On-line monitoring of apoptosis in insulin-secreting cells.
Diabetes
; 52(12): 2943-50, 2003 Dec.
Article
en En
| MEDLINE
| ID: mdl-14633855
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Islotes Pancreáticos
/
Apoptosis
/
Internet
/
Insulina
/
Monitoreo Fisiológico
/
Obesidad
Límite:
Animals
Idioma:
En
Revista:
Diabetes
Año:
2003
Tipo del documento:
Article
País de afiliación:
Suecia
Pais de publicación:
Estados Unidos