title: On-line monitoring of apoptosis in insulin-secreting cells.
abstract: Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.
authors.affiliation: Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden. martin.kohler@molmed.ki.se
authors.forename: Martin
authors.initials: M
authors.lastname: Köhler
authors.forename: Sergei V
authors.initials: SV
authors.lastname: Zaitsev
authors.forename: Irina I
authors.initials: II
authors.lastname: Zaitseva
authors.forename: Barbara
authors.initials: B
authors.lastname: Leibiger
authors.forename: Ingo B
authors.initials: IB
authors.lastname: Leibiger
authors.forename: Mikael
authors.initials: M
authors.lastname: Turunen
authors.forename: Iouri L
authors.initials: IL
authors.lastname: Kapelioukh
authors.forename: Linda
authors.initials: L
authors.lastname: Bakkman
authors.forename: Ioulia B
authors.initials: IB
authors.lastname: Appelskog
authors.forename: Jacques Boutet
authors.initials: JB
authors.lastname: de Monvel
authors.forename: Gabriela
authors.initials: G
authors.lastname: Imreh
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Berggren
mesh_terms: D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D020407:Internet; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008991:Monitoring, Physiologic; D009765:Obesity / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D017785:Photons
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D007202:Indicators and Reagents; D007328:Insulin; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases