Mouse models for ROS1-fusion-positive lung cancers and their application to the analysis of multikinase inhibitor efficiency.

ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated early-onset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1-fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer.

Novel mouse model for Gardner syndrome generated by a large-scale N-ethyl-N-nitrosourea mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We carried out large-scale mutagenesis using the chemical mutagen N-ethyl-N-nitrosourea. One specific aim of our mutagenesis project was to generate novel cancer models. We screened 7012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor, and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli (Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis. Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome.

Developmental potential of mouse tetraploid cells in diploid <--> tetraploid chimeric embryos.

Mouse 2n (lacZ-) <--> 4n (lacZ+) aggregation chimeras were examined 5 or 10 days after uterine transfer to test the potential of 4n cells to contribute to embryonic tissues. Recovered embryos corresponded to embryonic day 7.5 approximately 8.0 and 12.5, respectively. Ten days after transfer, 4n cells were never detected, as reported earlier, in embryonic tissues of chimeras produced by the standard procedure in which one 2n embryo at the8-cell stage is aggregated with a4n embryo at the4-cell stage. However, beta-gal positive cells were present in embryonic tissues, though in a low number, in chimeras produced by a 2n and a 4n embryo at the 4-cell stage. Similar results were obtained when one 2n embryo atthe 8-cell stage was aggregated with two 4n embryos atthe 4-cell stage. beta-gal positive cells were found in the heart, liver, skin and intestinal epithelium. The majority of chimeras 5 days after uterine transfer retained beta-gal positive cells in embryonic tissues. The complete lack of 4n cell contribution to chimeras produced by the standard procedure is therefore attributed to the initial low proportion of 4n cells allocated to epiblast and their severe elimination from embryonic tissues.

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.