Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis

被引:81
作者
Bullok, Kristin E.
Maxwell, Dustin
Kesarwala, Aparna H.
Gammon, Seth
Prior, Julie L.
Snow, Margaret
Stanley, Sam
Piwnica-Worms, David
机构
[1] Washington Univ, Sch Med, Mallinckrodt Inst Radiol, Mol Imaging Ctr,Dept Mol Microbiol, St Louis, MO 63110 USA
[2] Washington Univ, Sch Med, Dept Mol Biol & Pharmacol, St Louis, MO 63110 USA
关键词
D O I
10.1021/bi061959n
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
Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ(647), was synthesized and K-m, k(cat), and K-i values were biochemically characterized. Specific cleavage of TcapQ(647) by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ(647) with a k(cat) 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ(647) cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C-6-ceramide, TcapQ(647) detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ(647), a noncleavable all D-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ(647) to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ(647) represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.
引用
收藏
页码:4055 / 4065
页数:11
相关论文
共 40 条
[1]   ISOLATION AND GENETIC-CHARACTERIZATION OF HUMAN KB-CELL LINES RESISTANT TO MULTIPLE-DRUGS [J].
AKIYAMA, SI ;
FOJO, A ;
HANOVER, JA ;
PASTAN, I ;
GOTTESMAN, MM .
SOMATIC CELL AND MOLECULAR GENETICS, 1985, 11 (02) :117-126
[2]  
ALANIAN RV, 1997, J BIOL CHEM, V272, P9677
[3]  
Bernacchi S, 2001, Nucleic Acids Res, V29, pE62, DOI 10.1093/nar/29.13.e62
[4]   Synthesis and characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis [J].
Bullok, K ;
Piwnica-Worms, D .
JOURNAL OF MEDICINAL CHEMISTRY, 2005, 48 (17) :5404-5407
[5]   An intuitive look at the relationship of Ki and IC50:: A more general use for the Dixon plot [J].
Burlingham, BT ;
Widlanski, TS .
JOURNAL OF CHEMICAL EDUCATION, 2003, 80 (02) :214-218
[6]   Role of early plasma membrane events in chemotherapy-induced cell death [J].
Dimanche-Boitrel, MT ;
Meurette, O ;
Rebillard, A ;
Lacour, S .
DRUG RESISTANCE UPDATES, 2005, 8 (1-2) :5-14
[7]   Caspase inhibitors [J].
Ekert, PG ;
Silke, J ;
Vaux, DL .
CELL DEATH AND DIFFERENTIATION, 1999, 6 (11) :1081-1086
[8]   New approaches and therapeutics targeting apoptosis in disease [J].
Fischer, U ;
Schulze-Osthoff, K .
PHARMACOLOGICAL REVIEWS, 2005, 57 (02) :187-215
[9]  
Glantz SA., 1987, PRIMER BIOSTATISTICS, P379
[10]   Caspases:: key players in programmed cell death [J].
Grütter, MG .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 2000, 10 (06) :649-655