DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Abstract

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.

FIGURE 1.

Enhanced apoptosis in MMR-proficient cells after MNNG exposure. A, stable RKO6 (hMLH1^-/MMR^-) and genetically matched, hMLH1-transfected RKO7 (hMLH1⁺/MMR⁺) cells were generated and analyzed (46). MMR^- RKO6 versus MMR⁺ RKO7 cells were then exposed to MNNG (10 μm, 1 h) and monitored for apoptosis using TUNEL and G₁ subpopulations. Apoptosis was dramatically increased in RKO7 (MMR⁺) cells versus RKO6 (MMR^-) cells at 48 h. B, RKO6 (MMR^-) and RKO7 (MMR⁺) cells were treated with increasing MNNG doses (μm, 1 h), and apoptosis and survival end points were analyzed. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 2.

MMR-dependent apoptotic proteolysis of caspase-8, caspase-9, and PARP-1. A, RKO6 and RKO7 cells were treated with MNNG, and apoptotic proteolysis of caspase-8 and caspase-9 as well as PARP-1 was assessed by Western blot analyses. Caspase-9 cleavage (beginning at 48 h) occurred ∼20 h earlier than caspase-8 cleavage, initially observed at 72 h. MNNG-treated RKO6 cells showed no apoptotic proteolyses. B, pretreatment with the caspase inhibitor Z-VAD-fmk (20 μm) or Z-DEVD-fmk (50 μm) abrogated PARP-1 proteolytic cleavage induced by 10 μm MNNG treatment. GAPDH served as a loading control. Arrows indicate activating proteolytic cleavage fragments for caspase-9 (35 kDa), caspase-8 (43 kDa), and PARP-1 (89 kDa). UT, untreated.

FIGURE 3.

Functional abrogation of p53 does not affect MMR-dependent apoptosis. A, MMR^- RKO6 and MMR⁺ RKO7 were treated as described in the legend to Fig. 1, and Western blot analyses of total p53, phospho-p53 (Ser¹⁵), Bax, and p21 were performed. p53 was preferentially stabilized and phosphorylated in MMR⁺ RKO7 cells. GAPDH levels were monitored for equal loading. UT, untreated. B and C, stable MMR^- RKO6 and MMR⁺ RKO7 clones expressing human papillomavirus E6 or vector alone (B) or infected with lentiviral vectors containing shp53 or shSCR sequences (C) were treated with MNNG (10 μm, 1 h) and analyzed for apoptosis at 72 and 96 h. Survival confirmed that HCT116 3-6 and RKO7 cells were equally sensitive to MNNG regardless of p53 status (supplemental Fig. 4, A and B).

FIGURE 4.

MMR-dependent stabilization of p73α after MNNG exposure. A, MMR^- RKO6 and MMR⁺ RKO7 cells were exposed to MNNG (10 μm, 1 h), and the kinetics of p73 expression was examined by Western blot analyses. VO, vector only; UT, untreated. B, p73α levels were preferentially stabilized in RKO7 (MMR⁺) cells after MNNG treatment. Relative p73α and p73β levels before and after MNNG treatment were quantified from Western blots using NIH Image J software. C, c-Abl tyrosine kinase inhibition by STI571 (Gleevec™) abrogated p73α and p73β levels with or without MNNG (10 μm, 1 h). RKO6 and RKO7 cells were pretreated with a nontoxic dose of STI571 (25 μm, 2 h) and exposed to MNNG (10 μm, 1 h), and p73α or p73β expression was examined by Western blot assays. p73α/β-deficient human SK-N-AS neuroblastoma cells transfected with exogenous p73α or p73β were used as controls for expression. GAPDH levels were monitored for loading.

FIGURE 5.

Pharmacological or shABL inhibition dramatically decreases MMR-dependent apoptosis after MNNG exposure. A, RKO6-shSCR, RKO7-shSCR, RKO7-shABL-27, and RKO7-shABL-34 clones were exposed to MNNG (10 μm, 1 h), and apoptosis was monitored at 72 and 96 h. Specific knockdown of p73α decreased apoptosis. B, mock-, MNNG (10 μm, 1 h)-, or staurosporine (STS;10 μm, 24 h)-treated RKO7-shSCR versus RKO7-shABL-27 cells were analyzed for apoptosis. Half of the cells were pretreated with STI571 (25 μm, 2 h) prior to MNNG treatment. RKO7-shABL-27 knockdown cells were more resistant to apoptosis than RKO7-shSCR cells. Both cells were sensitive to staurosporine. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 6.

c-Abl-mediated stabilization of p73α regulates apoptosis in MMR⁺ cells after MNNG exposure. A, RKO7-shSCR, RKO7-shABL-27, and RKO7-shABL-34 clones were generated and exposed to MNNG (10 μm, 1 h), and p73α and p73β levels were analyzed before and after MNNG treatment (10 μm, 1 h). p73α/β-deficient SK-N-AS cells and p73α and p73β stable transfectants were analyzed along with GAPDH levels for loading. VO, vector only; UT, untreated. B, RKO7-shSCR, RKO7-shp73-4, and RKO7-shp73-12 cells were treated with or without various MNNG doses (μm, 1 h), and changes in survival were noted. Loss of p73α levels in RKO7-shp73-4 and RKO7-shp73-12 clones resulted in a significant reduction of MNNG-induced lethality. C, RKO6-shSCR, RKO7-shSCR, RKO7-shp73-4, and RKO7-shp73-12 clones were exposed to MNNG (10 μm, 1 h) and assessed for apoptosis. Specific knockdown of p73α decreased apoptosis. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 7.

c-Abl-mediated stabilization of GADD45α regulates apoptosis in MMR⁺ cells after MNNG treatment. A, RKO7-shSCR, RKO7-shGADD45α-2, and RKO7-shGADD45α-3 clones were treated with or without various MNNG doses (μm, 1 h), and changes in survival were noted. B, RKO6-shSCR, RKO7-shSCR, RKO7-shGADD45α-2, and RKO7-shGADD45α-3 clones were exposed to MNNG (10 μm, 1 h) and assessed for apoptosis. Specific knockdown of GADD45α decreased apoptosis. ^*, p < 0.05.

FIGURE 8.

hMLH1/c-Abl/p73α/GADD45α signaling controls G₂ arrest, apoptosis, and survival. Shown is the MMR detection of O⁶-methylguanine (O⁶-MeG) DNA lesions created after MNNG signals through c-Abl to increase p73α and GADD45α expression. c-Abl/GADD45α controls G₂ arrest, apoptosis, and survival, whereas c-Abl/p73α is needed in apoptosis and survival.