage: microarray analysis of dose- and time-dependent effects.

cDNA microarray technology can be used to establish associations between characteristic gene expression patterns and molecular responses to drug therapy. In this study, we used cDNA microarrays of 1694 cancer-related genes to monitor the gene expression consequences of the treatment of HCT116 colon cancer cells with the topoisomerase I inhibitor camptothecin (CPT). To obtain a more homogeneous cellular response, we synchronized the cells in S-phase using aphidicolin (APH) before CPT treatment. Brief incubation with 20 and 1000 nM CPT caused reversible and irreversible G(2) arrest, respectively, and the patterns of gene expression change (with reference to untreated controls) were strikingly different at the two concentrations. Thirty-three genes, mainly divided into three groups, showed characteristic changes in the first 20 h as a consequence of treatment. Northern blots performed for five of these genes (each under eight experimental conditions) were quite consistent with the microarray results (average correlation coefficient, 0.86). Several p53-activated stress response genes were up-regulated after treatment with 1000 nM CPT or prolonged exposure to APH, but it seemed that the up-regulation did not directly cause cell cycle arrest because the up-regulation induced by prolonged treatment with APH did not prevent cell cycle progression after removal of APH. In contrast, cell cycle-dependent up-regulation of a group of mitosis-related genes was delayed or blocked after CPT treatments. The interrupted up-regulation of this group of genes was directly associated with G(2) arrest. In addition, we observed down-regulation of gene expression in cells that were recovering from cell cycle delay. The observations reported here suggest a fundamental difference at the gene expression level between the molecular mechanism of reversible G(2) delay that follows mild DNA damage and the mechanism of permanent G(2) arrest that follows more extensive DNA damage.

Aphidicolin↗

PubMed2002

Importance of the fourth alpha-helix within the CAP homology domain of type II topoisomerase for DNA cleavage site recognition and quinolone action.

We report that point mutations causing alteration of the fourth alpha-helix (alpha4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser(740)Trp, Gln(743)Pro, and Thr(744)Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca(2+) or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr(744)Ala substitution had minimal effect on Ca(2+)- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the alpha4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser(83)Trp also changed the DNA cleavage pattern generated by Ca(2+) or quinolones. Finally, Thr(744)Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the alpha4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the alpha4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.

4-Quinolones↗