Activation of JNK was involved in TAp73α-mediated apoptosis induced by cisplatin.

<p>(A) TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) were treated with 20 μM SP600125 for different periods of time (indicated). The phosphorylation levels of JNK and c-Jun were measured. (B and C) TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) and the empty vector controls (V) were treated with 20 μM SP600125 or DMSO and then with cisplatin. The cell apoptosis were assessed by TUNEL assay (error bars indicated mean ± SD from three independent experiments; *: p<0.05) and the cleavage of PARP analysis. Inhibition of JNK attenuated TAp73α-mediated apoptosis in response to cisplatin. (D) TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) were treated with JNK siRNAs (Si-JNK) or the scrambled control siRNA (Control). The activations of JNK and c-Jun were absent upon cisplatin treatment, and associated with markedly reduced cell apoptosis (E and F).</p

GADD45α contributed to TAp73-mediated apoptosis in response to cisplatin.

<p>Cell apoptosis was diminished in TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) in response to cisplatin after GADD45α siRNAs treatment. The cell apoptosis was measured by (A) TUNEL assay (error bars indicate mean ± SD from three independent experiments; *: p<0.05; **: p<0.01) and (B) the cleavage of PARP assay.</p

A proposed model for the regulatory role of TAp73 in DNA damage response.

<p>DNA damage agent, cisplatin induces TAp73 accumulation and the subsequent up-regulation of its pro-apoptotic target genes to induce cell apoptosis. Simultaneously, up-regulation of TAp73 target gene, GADD45α activates the JNK apoptotic pathway via its interaction with MEKK4/MTK1 to induce apoptosis.</p

Overexpression of TAp73α enhanced cellular sensitivity to cisplatin.

<p>(A) The GFP-TAp73α overexpressing stable clones in SKOV3 (C8, C24 and C28) and OVCA433 (C1, C7 and C12) cells were verified by western blot analysis. (B and C) Both XTT viability assay and clonogenic assay showed significantly reduced cell proliferation in TAp73α-overexpressed cells of SKOV3 and OVCA433 compared to the empty vector controls (V) in response to cisplatin treatment. The percentage of cells/colonies surviving in cisplatin relative to cells/colonies in drug-free medium control was measured. Data was shown as mean ± SD from three independent experiments (*: p<0.05; **: p<0.01).</p

TAp73α activated the JNK pathway.

<p>(A) Increase of p-JNK and p-c-Jun was detected in TAp73α–overexpressed cells (SKOV3 C8, C24 and C28 and OVCA433 C1, C7 and C12) and further enhanced upon cisplatin treatment (4 μg/ml for 24 h), when compared to the controls (P: parental cells; V: empty vector control). (B) TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) were exposed to 4 μg/ml cisplatin for different periods of time, or to different doses of cisplatin for 12 h. The p-JNK level was measured by western blot analysis. (C) DNp73α (GFP-DNp73α) was over-expressed in SKOV3 (D2) and OVCA433 (D18) cells. (D) No JNK activation was observed in DNp73α-overexpressed cells (SKOV3 D2 and OVCA433 D18).</p

Overexpression of TAp73α promoted cell apoptosis in response to cisplatin.

<p>(A) TAp73α-overexpressed cells (SKOV3 C8, C24 and C28 and OVCA433 C1, C7 and C12) and the empty vector controls (V) of SKOV3 and OVCA433 were treated with 4 μg/ml cisplatin for 48 h. Apoptotic cells were assessed by TUNEL assay. More than 500 cells were counted for each group, the results presented were the relative of the apoptotic cells to total cells and at least three independent experiments were performed (**: p<0.01). (B) TAp73α-overexpressed cells and empty vector controls were treated with different doses (indicated) of cisplatin for 24 h, and the cleavage of PARP was detected by western blot analysis.</p

TAp73α activated the JNK pathway through up-regulating GADD45α and the subsequent activation of MKK4.

<p>(A) Increase of GADD45α mRNA expression in TAp73α-overexpressed cells (SKOV3 C8, C24 and C28 and OVCA433 C1, C7 and C12). (B) Increase of GADD45α protein expression and the MKK4 phosphorylation level in TAp73α-overexpressed cells (SKOV3 C8, C24 and C28 and OVCA433 C1, C7 and C12). (C) GADD45α was knocked down in TAp73α-overexpressed cells (SKOV3 C8 and OVCA433 C1) by siRNAs (Si1-GADD45α and Si2-GADD45α) treatment. The activation of MKK4, JNK and c-Jun were diminished, even under the cisplatin treatment.</p