3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

被引:11
作者
Isobe, I
Yanagisawa, K
Michikawa, M
机构
[1] Nagoya City Univ, Sch Med, Dept Legal Med, Mizuho Ku, Nagoya, Aichi 4678601, Japan
[2] Nagoya City Univ, Sch Med, Dept Dementia Res, Natl Inst Longev Sci, Nagoya, Aichi 467, Japan
关键词
AM; astrocyte; 3-(4; 5-dimethylthiazol-2-yl)-2; 5-diphenyltetrazolium bromide; endosome; phosphatidylinositol-3-OH kinase; tetrazolium salt;
D O I
10.1046/j.1471-4159.2001.00237.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of AM probably through a phosphatidylinositol-3-OH kinase [P1(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or P1(3)K is activated.
引用
收藏
页码:274 / 280
页数:7
相关论文
共 30 条
[21]   THE INTRACELLULAR COMPONENT OF CELLULAR 3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5-DIPHENYLTETRAZOLIUM BROMIDE (MTT) REDUCTION IS SPECIFICALLY INHIBITED BY BETA-AMYLOID PEPTIDES [J].
SHEARMAN, MS ;
HAWTIN, SR ;
TAILOR, VJ .
JOURNAL OF NEUROCHEMISTRY, 1995, 65 (01) :218-227
[22]   Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling [J].
Shepherd, PR ;
Withers, DJ ;
Siddle, K .
BIOCHEMICAL JOURNAL, 1998, 333 :471-490
[23]   Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin [J].
Shpetner, H ;
Joly, M ;
Hartley, D ;
Corvera, S .
JOURNAL OF CELL BIOLOGY, 1996, 132 (04) :595-605
[24]   EEA1 links PI(3)K function to Rab5 regulation of endosome fusion [J].
Simonsen, A ;
Lippé, R ;
Christoforidis, S ;
Gaullier, JM ;
Brech, A ;
Callaghan, J ;
Toh, BH ;
Murphy, C ;
Zerial, M ;
Stenmark, H .
NATURE, 1998, 394 (6692) :494-498
[25]   RABAPTIN-5 IS A DIRECT EFFECTOR OF THE SMALL GTPASE RAB5 IN ENDOCYTIC MEMBRANE-FUSION [J].
STENMARK, H ;
VITALE, G ;
ULLRICH, O ;
ZERIAL, M .
CELL, 1995, 83 (03) :423-432
[26]   Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger [J].
Stenmark, H ;
Aasland, R ;
Toh, BH ;
DArrigo, A .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (39) :24048-24054
[27]   Signalling through the lipid products of phosphoinositide-3-OH kinase [J].
Toker, A ;
Cantley, LC .
NATURE, 1997, 387 (6634) :673-676
[28]   Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells [J].
Ushio-Fukai, M ;
Alexander, RW ;
Akers, M ;
Yin, QQ ;
Fujio, Y ;
Walsh, K ;
Griendling, KK .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (32) :22699-22704
[29]   Subcellular localization of the small GTPase Rab5a in resting and stimulated human neutrophils [J].
Vita, F ;
Soranzo, MR ;
Borelli, V ;
Bertoncin, P ;
Zabucchi, G .
EXPERIMENTAL CELL RESEARCH, 1996, 227 (02) :367-373
[30]  
VLAHOS CJ, 1994, J BIOL CHEM, V269, P5241