TAp73alpha protects small cell lung carcinoma cells from caspase-2 induced mitochondrial mediated apoptotic cell death

Abstract

Caspase-2 is ubiquitously expressed and the most evolutionarily conserved mammalian caspase. It can be activated by a range of death stimuli prior to Bax activation and the occurrence of apoptotic mitochondrial dysfunctions. Caspase-2 has also been reported to exert tumour suppressor function in vivo. The full length TAp73alpha isoform is found up-regulated in various tumour types, and is reported in a cell-type specific manner to repress drug-induced apoptosis. Here, we report that TAp73alpha represses caspase-2 enzymatic activity and by this means reduce caspase-2 induced Bax activation, loss of mitochondrial transmembrane potential and resulting apoptosis. The inhibitory effect on caspase-2 requires the presence of the DNA binding domain and SAM domain region of TAp73alpha. In conclusion, the ability of TAp73alpha to act as an inhibitor of caspase-2-induced cell death together with its up-regulation in certain tumour types strengthen the potential oncogenic activities for this protein.

Figure 1. TAp73alpha inhibits apoptosis induced by caspase-2 over-expression in SCLC NCI-H82 cells

SCLC NCI-H82 cells (Figure 1A-D) were co-transfected with EGFP together with either an empty (control) vector or plasmids encoding TAp73alpha, TAp73beta and/or caspase-2. Twenty-four hours post-transfection, cells were fixed in 4% PFA, stained with Hoechst and scored under a fluorescence microscope as percentage of EGFP expressing cells displaying condensed or fragmented nuclei. Three hundred EGFP transfected cells were analyzed and each experiment was repeated three times. Figures are mean + SD of three independent experiments, where ***p-value <0.001, **p-value <0.01

Figure 2. TAp73alpha expression inhibits caspase-2 activity

SCLC NCI-H82 cells (Figure 2) were transfected with TAp73alpha, caspase-2 and/or empty expression vectors, and caspase-2 activity was monitored. The enzyme activity of caspase-2 (VDVADase activity) was evaluated as the release of AMC from the substrate Ac-VDVAD-AMC (Ac-Val-Asp-Val-Ala-Asp-7-amino-4-methylcoumarin). The data represents three independent experiments.

Figure 3. TAp73alpha prevents caspase-2 induced mitochondrial dysfunction in SCLC NCI-H82 cells

SCLC NCI-H82 cells (Figure 3) were co-transfected with TAp73alpha, caspase-2 and/or empty expression vectors. Twenty-four hours post-transfection, cells were stained with TMRE and loss of mitochondrial trans-membrane potential (TMRE-negative cells) was assayed by scoring of EGFP transfected cells without TMRE by FACS analysis. Images are representatives of three independent experiments.

Figure 4. TAp73alpha counteracts caspase-2 induced Bax activation in SCLC NCI-H82 cells

SCLC NCI-H82 cells (Figure 4) were co-transfected with EGFP and plasmids encoding TAp73alpha, caspase-2 and/or empty expression vectors. Twenty-four hours post-transfection, SCLC NCI-H82 cells were collected, fixed in 4% PFA and incubated with the 6A7 anti-Bax antibody at 4°C, overnight, followed by secondary Alexa Fluor 635-conjugated antibody. For each sample 10,000 cells were sorted for green (EGFP) fluorescence using BD LSR II flow cytometer, and assessed for intensity of active Bax staining.

Figure 5. The DNA binding domain and SAM domain are required for TAp73alpha inhibition of caspase-2 induced apoptosis

SCLC NCI-H82 cells (Figure 5) were co-transfected with EGFP together with either an empty (control) vector, TAp73alpha or plasmids encoding different mutant forms of TAp73alpha (DBDmutp73alpha/p73alphaSAMdel), and/or caspase-2. Twenty-four hours post-transfection, cells were fixed, stained with Hoechst and scored under a fluorescence microscope as percentage of EGFP expressing cells displaying condensed or fragmented nuclei. Three hundred EGFP transfected cells were analyzed and each experiment was repeated three times. Figures are mean + SD of three independent experiments, where *p <0.05.