The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes

Blommaart, E. F. C.; Krause, U.; Schellens, J. P. M.; Vreeling-Sindelárová, Helena; Meijer, Alfred J.

doi:10.1111/j.1432-1033.1997.0240a.x

Cited by 783 publications

(630 citation statements)

References 42 publications

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“…8). This is in accordance with data previously reported for hepatocytes [20,42] and other cells [43,44]. This is conceivably because the IC 50 value (the concentration causing the half-maximal inhibition) is 10-fold smaller for cell-free preparations than for intact-cell preparations as a result of limited permeability of plasma membranes to the inhibitor [24].…”

Section: Involvement Of Specific Receptors In the Egf Actions Observesupporting

confidence: 92%

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture

Fujioka

2001

European Journal of Biochemistry

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Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1±18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15±30 s and with similar EC 50 s in the nm range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor b-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.Keywords: signal transduction; tyrosine kinase receptor; tyrosine phosphorylation.Membrane receptors having intrinsic protein tyrosine kinase activity are known to be responsible for promoting proliferation of hepatocytes [1] as well as many other types of cells [2]. If kept in a serum-free medium, DNA synthesis in hepatocytes occurs with the addition of growth factors, among which epidermal growth factor (EGF) is effective in vivo and in culture [3,4] as a result of its binding to typical tyrosine kinase related receptors. The EGF receptor (EGFR) is the prototype of the type I ErbB subfamily, which includes three additional members, ErbB-2/Neu, ErbB3 and ErbB4 [5]. All members of the ErbB subfamily have a domain that serves as a docking site for specific SH-2 domains, which are present in signalling proteins such as the p85 regulatory subunit of phosphatidylinositol (PtdIns) 3-kinase. EGF has been shown to activate PtdIns 3-kinase, although it does not bind directly to the EGFR but is instead activated via association with p120 cbl [6,7] or Gab1 [8], which are phosphorylated on tyrosine residues by the growth factor. PtdIns 3-kinase may also interact with heterodimers of other members of the EGFR family, such as ErbB3, in response to EGF [9,10].PtdIns 3-kinase plays important roles in a wide variety of biological responses to hormones, growth factors and cytokines [11]. The typical responses s...

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Section: Involvement Of Specific Receptors In the Egf Actions Observesupporting

confidence: 92%

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture

Fujioka

2001

European Journal of Biochemistry

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“…It has long been known that 3-MA inhibits autophagy, 26 and it was recently found to be an inhibitor of PI3K. 27 PI3K is a conserved family of lipid kinases that catalyze the phosphorylation of the position 3 of the inositol ring of phosphoinositides. [28][29][30] They produce lipids that are involved in cell proliferation, differentiation, apoptosis, autophagy, cytoskeletal organization, and membrane trafficking.…”

Section: Discussionmentioning

confidence: 99%

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

et al. 2004

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Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 lM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein lightchain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H þ -ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.

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“…13 Amino-acid stimulation of S6 phosphorylation was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002. 26 Unexpectedly, and in contrast to the effect of rapamycin, these two compounds inhibited autophagy in the absence of amino acids. 26 The explanation of this apparent anomaly was found by a study carried out with HT-29 cells, a human colon cancer cell line.…”

Section: Inhibition Of Autophagy By Amino Acidsmentioning

confidence: 94%

“…26 Unexpectedly, and in contrast to the effect of rapamycin, these two compounds inhibited autophagy in the absence of amino acids. 26 The explanation of this apparent anomaly was found by a study carried out with HT-29 cells, a human colon cancer cell line. These experiments have shown that the product of class-III PI3K, phosphatidylinositol 3-phosphate (PI(3)P), is required for autophagy, whereas the products of class-I PI3K, phosphatidylinositol 3,4, bisphosphate (PI(3,4)P 2 ), and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ), have inhibitory effects.…”

Section: Inhibition Of Autophagy By Amino Acidsmentioning

confidence: 94%

Autophagy and signaling: their role in cell survival and cell death

2005

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Macroautophagy is a vacuolar, self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by the lysosome. The discovery of the ATG genes has provided key information about the formation of the autophagosome, and about the role of macroautophagy in allowing cells to survive during nutrient depletion and/or in the absence of growth factors. Two connected signaling pathways encompassing class-I phosphatidylinositol 3-kinase and (mammalian) target of rapamycin play a central role in controlling macroautophagy in response to starvation. However, a considerable body of literature reports that macroautophagy is also a cell death mechanism that can occur either in the absence of detectable signs of apoptosis (via autophagic cell death) or concomitantly with apoptosis. Macroautophagy is activated by signaling pathways that also control apoptosis. The aim of this review is to discuss the signaling pathways that control macroautophagy during cell survival and cell death.

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The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes

Cited by 783 publications

References 42 publications

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

Autophagy and signaling: their role in cell survival and cell death

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