Targeting Wee1 kinase induces apoptosis in HCC cells.

<p>A, The number of sub-G1 HuH7 cells significantly increased after PD166285 treatment (200 nM). PD166285-mediated apoptosis of HuH7 cells was completely inhibited by pretreatment with 20 µM roscovitine (Ros). * indicates significant differences between each group (P<0.05). The results are presented as means ± S.D. of three experiments. <b>B,</b> PD166285 decreased the number of HuH7 cells in a concentration-dependent manner after 72 h. * indicates significant differences between each group (P<0.05). The representative results of three independent experiments are shown. <b>C</b> and D, Wee1 kinase-specific siRNAs (18-2 or 18-3) significantly reduced the total number of cells and increased the sub-G1 cell population 48 h after transfection in a concentration-dependent manner compared with control-vector transfectants. * indicates significant differences between each group (P<0.05).</p

Wee1 kinase is expressed in moderately to poorly differentiated HCC.

<p><b>A-C,</b> Wee1 kinase was stained with anti-Wee1 antibodies in surgically resected HCC samples (▾ HCC lesion). Anti-Wee1 antibody staining revealed mostly nuclear localization in Wee1 kinase-positive cells. The magnification is 40 x (A), 100 x (B), or 200 x (C). Scale bars  = 200 µm. <b>D,</b> Wee1 kinase is expressed in moderately to poorly differentiated HCC. A chi-square test revealed significant differences (p = 0.0026). (NC; Non-cancerous lesion, Mod.; Moderately).</p

TGF-B1 induces apoptosis through cdc2 activation.

<p><b>A,</b> FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. <b>C,</b> After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. <b>D,</b> Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).</p