Journal: Biology of Reproduction

Article Title: Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis ^†

doi: 10.1093/biolre/ioaa097

Figure Lengend Snippet: Functional and biological evaluation of WEE inhibitors in oocytes and somatic cells. (A) ELISA analysis for phosphorylation activity of WEE1 and WEE2 in situ. MK-1775 significantly inhibits WEE1 and WEE2, reducing a greater proportion of WEE2 kinase activity. Error bars represent SEM with P > 0.05 by Student t -test. (B) Fertilization rates of bovine oocytes co-cultured with inhibitor or DMSO (control) during IVF ( N = 126). MK-1775 reduced MII exit compared to DMSO by 16%. (C) Somatic cell division activity (mitosis) was tracked over time by flow cytometry analysis. MK-1775 induced a decrease in mitosis at 0.1 and 0.01 μM and arrested the cell culture at 1 μM.

Article Snippet: Inhibitor activity against WEE1 and WEE2 were assessed using a CycLex WEE1 Kinase Assay/Inhibitor Screening Kit (Cat# CY-1172; MBL International, Woburn, MA) as previously described [ ].

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, In Situ, Cell Culture, Flow Cytometry

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Mitotic progression in cells synchronized at S/G2 and treated with Wee1/Myt1 and Cdc25 inhibitors. (A, B) HeLa cells stably expressing fluorescent histone H2B fused to GFP were synchronized by the double thymidine block at the S/G2 border and treated with the Wee1/Myt1 inhibitor, PD0166285, alone (A) or in combination with Cdc25 inhibitor, NSC663284 (B). While the Wee1/Myt1 inhibitor alone rapidly triggers mitosis in the majority of cells, the combination of the Wee1/Myt1 and Cdc25 inhibitors results in slow mitotic entry followed by mitotic collapse. The complete time-lapse sequence is shown in Supplemental Videos 7 and 8. Bar, 10 μm. (C) Synchronized HeLa cells were treated with the Wee1/Myt1 inhibitor, PD0166285, alone or in combination with Cdc25 inhibitor, NSC663284, for 90 min. Cells were then fixed and processed by immunofluorescence for alpha-tubulin and phosphorylated-histone H3 on S10 (mitotic marker). Labeling shows disorganized mitotic spindle, and in some cells, reduced mitotic marker.

Article Snippet: The Wee1/Myt1 inhibitor PD0166285 (Pfizer, New York, NY) was used at 0.5 μM.

Techniques: Stable Transfection, Expressing, Blocking Assay, Sequencing, Immunofluorescence, Marker, Labeling

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Inhibition of Wee1/Myt1 and Cdc25 in synchronized cells causes mitotic collapse. (A) HeLa cells were synchronized at the S/G2 border after double thymidine block and then treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the two drugs. Nocodazole was added to the medium to prevent mitotic exit. Cells were then collected at indicated time points, fixed and stained with antibody to phospho-histone H3 (mitotic marker) conjugated with Alexa Fluor 647, and processed by flow cytometry. In cells treated with vehicle only (DMSO, blue line), the mitotic index progressively increased, with more than half the cells being in mitosis by the end of the experiment. Cdc25 inhibitor, NSC663284, blocked mitotic entry (brown line). Wee1 inhibitor, PD0166285 (green line), caused rapid mitotic entry during the first hour after its addition. In cells treated with both PD0166285 and NSC663284 (orange line), the mitotic index first increased then fell. (B) HeLa cells were treated as in (A), lysed and analyzed by SDS–PAGE. In cells not treated with inhibitors (blue lanes), phosphorylations on histone H3 and nucleolin appeared by 8 h after second thymidine release and increased for the duration of the experiment. Phosphorylation of Cdk1 on inhibitory T14 and Y15 decreased over time, indicating the activation of the Cdk1/cyclin B complex. As cells were entering mitosis, a portion of Wee1, Myt1 Cdc25C, Cdc27, and MastL acquired an electrophoretic mobility shift. Cyclin B1 levels were increasing, and cyclin A2 levels dropped slightly as cells accumulated in mitosis. Inhibition of Wee1 and Myt1 kinases with PD0166285 (green lanes) resulted in rapid phosphorylation of Nucleolin and histone H3 that peaked 2 h after the drug addition and remained steadily high for the duration of the experiment. Cdk1 was rapidly dephosphorylated on inhibitory T14 and Y15. Wee1, Myt1, Cdc25, and Cdc27 rapidly shifted up. By 1 h after drug addition, Cyclin A2 was largely degraded and cyclin B1 was stable. Inhibition of Wee1 and Myt1 together with Cdc25 by addition of both PD0166285 and NSC 663284 (orange lanes) triggered the a weak phosphorylation on Nucleolin and histone H3 that peaked at 1–2 h and disappeared at 3–4 h after addition of the two drugs. Reduced mitotic phosphorylation shifts of Wee1, Myt1, Cdc25, and Cdc27 indicated that these proteins were not fully phosphorylated. Note that cyclin B and most of the cyclin A were not degraded in these cells. Panels on the right show quantifications of indicated Western blots. All values were adjusted for loading and normalized to the 4-h time point of DMSO-treated cells.

