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

the mice did not receive any doxycycline, one third of the mice were given doxycycline in their drinking
water from day 0 onwards, and one third of the mice received doxycycline in their drinking water
from day 7 onwards. Clearly, tumor growth was reduced in the mice that received doxycycline in their
drinking water (Fig.3A). Tumor growth was initially most efficiently inhibited in the group that received
doxycycline on day 0, but almost all of the mice displayed progressive tumor growth (Fig.3A). Thus,
the conditional inactivation of Mps1 that is achieved in this system is capable of slowing down tumor
growth, but it is unable to fully arrest tumor growth. Whether this is due to incomplete inactivation of
Mps1 in vivo is currently unclear. Alternatively, tumor cells in vivo might be less dependent on Mps1
function for proper chromosome segregation than tumor cells grown in vitro. To analyze if tumors
in vivo had lost the ability to shut down Mps1 expression in response to doxycycline, we isolated
tumors from the mice that received doxycycline in their drinking water and analyzed their growth in
vitro. Importantly, their viability and mitotic checkpoint function was again severely compromised
when doxycycline was added to the culture medium, indicating that these cells had not simply lost
conditional expression of the Mps1 shRNA (Fig.3B). However, this observation does not discriminate
between the possibility that doxycycline does not efficiently reach the tumors in situ, or the possibility
that in vivo tumor growth is less dependent on Mps1 function. In order to resolve this we will have
to examine the effect of doxycycline on chromosome segregation in vivo. For this purpose we wish to
perform intravital imaging of cell division of the xenografted tumor cells. Nonetheless, our data show
that inactivation of Mps1 can partially inhibit tumor growth and suggest that targeting Mps1 function
might be a useful anti-cancer strategy.
Materials and Methods
Tissue Culture, Transfections and Treatments
LS174T cells were grown in DMEM (Lonza) with 10% Tet-approved FCS (Clontech), supplemented with
pen/strep (Invitrogen) and ultraglutamine (Lonza). Taxol, MG132 and doxycycline (used at 1mg/ml) were
from Sigma. LS174T cells expressing TetR were infected with retrovirus carrying pSuperior-retro-puro-
Mps1 and selected with 2µg/ml puromycin. Single colonies were selected after replating 1-2 cells/well.
Immunofluorescence Microscopy
Cells plated on 12mm coverslips were harvested after 90 minutes MG132 treatment. Fixation
was done using 4% PFA in PEM buffer. CREST was incubated O/N in PBS 3% BSA. Anti-human
Alexafluor647 and DAPI were incubated in PBS 0, 1% Tween. Stained coverslips were mounted with
Vectashield Mounting Medium (Vector). Images were acquired on a Zeiss 510 Meta confocal laser
scanning microscope with a 63X/1.4NA Plan-ApoChromat objective using the Zeiss LSM software.
Chromosome spreads
Nocodazole was added for 4 hours to the medium to enrich for mitotic cells. Cells were treated with
0.75 M KCl at 37ºC for 10 minutes, centrifuged at 2000 rpm and fixed for 20 minutes with Methanol:
Acetic Acid (3:1). Fixation procedure was repeated 3 times. Samples were collected in Methanol
and DAPI to stain for DNA. Chromosome spreads were created by allowing the drops to fall from
30 cm height onto glass slides. Images were acquired as described above for Immunofluorescence.
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Chromosome missegregations kill tumor cells
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