Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes.

A 3-h exposure of V79 Chinese hamster cells with the sensory irritant 2-chlorobenzylidene malonitrile (CS) caused apolar mitoses in a dose-dependent manner. With a preparations and staining technique that allows for the visualization of the spindle apparatus and the chromosomes it was found that unlike in Colcemid-induced c-metaphases residual spindle fibers or microtubule material were present in the majority of CS-induced c-metaphases. The observation suggests different mechanisms for the induction of the c-mitotic effect by the two spindle poisons

Centromere Detection in Vinblastine- and Radiation-induced Micronuclei of Cytokinesis-blocked Mouse Cells Using in Situ Hybridization with a Mouse gamma (major) Satellite DNA Probe.

Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70-90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents

Dicentric and translocation analysis for retrospective dose estimation in humans exposed to ionising radiation during the Chernobyl nuclear power plant accident.

Chromosome analyses were carried out in peripheral blood lymphocytes obtained between September 1991 and March 1992 from 15 persons exposed to ionising radiation during the Chernobyl nuclear power plant accident; At present, all are being treated for symptoms of the delayed stage of the cutaneous radiation syndrome. Biological dose-equivalent estimates were determined, either by measuring the frequency of dicentric and ring chromosomes in first division unstable cells from conventional preparations (Qdr method), or by measuring the frequency of stable translocations using two-colour fluorescence in situ hybridisation (FISH) with composite whole chromosome-specific DNA libraries for human chromosomes 1, 4 and 12 (chromosome painting) and a degenerate a-satellite pancentromeric DNA probe. With both methods fairly comparable individual estimates between 1.1 and 5.8 Gy were obtained for 12 of 15 individuals. Three individuals exhibited no elevated aberration frequencies. Perspectives and limitations of chromosome painting for dose reconstruction of past radiation exposures are discussed

Induction of Chromosome Aberrations and SCE by Indirectly Acting Mutagens in Immortal Mouse and Rat Hepatocyte Lines.

Two immortalized, differentiated mouse and rat hepatocyte lines were examined for their capacity to activate the indirectly acting mutagens aflatoxin B1 (AFB1), cyclophosphamide (CP), benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA) into DNA reactive metabolites as determined by the induction of structural chromosome aberrations (CA) and sister chromatid exchange (SCE). The rat and mouse hepatocyte lines were able to efficiently activate either AFB1, BaP, DMBA, or BaP and DMBA, respectively, as shown by significant clastogenic responses. SCE induction was apparent in both cell lines in response to each of the compounds. Due to the observed longterm maintenance of various liver specific functions as well as the capability to metabolize xenobiotics these cells may be a suitable assay system for the detection of indirectly acting mutagens

Translocation t(10;14)(q11.2:q22.1) fusing the <em>kinectin</em> to the <em>RET</em> gene creates a novel rearranged form (PTC8) of the <em>RET</em> proto-oncogene in radiation-induced childhood papillary thyroid carcinoma.

Evaluation of 20 cases of radiation-induced childhood papillary thyroid carcinoma using fluorescence in situ hybridization demonstrated the presence of clonal translocations affecting the RET locus. Semiquantitative reverse transcription-PCR indicated overexpression of the RET tyrosine kinase (TK) domain in four cases. In two cases, the RET rearrangements PTC6 and PTC7 were identified and assigned to balanced translocations t(7;10)(q32;q11.2) and t(1;10)(p13;q11.2), respectively. In one case with a balanced translocation t(10;14)(q11.2;q22.1), 5&#39; rapid amplification of cDNA ends revealed a novel type of RET oncogenic activation (PTC8), arising from a fusion of the 5&#39; part of the kinectin (KTN1) gene to the TK domain of the RET gene. The presence of coiled-coil domains in the resulting ktn1/ret fusion protein suggests ligand-independent dimerization and thus constitutive activation of the ret TK domain

An efficient proteomics method to identify the cellular targets of protein kinase inhibitors

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38α whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action