Effect of 3-methyladenine on the Fusion Process of Macropinosomes in EGF-stimulated A431 Cells

Araki, Nobuhito; Hamasaki, Masao; Egami, Youhei; Hatae, Tanenori

doi:10.1247/csf.06029

Cited by 60 publications

(55 citation statements)

References 50 publications

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“…Consistently, the local turnover of phosphoinositides by PLC and PI3-kinase activities has been suggested to regulate membrane curvature and related processes during bulk-membrane endocytosis (Araki et al, 2006). We have proposed that RBO might modify PtdIns(4,5)P 2 acyl side chains, making it inaccessible to subsequent conversion to other phospholipid species, which may in turn be required for bulk endocytosis.…”

Section: Discussionmentioning

confidence: 75%

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Vijayakrishnan

Woodruff

Broadie

2009

Journal of Cell Science

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Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis within minutes following a temperature shift from 25°C to 37°C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbots displays a strong synergistic interaction with the Syntaxin-1A TS allele syx3-69, recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbots showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbots;syx3-69 double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbots synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbots displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

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Section: Discussionmentioning

confidence: 75%

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Vijayakrishnan

Woodruff

Broadie

2009

Journal of Cell Science

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“…3-MA specifically inhibits the ability of class III phosphatidylinositol 3-kinase (PI3KC) to form an autophagosome initiation complex with BECN1, thereby preventing autophagosome formation. 1 Moreover, 3-MA inhibits the ER UPR by suppressing the activation of the ER stress transducers PERK, IRE1, and ATF-6 and their downstream targets GRP78, ATF-4, CHOP/GADD-153, EIF-2a, JNK, and XBP-1.…”

Section: Grp78-dependent Autophagy Modulates Temozolomide Cytotoxicitymentioning

confidence: 99%

Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy

Golden

Cho²,

Jahanian

et al. 2014

FOC

153

117

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Object In a recent clinical trial, patients with newly diagnosed glioblastoma multiforme benefited from chloroquine (CQ) in combination with conventional therapy (resection, temozolomide [TMZ], and radiation therapy). In the present study, the authors report the mechanism by which CQ enhances the therapeutic efficacy of TMZ to aid future studies aimed at improving this therapeutic regimen. Methods Using in vitro and in vivo experiments, the authors determined the mechanism by which CQ enhances TMZ cytotoxicity. They focused on the inhibition-of-autophagy mechanism of CQ by knockdown of the autophagy-associated proteins or treatment with autophagy inhibitors. This mechanism was tested using an in vivo model with subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ. Results Knockdown of the autophagy-associated proteins (GRP78 and Beclin) or treatment with the autophagy inhibitor, 3-methyl adenine (3-MA), blocked autophagosome formation and reduced CQ cytotoxicity, suggesting that autophagosome accumulation precedes CQ-induced cell death. In contrast, blocking autophagosome formation with knockdown of GRP78 or treatment with 3-MA enhanced TMZ cytotoxicity, suggesting that the autophagy pathway protects from TMZ-induced cytotoxicity. CQ in combination with TMZ significantly increased the amounts of LC3B-II (a marker for autophagosome levels), CHOP/GADD-153, and cleaved PARP (a marker for apoptosis) over those with untreated or individual drug-treated glioma cells. These molecular mechanisms seemed to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ displayed higher levels of CHOP/GADD-153 than did untreated or individual drug-treated tumors. Conclusions Taken together, these results demonstrate that CQ blocks autophagy and triggers endoplasmic reticulum stress, thereby increasing the chemosensitivity of glioma cells to TMZ.

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“…20 Initiation of the autophagy process requires class III PI3K (PI3KC3) and its complex formation with Beclin1 and the myristylation protein kinase p150. This initiation process could be suppressed by 3-methyladenine (3-MA), a specific inhibitor of endogenous lysosomal protein degradation that targets PI3KC3 but not the other PI3Ks, 21 as well as wortmannin, another PI3K inhibitor. The further elongation of the autophagosome membrane is mediated by two ubiquitin-like conjugation systems.…”

mentioning

confidence: 99%

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

et al. 2008

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In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3 0 -kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

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Effect of 3-methyladenine on the Fusion Process of Macropinosomes in EGF-stimulated A431 Cells

Cited by 60 publications

References 50 publications

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

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