Chromosome segregation errors: a double-edged sword - TI Pharma

treatment respectively. In line with these data, C26 cells also showed a significant increase in CFP/
YFP ratio following docetaxel treatment (a ratio of 1.0 before and 1.3 and 1.7 after 20 and 48 hours
of docetaxel treatment respectively) (Fig.3B). To confirm our FRET results, we also performed
fluorescence lifetime imaging microscopy (FLIM) (Fig.3C). Caspase-3 activation results in loss of
FRET due to cleavage of the DEVD motif (Fig.2A) and therefore should cause an increase in the CFP
fluorescence lifetime 610 . In line with our CFP/YFP ratio measurements, FLIM analysis of C26 cells after
docetaxel treatment revealed a significant increase in CFP lifetime from, on average, 1.3 ns before
docetaxel treatment to 1.8 ns at two days after docetaxel treatment (Fig.3C, D). The observed increase
in caspase-3 activity was not caused by insertion of the imaging window or by light exposure during
imaging, since FLIM analysis of tumor sections from within the tumor (not reached by either the
window or the laser beam) showed that FRET was even more frequently lost in these areas (Fig.S2A).
Together, these data show that caspase-3 activity, and therefore the onset of apoptosis, is induced
upon treatment with docetaxel both in vitro and in vivo.
A In vitro B
In vivo
7
6
5
4
6
SW480
*
*
*
3
2
4
1
2
0
-
0
+
16
+
24
+
24*
Docetaxel
hours
0
Before Day1 Day2
After Dtx
C C26
25mg/kg Dtx: D
CFP/YFP ratio
Increase in CFP-Fluoresc.
Lifetime
1.25 ns
Alive
2.5 ns
Dead
CFP/YFP ratio
-
+
Fluorescence Lifetime (nsec)
Figure 3. Docetaxel increases caspase-3 activity in vitro and in vivo
A) Quantification of the CFP-YFP ratio of SW480 cells in vitro under indicated conditions. 24* indicates that only apoptotic cells
were analyzed, in all other cases (0, 16, 24) mitotic cells were quantified. Apoptotic and mitotic cells were determined according
to morphology. Apoptotic: membrane blebbing, fragmented DNA. Mitotic: condensed DNA, rounded membrane morphology.
10 cells were quantified per condition. Average + SEM is shown. B) Quantification of CFP-YFP ratios in single cells in vivo of the
same tumor fields before, 20 hours (day1) and 48 hours (day2) after 25mg/kg docetaxel treatment (SW480-left, C26-right).
Representative experiment of 3 independent experiments per cell line is shown. One dot represents one cell. Line indicates average
+ SD. n.s.: not-significant (student t-test, paired, P>0.05), *: significant (student t-test, unpaired, p0.05), *: significant (student
t-test, unpaired, p