Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death.

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre-apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)-sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2alpha, followed by partial activation of caspase-8 (but not caspase-3), caspase-8-mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE-dependent exocytosis. Knock-in mutation of eIF2alpha (to make it non-phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase-8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase-8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.

Vacuolar functions determine the mode of cell death.

The yeast vacuole plays a crucial role in cell homeostasis including pH regulation and degradation of proteins and organelles. Class C VPS genes code for proteins essential for vacuolar and endosomal vesicle fusion, their deletion results in the absence of a detectable vacuole. We found that single gene deletions of class C VPS genes result in a drastically enhanced sensitivity to treatment with acetic acid whereas sensitivity towards H2O2 remains largely unaffected. Interestingly acetic acid treatment known as an established inducer of yeast apoptosis leads to necrosis in class C VPS deletion strains. Their intracellular pH drops from 6.7 to 5.5 after acetic acid treatment, while in wild type the pH drops to just 6.3. When the intracellular pH in wild type is lowered below pH 5.5 using a higher concentration of acetic acid, the survival rate is similarly low as in the class C VPS mutants, however, the death phenotype is predominantly apoptotic. Hence, the vacuole not only prevents acetic acid induced cell death by buffering the cytosolic pH, but it also has a proapoptotic function.

The redox-sensing quinone reductase Lot6p acts as an inducer of yeast apoptosis.

Mammalian NAD(P)H:quinone oxidoreductases such as human NQO1 act as inducers of apoptosis. Quinone reductases generated interest over the last decade due to their proposed function in the oxidative stress response. Furthermore, human NQO1 was reported to regulate p53 stability and p53-dependent apoptosis through regulation of cellular oxidation-reduction events. In this study, we have used low concentrations of hydrogen peroxide (0.4 and 0.6 mM) to induce apoptosis-like cell death in wild type, an LOT6 overexpressing and a Deltalot6 yeast strain to monitor cell survival. Using this approach, we demonstrate that yeast quinone reductase Lot6p, an orthologue of mammalian quinone reductases, plays a pivotal role in apoptosis-like cell death in Saccharomyces cerevisiae. Overexpression of LOT6 results in enhanced cell death, as shown by an investigation of the morphological hallmarks of apoptosis-like fragmentation of DNA and externalization of phosphatidylserine, whereas the deletion strain displays a deficiency in apoptosis-like cell death as compared with the wild type. Thus, we propose that Lot6p is directly involved in the control of the apoptosis-like cell death in yeast.

Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit.

Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3'-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.

Phylogenetic conservation of the preapoptotic calreticulin exposure pathway from yeast to mammals.

The pre-apoptotic exposure of calreticulin (CRT) on the cell surface determines the efficient engulfment of mouse or human tumor cells by antigen-presenting dendritic cells. CRT exposure is rapidly induced by anthracyclins and ionizing irradiation and follows a complex signal transduction pathway that is interrupted by depletion of PERK, caspase-8, BAP31, Bax, Bak or SNAREs, as well as by knock-in mutation of eIF2alpha (to make it non-phosphorylable by PERK) or BAP31 (to render it uncleavable by caspase-8). Here, we show that yeast (Saccharomyces cerevisiae) can expose the CRT orthologue CNE1 on the surface in response to cell death induced by the anthracylin mitoxantrone (MTX). This MTX-triggered CNE1 translocation is abolished by knockout of the yeast orthologues of PERK (Gcn2), BAP31 (Yet3) and SNAREs (Nyv1, Sso1). Altogether, our data point to the existence of an ancestral and cell death-related CRT exposure pathway with conserved elements shared between unicellular fungi and mammals.