Methuosis Nonapoptotic Cell Death Associated with Vacuolization of Macropinosome and Endosome Compartments

Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death

Mutant Rab24 GTPase is targeted to nuclear inclusions

BACKGROUND: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity. RESULTS: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-β and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24. CONCLUSION: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24

A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

<p>Abstract</p> <p>Background</p> <p>Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell.</p> <p>Results</p> <p>Here we describe a novel chalcone-like molecule, 3-(2-<b>m</b>ethyl-1H- <b>i</b>ndol-3-yl)-1-(4-<b>p</b>yridinyl)-2-<b>p</b>ropen-1-one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1), they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6) are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line.</p> <p>Conclusions</p> <p>MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.</p

Presenilin-1 mutations associated with familial Alzheimer’s disease do not disrupt protein transport from the endoplasmic reticulum to the Golgi apparatus

AbstractMutations in genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) have been linked to familial forms of Alzheimer’s disease (AD). Cells expressing mutant presenilins produce elevated levels of Aβ42, the major amyloid peptide found in AD plaques. The mechanism whereby this occurs remains unknown, but the localization of presenilins to endoplasmic reticulum (ER) and Golgi compartments has suggested that they may function in intracellular trafficking pathways involved in processing β-amyloid precursor proteins (APP). To test this possibility, we coexpressed PS1(wt), PS1(M146L), or PS1(L286V) in HEK293 cells together with the LDL receptor, a classic glycoprotein marker that undergoes post-translational O-glycosylation in the Golgi compartment. Pulse-chase analysis of the receptor indicated that mutant presenilins had no effect on ER→Golgi transport. Similar results were obtained when the studies were carried out with cells expressing the Swedish variant of APP (SWAPP751) instead of the LDL receptor. Moreover, secretion of the soluble exodomain polypeptide fragments of SWAPP751 that arise from α-secretase and β-secretase cleavage was not markedly affected by the PS1 mutants. Despite the lack of discernible effect of the PS1 mutants on trafficking of proteins through the Golgi apparatus, they caused a substantial increase in the proportion of Aβ42 relative to total Aβ in the culture medium. The results suggest that mutant forms of PS1 cause elevated production of Aβ42 by a mechanism that is independent of a major disruption of exocytic trafficking of APP

Killing of Cancer Cells by the Photoactivatable Protein Kinase C Inhibitor, Calphostin C, Involves Induction of Endoplasmic Reticulum Stress1

Calphostin C (cal-C) is a photoactivatable inhibitor that binds to the regulatory domain of protein kinase C (PKC) and to other proteins that contain diacylglycerol/phorbol ester binding sites. Cal-C is cytotoxic against many types of cancer cells, yet the basis for this activity remains poorly understood. Here, we show that one of the earliest effects of cal-C is an impairment of glycoprotein export from the endoplasmic reticulum (ER), accompanied by formation of ER-derived vacuoles. Vacuolization of the ER is correlated with induction of an ER stress response that includes activation of c-Jun N-terminal kinase and protein kinase R-like ER kinase, as well as increased expression of CCAAT/enhancer binding protein homologous transcription factor (CHOP; GADD153). These effects of cal-C are not mimicked by staurosporine, an inhibitor of PKC catalytic activity, indicating that ER stress is due to interaction of cal-C with targets other than PKC. In conjunction with the induction of ER stress, breast carcinoma cells undergo caspase-dependent cell death with early activation of caspases 9 and 7 and cleavage of poly(ADP-ribose)polymerase. Reduction of CHOP expression by short hairpin RNA decreases the sensitivity of the cells to cal-C, suggesting that induction of apoptosis by cal-C is related, at least in part, to ER stress triggered by disruption of ER morphology and transport function. Antineoplastic drugs that work by inducting ER stress have shown promise in preclinical and clinical trials. Thus, the present findings raise the possibility that cal-C may be useful for photodynamic therapy based on induction of ER stress in some forms of cancer