E1A oncogene induced sensitization to NK cell induced apoptosis requires PIDD and Caspase-2

Abstract

Expression of the adenovirus E1A oncogene sensitizes tumor cells to innate immune rejection by NK cells. This increased NK sensitivity is only partly explained by an E1A-induced increase in target cell surface expression of NKG2D ligands. The post-recognition mechanisms by which E1A sensitizes cells to the apoptotic cell death response to NK injury remains to be defined. E1A sensitizes cells to apoptotic stimuli through two distinct mechanisms-repression of NF-κB-dependent antiapoptotic responses and enhancement of caspase-2 activation and related mitochondrial injury. The current studies examined the roles of each of these post-NKG2D-recognition pathways in the increased sensitivity of E1A-positive target cells to NK killing. Sensitization to NK-induced apoptosis was independent of E1A-mediated repression of cellular NF-κB responses but was dependent on the expression of both caspase-2 and the upstream, caspase-2 activating molecule, PIDD. Target cells lacking caspase-2 or PIDD expression retained E1A-induced increased expression of the NKG2D ligand, RAE-1. NK cell-induced mitochondrial injury of E1A-expressing cells did not require expression of the mitochondrial molecules, Bak or Bax. These results define a PIDD/caspase-2-dependent pathway, through which E1A sensitizes cells to NK-mediated cytolysis independently of and complementarily to E1A-enhanced NKG2D/RAE-1 ligand expression.

Keywords: Apoptosis; Innate immunity.

Fig. 1. Role of caspases in NK-mediated cytolysis of E1A-expressing cells.

a [Cr⁵¹]-labeled MCA102 (circle), MCA102 E1A (triangle) cells were incubated with RNK-16 cells at the indicated RNK:target ratios in the absence or presence (inverted triangle) of 100 μM zVAD-fmk. After 6 h, supernatants were collected and % specific Cr⁵¹ release was assessed (mean ± SEM; n = 4, ***P < 0.0001, **P ≤ 0.0084, one-way ANOVA). b [Cr⁵¹]-labeled BMK (circle) or BMK-E1A (triangle) cells were incubated with RNK-16 cells at the indicated RNK:target ratios in the absence or presence (inverted triangle) of 100 μM zVAD-fmk. After 6 h, supernatants were collected and % specific Cr⁵¹ release was assessed (mean ± SEM; n = 4, ***P ≤ 0.0001, one-way ANOVA)

Fig. 2. Role of NF-κB-dependent cellular responses in susceptibility of E1A-expressing cells to NK.

[Cr⁵¹]-labeled NIH-3T3 (circle), 3T3-E1A (square), 3T3-E1A TNF resistant (TNFr) (triangle) or 3T3-E1A p65 RelA (p65) (diamond) cells were incubated with RNK-16 cells at the indicated RNK:target ratios. After 6 h, supernatants were collected and % specific Cr⁵¹ release was assessed (mean ± SEM; n = 3, **P = 0.0015, *P = 0.0126, one-way ANOVA)

Fig. 3. NK cell induced mitochondrial injury.

3T3-E1A cells were incubated with cell tracker blue-stained RNK-16 cells at the indicated ratios of RNK:E1A cells for 4 h and then stained with TMRE. Histograms show non-blue gated cells (3T3-E1A cells) and are representative of four individual experiments. Percentage of non-blue cells are shown above the markers

Fig. 4. Role of Bak and Bax in RNK cytolysis of E1A-expressing cells.

a [H³]-thymidine-labeled BMK-E1A, BMK-E1A-Bak^−/− (BMK Bak), BMK-E1A-Bax^−/− (BMK Bax), or BMK-E1A-Bak/Bax^−/−/^−/− (BMK DKO) cells were incubated with ceramide (100 µM) overnight. Supernatants were collected and % specific thymidine release was assessed (mean ± SEM; n = 5, ***P = 0.001, one-way ANOVA). b [Cr⁵¹]-labeled BMK (circle), BMK-E1A (triangle), BMK-E1A-Bak^−/− (square), BMK-E1A-Bax^−/− (inverted triangle) or BMK-E1A-Bak/Bax^−/−/^−/− (double knockout = DKO, diamond) cells were incubated with RNK-16 cells at the indicated RNK:target ratios. After 6 h, supernatants were collected and % specific ⁵¹Cr release was assessed (mean ± SEM; n = 4, ***P ≤ 0.0003, one-way ANOVA). c [Cr⁵¹]-labeled BMK (circle) or BMK-E1A-Bak/Bax^−/−/^−/− (double knockout = DKO, diamonds) cells were incubated with RNK-16 cells at the indicated RNK:target ratios in the absence (filled diamond) or presence (open diamond) of 100 μM zVAD-fmk. After 6 h, supernatants were collected and % specific ⁵¹Cr release was assessed (mean ± SEM; n = 4, ***P ≤ 0.0001, **P = 0.0016, one-way ANOVA)

Fig. 5. Role of caspase-2, PIDD and RAE-1 in the sensitivity of E1A-expressing cells to NK cell lysis.

a Expression of Casp-2, E1A and actin in 3T3, 3T3-E1A and E1A-iC2 cell lines (originally published in. b Expression of PIDD, E1A and actin in 3T3, 3T3-E1A and E1A-iPIDD cell lines. c [Cr⁵¹]-labeled 3T3 (circle), 3T3-E1A (square), E1A-iC2 (inverted triangle), and E1A-iPIDD (triangle) cells were incubated with nude rat splenic NK cells at the indicated spleen cell:target ratios. % specific ⁵¹Cr release was assessed (mean ± SEM; n = 4, ***P ≤ 0.0002, **P = 0.0012, one-way ANOVA). d [Cr⁵¹]-labeled 3T3 (circle), 3T3-E1A (square), E1A-iC2 (inverted triangle), and E1A-iPIDD (triangle) cells were incubated with RNK-16 cells at the indicated RNK:target ratios. % specific ⁵¹Cr release was assessed (mean ± SEM; n = 4, ***P ≤ 0.0008, one-way ANOVA). d RAE-1 expression on 3T3 (filled histogram) compared to 3T3-E1A (open histogram), E1A-iC2 (open histogram), and E1A-iPIDD (open histogram) cells. Data shown are representative of four individual experiments