Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

BACKGROUND: Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. METHODS: Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. RESULTS: In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. CONCLUSION: We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy

α1,6-Fucosyltransferase-deficient Mice Exhibit Multiple Behavioral Abnormalities Associated with a Schizophrenia-like Phenotype: IMPORTANCE OF THE BALANCE BETWEEN THE DOPAMINE AND SEROTONIN SYSTEMS*

Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8−/−) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-β1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8−/− mice using a combination of neurological and behavioral tests. Fut8−/− mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8−/− mice displayed increased locomotion compared with wild-type (Fut8+/+) and heterozygous (Fut8+/−) mice. In particular, Fut8−/− mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8−/− mice as evidenced by the Y-maze tests. Furthermore, Fut8−/− mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8+/+ and Fut8+/− mice in the PPI test under normal conditions, Fut8+/− mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8−/− mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders