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. 2003 Dec 19;278(51):51587-93.
doi: 10.1074/jbc.M309165200. Epub 2003 Oct 6.

Antisense depletion of death-associated protein kinase promotes apoptosis

Yijun Jin  1 , Patricia J Gallagher
Affiliations

Antisense depletion of death-associated protein kinase promotes apoptosis

Yijun Jin et al. J Biol Chem. .

Abstract

Death-associated protein kinases (DAPK) are serine/threonine protein kinases that have an important role in regulating cell death. In this study two antisense approaches were employed to down-regulate expression of the endogenous DAPK-alpha and DAPK-beta proteins. Transient expression of an antisense DAPK cDNA or antisense morpholino oligonucleotides in HeLa, 3T3, or primary human vascular smooth muscle cells demonstrate that decreased DAPK expression promotes a spontaneous, caspase-mediated apoptosis as evidenced by increased activities of caspases-3 and -9. Clonal HeLa cell lines with attenuated levels of DAPK expression, obtained following selection in the presence of antisense DAPK cDNA, are more sensitive to tumor necrosis factor-induced caspase-mediated apoptosis, and their sensitivity is inversely related to DAPK expression. In contrast, HeLa cells with reduced DAPK expression are moderately resistant to cell death induced by interferon-gamma. This finding is consistent with previous studies showing that DAPK has a role in promoting caspase-independent cell death. Together, these studies demonstrate that the cellular activities of DAPK are critical for antagonizing caspase-dependent apoptosis to promote cell survival under normal cell growth conditions.

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Figures

Fig. 1
Fig. 1. Reduction of endogenous DAPK by antisense DAPK cDNA
A, fluorescent micrographs showing representative HeLa cells co-transfected with GFP (green) and sense DAPK-α or antisense (AS) DAPK-α cDNA plasmid, at ratios of 1:5. Control cells were co-transfected with GFP and LacZ. A monoclonal antibody that recognizes both α and β forms of DAPK was used to examine the levels of expression of DAPK (red). B, Western blotting to examine total DAPK (DAPK-α+β) or DAPK-β expression levels in HeLa cells transfected with sense DAPK-α or antisense (AS) DAPK-α plasmid and selected with G418 for 7 days. A monoclonal anti-DAPK antibody was used to detect the total expression level of DAPK (upper panel), and a polyclonal anti-DAPK-β was used to specifically detect DAPK-β (middle panel). The same blot was reacted with an anti-vinculin antibody to control (Cont) for sample loading (lower panel).
Fig. 2
Fig. 2. Antisense depletion of DAPK induces spontaneous apoptosis
A, flow cytometry analysis of DNA content (propidium iodide staining) in antisense DAPK transfected cells. HeLa or 3T3 cells were co-transfected with GFP and sense DAPK-α or antisense (AS) DAPK-α plasmid at ratios of 1:5. Control cells were co-transfected with GFP and LacZ. Only GFP-positive cells were selected and analyzed. Percentages of cells gated with sub-G1 DNA were quantified. B, Western blot of total DAPK (DAPK-α+β) or DAPK-β, PARP, and vinculin (loading control) in transfected 3T3 or HeLa cells as described for A. GFP-positive cells were enriched by FACS of transfected cells. Data shown are representative of four independent experiments.
Fig. 3
Fig. 3. Loss of DAPK expression induces apoptosis in differentiated HASMC
A, Western blot showing the levels of total DAPK (DAPK-α+β) and PARP in HASMC treated with varying concentration of antisense DAPK M-oligonucleotides (AS M-Oligo). A total of 2 μM oligonucleotides was transfected in each experiment by addition of control M-oligonucleotides. The blot is representative of three independent experiments. B, quantification of apoptosis in HASMC treated with AS or control M-oligonucleotides. Levels of apoptosis were determined as described under “Materials and Methods.” C, analysis of caspase activity. For each experiment, at 24 h post-transfection with AS or control M-oligonucleotides (2 μM) cells were lysed, and caspase activity was determined by quantitating cleavage of IETD-pNA (Caspase-8), LEHD-pNA (Caspase-9), or DEVD-pNA (Caspase-3).
Fig. 4
Fig. 4. Relationship between DAPK expression level and sensitivity to TNF-induced apoptosis
Clonal 3T3 cell lines incorporating sense DAPK-α (S1) or antisense DAPK-α (AS3 through AS7) plasmid were selected with G418 for 7 days and then expanded. β-Galactosidase was expressed in G418-resistant control cells (LacZ). A, Western blotting examining the levels of total DAPK (DAPK-α+β) or DAPK-β in these cell lines. Western blots were also reacted with an anti-vinculin antibody to control for sample loading. B, levels of apoptosis determined for cell lines expressing either sense or antisense DAPK cDNAs following treatment with TNF and cycloheximide (CHX) at 10 ng/ml and 10 μg/ml, respectively, for 4 h. Levels of apoptosis were calculated as described under “Materials and Methods” after counting the number of trypan blue-excluding viable cells in TNF/CHX-treated and CHX-only-treated control experiments. C, linear regression dot plot of levels of apoptosis (y axis) compared with total DAPK levels (x axis; determined by densitometry measurement of Western blots to detect total DAPK). Both an R2 value of 0.74 and a p value of <0.0001 are indicative of a high degree of correlation. D, analysis of caspase activity. For each experiment, the cells were treated with TNF (10 ng/ml) and CHX (10 μg/ml) for 4 h and lysed, and caspase activity was determined by quantitating cleavage of IETD-pNA (Caspase-8), LEHD-pNA (Caspase-9), or DEVD-pNA (Caspase-3).
Fig. 5
Fig. 5. Interferon-γ does not promote caspase-dependent apoptosis in DAPK-depleted cells
A, analysis of survival efficiency in HeLa cells transfected with DAPK-AS, DAPK-S, or control plasmids and treated with IFN-γ. At 24 h following transfection cells were selected in the presence of G418 (500 μg/ml) and IFN-γ (1000 units/ml). Viable cells were quantified at the indicated times using trypan blue exclusion. B, Western blotting to examine DAPK expression and PARP cleavage in G418R cells following treatment with IFN-γ for 5 days. C, analysis of caspase activity. For each experiment, cells were treated with G418 and IFN-γ for 5 days and lysed, and caspase activity was determined by quantitating cleavage of IETD-pNA (Caspase-8), LEHD-pNA (Caspase-9), or DEVD-pNA (Caspase-3) as described under “Materials and Methods.” D, flow cytometry analysis of DNA content (propidium iodide staining) in transfected cells. HeLa cells were transfected with sense DAPK-α or antisense (AS) DAPK-α plasmid and selected with G418/IFN-γ for 5 days. Control cells were transfected with LacZ. Percentages of cells gated with sub-G1 DNA were quantified.
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