Article Snippet: The Wee1/Myt1 inhibitor PD0166285 (Pfizer, New York, NY) was used at 0.5 μM.

Techniques: Inhibition, Blocking Assay, Staining, Marker, Flow Cytometry, SDS Page, Phospho-proteomics, Activation Assay, Electrophoretic Mobility Shift Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Deposphorylation of mitotic substrates in “collapsed” cells is a result of incomplete inhibition of Cdk-opposing phosphatases. (A) Cdk1/cyclin B1 activity does not drop in mitotic collapse cells. HeLa cells were synchronized at the S/G2 border and treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the two in the presence of nocodazole. Cells were then collected at indicated time points and lysed. An aliquot of the lysate was analyzed by Western blotting for Nucleolin phosphorylation. β-Actin served as a loading control. Cyclin B1/Cdk1 complex was immunoprecipitated from the rest of the lysate and subjected to an in vitro kinase assay using histone H1 as a substrate. The kinase reaction mixture was resolved by SDS–PAGE, and the gel was exposed to phosphor-screen, which was then scanned with phosphor-imager. For a control, samples derived from the 4-h time point of DMSO-treated cells were treated with Cdk inhibitor (lane labeled “+Flavopiridol”), or processed omitting cyclin B1 antibody from immunoprecipitation (lane labeled “mock”). The gel was subsequently stained with Coomassie blue for loading. Panel on the right shows quantifications of histone H1 phosphorylation normalized to the 4 h time point of DMSO-treated cells. An average of three independent assays is shown. Error bars denote SD. (B) Simultaneous inhibition of Wee1/Myt1 and Cdc25 in cells already in mitosis does not cause mitotic substrate dephosphorylation. Mitotic HeLa cells were collected in nocodazole and then treated with Wee1/Myt1 and Cdc25 inhibitors for the indicated time, lysed, and analyzed by Western blotting. Mitotic substrates nucleolin and histone H3 remained phosphorylated throughout the experiment. (C) The phosphatase inhibitor, okadaic acid, prevents dephosphorylation of mitotic substrates in cells treated with a combination of Wee1/Myt1 and Cdc25 inhibitors. HeLa cells were synchronized at the S/G2 border after double thymidine block and treated with the Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, for the indicated time in the presence or absence of okadaic acid. Addition of the okadaic acid resulted in robust and sustained phosphorylation of mitotic substrates.

Article Snippet: The Wee1/Myt1 inhibitor PD0166285 (Pfizer, New York, NY) was used at 0.5 μM.

Techniques: Inhibition, Activity Assay, Western Blot, Phospho-proteomics, Control, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page, Derivative Assay, Labeling, Staining, De-Phosphorylation Assay, Blocking Assay