The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death

Abstract

Autophagic cell death is morphologically characterized by an accumulation of autophagic vacuoles. Here, we show that inactivation of LAMP2 by RNA interference or by homologous recombination leads to autophagic vacuolization in nutrient-depleted cells. Cells that lack LAMP2 expression showed an enhanced accumulation of vacuoles carrying the marker LC3, yet a decreased colocalization of LC3 and lysosomes, suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. While a fraction of mitochondria from starved LAMP2-expressing cells colocalized with lysosomal markers, within autophagolysosomes, no such colocalization was found on removal of LAMP2 from the experimental system. Of note, LAMP1 depletion had no such effects and did not aggravate the phenotype induced by LAMP2-specific small interfering RNA. Serum and amino acid-starved LAMP2-negative cells exhibited an accumulation of autophagic vacuoles and then succumbed to cell death with hallmarks of apoptosis such as loss of the mitochondrial transmembrane potential, caspase activation and chromatin condensation. While caspase inhibition retarded cell death, it had no protective effect on mitochondria. Stabilization of mitochondria by overexpression of Bcl-2 or the mitochondrion-targeted cytomegalovirus protein vMIA, however, blocked all signs of apoptosis. Neither caspase inhibition nor mitochondrial stabilization antagonized autophagic vacuolization in LAMP2-deficient cells. Altogether, these data indicate that accumulation of autophagic vacuoles can precede apoptotic cell death. These findings argue against the clear-cut distinction between type 1 (apoptotic) and type 2 (autophagic) cell death.

Molecular mechanisms of ATP secretion during immunogenic cell death

“…Further substantiating this possibility, we found that the depletion of LAMP1, a lysosomal protein that is dispensable for autophagy, 50 abolishes ATP secretion by U2OS cells exposed to MTX or OXA (Figures 7a-c). The knockdown of vesicle-associated membrane protein 1 (VAMP1, also known as synaptobrevin 1), which is known to have a non-redundant role in exocytosis, 51 also inhibited ATP release in this system, similar to the depletion of ATG5 and BCN1 (Figure 7b).…”

NF-κB inhibition sensitizes to starvation-induced cell death in high-risk myelodysplastic syndrome and acute myeloid leukemia

“…Next, we determined whether the inhibition of autophagy by targeting essential autophagy genes (ATG6/Beclin-1, ATG5, ATG10, ATG12) with small interfering RNAs (siRNA) would reduce cell killing by BAY11-7082 plus starvation. Transfection of cells with siRNAs that inhibit autophagy Gonzalez-Polo et al, 2005;Lum et al, 2005) and the depletion of essential ATG proteins (as shown for ATG6/Beclin-1, Figure 11a) had no protective effect on cell death induction by BAY11-7082 plus starvation, even in the presence of z-VAD-fmk (Figure 11b). Similarly, the knockdown of AIF (Figure 12a) failed to abolish cell killing induced by BAY11-7082 plus starvation (Figure 12b).…”

“…In many paradigms, autophagy actually constitutes a mechanism of adaptation to nutrient stress (in which case autophagy allows macromolecules to catabolize, to generate ATP and other building blocks of biogenesis) or to organelle stress (in which damaged organelles are removed by the autophagic machinery) (Shintani and Klionsky, 2004;Levine and Yuan, 2005). Moreover, cell death that has initially adopted a type 2 morphology may switch to type 1 at a late stage of the process, in a caspasedependent fashion (Martin and Baehrecke, 2004;Gonzalez-Polo et al, 2005).…”

Apoptosis and autophagy: regulatory connections between two supposedly different processes

“…Atg9 is a transmembrane protein that shuttles between the Golgi and endosomes and may identify the membranes used to form the autophagic vesicle. Fusion of the autophagosome with the lysosome requires the Rab7 GTPase (39,40) and the lysosome-associated membrane protein-2 (LAMP2) (41). By interfering with specific regulators it is feasible to block autophagy at different stages in the process.…”

“…Similar mechanisms apply in mammalian cells too; nutrient deprivation in cells that lack LAMP2 and thus cannot fuse autophagosomes with lysosomes causes a cell death response with morphological characteristics of autophagic cell death. However, the actual killing of the cells involves a later activation of apoptosis with caspase activation occurring subsequent to MOMP (41). These examples suggest that the idea that there are two clearly different programmed cell death mechanisms (Type I and Type II, apoptosis and autophagic cell death) is oversimplified.…”