Regulation of zebrafish hatching by tetraspanin cd63.

Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

Drug-resistant aurora A mutants for cellular target validation of the small molecule kinase inhibitors MLN8054 and MLN8237.

The Aurora kinases regulate multiple aspects of mitotic progression, and their overexpression in diverse tumor types makes them appealing oncology targets. An intensive research effort over the past decade has led to the discovery of chemically distinct families of small molecule Aurora kinase inhibitors, many of which have demonstrated therapeutic potential in model systems. These agents are also important tools to help dissect signaling pathways that are orchestrated by Aurora kinases, and the antiproliferative target of pan-Aurora inhibitors such as VX-680 has been validated using chemical genetic techniques. In many cases the nonspecific nature of Aurora inhibitors toward unrelated kinases is well established, potentially broadening the spectrum of cancers to which these compounds might be applied. However, unambiguously demonstrating the molecular target(s) for clinical kinase inhibitors is an important challenge, one that is absolutely critical for deciphering the molecular basis of compound specificity, resistance, and efficacy. In this paper, we have investigated amino acid requirements for Aurora A sensitivity to the benzazepine-based Aurora inhibitor MLN8054 and the close analogue MLN8237, a second-generation compound that is in phase II clinical trials. A crystallographic analysis facilitated the design and biochemical investigation of a panel of resistant Aurora A mutants, a subset of which were then selected as candidate drug-resistance targets for further evaluation. Using inducible human cell lines, we show that cells expressing near-physiological levels of a functional but partially drug-resistant Aurora A T217D mutant survive in the presence of MLN8054 or MLN8237, authenticating Aurora A as a critical antiproliferative target of these compounds.

A pilot study examining germline minisatellite mutations in the offspring of Danish childhood and adolescent cancer survivors treated with radiotherapy.

PURPOSE: To investigate germline mutation rate at eight minisatellite loci in 24 Danish families, where one parent is the survivor of childhood or adolescent cancer treated with radiotherapy. MATERIALS AND METHODS: Parents and offspring were profiled for eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern blotting. RESULTS: Seven paternal mutations were observed for 130 informative alleles in 18 offspring from 11 radiation-exposed fathers (mean preconceptional dose for offspring 0.29 Gy, range<0.01-1.2 Gy), compared to six mutations for 146 informative alleles in 21 offspring from 13 unexposed fathers. No statistically significant difference between the total paternal mutation rates was observed (5.4% for exposed fathers and 4.1% for unexposed fathers). Three maternal mutations were observed for 148 informative alleles in 21 offspring from 13 radiation-exposed mothers (mean preconceptional dose for offspring 0.71 Gy, range <0.01-9.2 Gy), compared to one mutation for 130 informative alleles in 18 offspring from 11 unexposed mothers. Again, no statistically significant difference was observed between the total maternal mutation rates (2.0% for exposed mothers and 0.8% for unexposed mothers). CONCLUSIONS: The data from this pilot study demonstrate no statistically significant increase in germline minisatellite mutation rate associated with radiotherapy for childhood and adolescent cancer.

Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida.

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.