WEE1 kinase regulates the onset of mitosis and is expressed in human OS. (A) The Cyclin-B/CDC2 complex is considered the master switch for the G2/M transition and CDC2 is designated the general controller in the onset of mitosis. Inhibition of the Cyclin-B/CDC2 complex prevents mitotic entry. CDC2 activation is essential for G2/M transition and thus progression through the cell cycle and is regulated on multiple levels. Inhibitory phosphorylation of CDC2 is achieved by WEE1 and prevents proper association with Cyclin-B. Consequently, Cyclin-B and CDC2 cannot form a complex and the complex cannot be activated. This induces a G2 arrest and prevents the cell cycle to progress until DNA repair is completed. Dephosphorylation of CDC2 by the phosphatase CDC25C activates the Cyclin-B/CDC2 complex, allowing cell cycle progression. The activation status of the Cyclin-B/CDC2 complex is dependent on the balance between WEE1 kinase and CDC25C phosphatase activity, in which WEE1 kinase is the rate limiting, dominant molecule. (B) WEE1 mRNA expression is significantly higher in the OS samples when compared to the normal tissue samples (ANOVA: p < 0.0001). (C) Panel C shows immunohistochemical staining of sections of primary OS (extremity: II, III and axial: I, V) and OS lung metastasis (IVa,b). The brown nuclear staining indicates WEE1 expression in OS tumor tissue. (D) Western blot analysis of the effect of PD0166285 on WEE1 function. Irradiated (IR) cells show increased expression levels of CDC2-pY15 (CDC2-p) compared to untreated cells. After subsequent treatment with WEE1-inhibitor PD0166285 (WEE1-i), CDC2-pY15 expression levels are diminished, indicative of inhibition of WEE1 kinase activity. (E) Baseline expression levels of phosphorylated CDC (CDC2-p), the most important molecular target of WEE1 kinase, are distinctly lower in human primary osteoblasts compared to human OS cells.