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 β-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 β-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 μmol/l), showing that this method worked in insulin-producing cells.
| Original language | English |
|---|---|
| Pages (from-to) | 2943-2950 |
| Number of pages | 8 |
| Journal | Diabetes |
| Volume | 52 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 1 2003 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Internal Medicine
- Endocrinology, Diabetes and Metabolism
Cite this
On-Line Monitoring of Apoptosis in Insulin-Secreting Cells. / Köhler, Martin; Zaitsev, Sergel V.; Zaitseva, Irina I.; Leibiger, Barbara; Leibiger, Ingo B.; Turunen, Mikael; Kapelioukh, Iouri L.; Bakkman, Linda; Appelskog, Ioulia B.; De Monvel, Jacques Boutet; Imreh, Gabriela; Berggren, Per Olof.
In: Diabetes, Vol. 52, No. 12, 01.12.2003, p. 2943-2950.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - On-Line Monitoring of Apoptosis in Insulin-Secreting Cells
AU - Köhler, Martin
AU - Zaitsev, Sergel V.
AU - Zaitseva, Irina I.
AU - Leibiger, Barbara
AU - Leibiger, Ingo B.
AU - Turunen, Mikael
AU - Kapelioukh, Iouri L.
AU - Bakkman, Linda
AU - Appelskog, Ioulia B.
AU - De Monvel, Jacques Boutet
AU - Imreh, Gabriela
AU - Berggren, Per Olof
PY - 2003/12/1
Y1 - 2003/12/1
N2 - 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 β-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 β-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 μmol/l), showing that this method worked in insulin-producing cells.
AB - 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 β-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 β-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 μmol/l), showing that this method worked in insulin-producing cells.
UR - http://www.scopus.com/inward/record.url?scp=10744224294&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=10744224294&partnerID=8YFLogxK
U2 - 10.2337/diabetes.52.12.2943
DO - 10.2337/diabetes.52.12.2943
M3 - Article
VL - 52
SP - 2943
EP - 2950
JO - Diabetes
JF - Diabetes
SN - 0012-1797
IS - 12
ER -