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

A B
U2OS + H2B-YFP
percentage of cells
0’ 10’ 15’ 20’ 25’ 30’ 35’
100
0’ 5’ 10’ 15’ 20’ 25’ 30’
80
60
40
20
0
n=34
n=55
- +
RPE-1
n=46
- +
U2OS
n=72
n=52
n=61
- +
Hela
no errors
mild errors (1-5)
severe errors (>5)
100nM Cpd-5
Control
Compound-5
(100nM)
Mps1, we treated U2OS, Hela and RPE-1 cells with low doses of compound-5 and simultaneously
treated them with sub-lethal doses of taxol. Indeed, combined inhibition of Mps1 and treatment
with taxol resulted in a synergistic effect on cell viability in both Hela and U2OS cells (Fig.S2A, B)
whereas such synergy was not evident in the untransformed cell line RPE-1 (Fig.S2A). Moreover,
when we treated various tumor and normal, untransformed cell lines with increasing doses of the
Mps1 inhibitor compound-5, we also observed selectivity towards tumor cells with low doses of
compound-5 in the absence of taxol (Fig.4A). All tumor cell lines lost the capacity to form colonies
upon treatment with 20-30 nM compound-5, whereas viability of both RPE-1 and BJ-Tert cells
was not affected in the presence of 50 nM compound-5 (Fig.4A). The effect on cell viability was
specifically due to Mps1 inhibition, since the Mps1-602Q mutant could overcome the lethal effects
of compound-5 in U2OS cells (Fig.S2C). The difference in sensitivity between cell lines is not due to
differences in compound-5 uptake, since treatment of RPE-1 cells with compound-5 affected the
ability to delay mitosis in the presence of nocodazole as potently as in U2OS and Hela cells (Fig.2A).
We have hypothesized before that untransformed, healthy cells could be less prone to missegregate
their chromosomes upon Mps1 depletion or inhibition when compared to aneuploid tumor
cells 299,447 . Indeed, when we followed chromosome segregation using time-lapse imaging of
various cell lines stably expressing tagged H2B, we observed that tumor cell lines were more
prone to missegregate their chromosomes upon low-dose compound-5 treatment (30 nM) when
compared to untransformed RPE-1 cells (Fig.4B). In unperturbed anaphases, tumor cell lines
showed a chromosome missegregation rate of 20-50% (including both mild and severe segregation
errors), which increased to 80-95% when 30 nM compound-5 was added (Fig.4B). In contrast to
the tumor cell lines, RPE-1 cells showed mild missegregation events in only 20% of anaphases
when treated with low dose compound-5 versus 10% in controls and almost never displayed
percentage of cells
100
80
60
40
20
0
U2OS:
n=87
n=38
- +
-
n=25
aligned
mild misalignment (1-5)
severe misalignment (>5)
n=20
- +
Mps1-602Q
30nM Cpd-5
Figure 3. Selective inhibition of Mps1 results in the induction of chromosome misalignments and segregation errors
A) (top) Representative images of U2OS cells stably expressing H2B-GFP undergoing division in the absence or presence of
missegregating chromosomes. (bottom) Quantification of time-lapse movies of RPE-1, U2OS and Hela cells stably expressing tagged
H2B. Three phenotypes were scored; anaphase without missegregations, mild missegregations (1-5 chromosomes) and severe
missegregations (>5 chromosomes). Time indicates minutes after chromosome condensation. N indicates number of cells analyzed.
Compound-5 was added 10 minutes prior to filming. B) (top) Representative images of fixed U2OS cells showing no, mild (1-5
misaligned chromosomes) and severe misalignments (>5 misaligned chromosomes). DNA was visualized using DAPI. (bottom)
Quantification of chromosome alignment of indicated U2OS cell lines after 30 minutes of treatment with MG132 in the presence
or absence of 30nM compound-5.
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Mps1 inhibition kills aneuploid cells
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