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|Title: ||The Role of Apc in Medulloblastoma|
|Authors: ||McCaffery, Dennis|
|Supervisor(s): ||Mason, John|
|Issue Date: ||2008|
|Abstract: ||Medulloblastomas represent the most frequent malignant brain tumour in children, and are
thought to develop in the posterior fossa of the cerebellum. Some patients with
medulloblastomas have a deficiency in the tumour suppressor gene Apc (Turcot’s syndrome).
Although the majority of medulloblastomas arise sporadically, people with Apc mutations are
92 times more likely to develop medulloblastomas. Apc encodes a very large protein known to
function as a regulator of the Wnt signalling pathway. Activation of the canonical Wnt
pathway leads to the stabilisation of beta-catenin. In response to Wnt signals, beta-catenin
translocates to the nucleus where it interacts with the LEF/TCF family of transcription factors
to activate transcription of target genes such as c-myc and cyclinD1. Mutations of Apc that
cause an increase in beta-catenin are found to be tumourgenic, whereas other mutations are not.
Therefore it is thought that the main tumour suppression function of Apc is in its ability to
destabilise and hence reduce cytoplasmic beta-catenin.
The central hypothesis of this thesis is that the loss of Apc can lead to the development of
medulloblastoma. Work from other groups has reported activation of Wnt signalling in a
proportion of primary medulloblastomas. We undertook a study to assess this by using the
cre-loxP recombination system to mutate Apc in a temporal and spatial manner. This approach
is necessary as Apc has many functions in development and Apc mutant mice (Apcmin) do not
develop past embryonic day 6.5 (E6.5). To date, there are no known cre-strains available to
mutate Apc specifically in the cerebellum at early postnatal stages, so we combined the creloxP
method with an avian retrovirus mediated method for tissue specific gene delivery
(RCAS/tv-a system), in an attempt to create a strain of mice which carried the genotype Ntv-a
+;ApcLoxP/LoxP. This would allow us to infect an RCAS-cre virus directly into the hindbrain at postnatal day 4 (P4). However subsequent genotyping of these animals showed that none
carried the desired genotype of Ntv-a +;ApcLoxP/LoxP, making it impossible for both copies of
Apc to be mutated in a mouse most likely because both the Ntv-a and Apc transgenes were
located on the same chromosome. Consistent with this, out of a total of 265 mice none were
found to have the Ntv-a +;ApcLoxP/LoxP genotype.
We then adopted an alternative method for mutating Apc by infecting ApcLoxP/LoxP mice directly
with an AdCre virus. PCR analysis showed that Apc was mutated, however the AdCre virus
did not infect cells of the cerebellum, and instead only infected the choroid plexus. In these
animals, 7 of 94 (7%) developed hydrocephalus indicating that losing Apc in the choroid
plexus may promote hydrocephalus.
Finally, to address the role of Apc in normal hindbrain development, we crossed our
ApcLoxP/LoxP mice to an En1cre strain which caused mutation in Apc from E8.5 in the midhindbrain
region. The resulting En1cre+;ApcLoxP/LoxP mutants displayed hydrocephalus in all
ventricles and an in-growth of mesenchyme tissue at the mid-hindbrain border, closely
associated with a tumour-like area of cells showing activated Wnt signalling. No mice were
found to live past E18.5.
In conclusion, the role of Apc in medulloblastoma remains unclear. Future studies could use a
different technique to mutate Apc such as crossing ApcLoxP/LoxP mice to the new nestin-creER
strain and inducting cre with administration of tamoxifen.|
|Keywords: ||Biomedical science|
|Appears in Collections:||School of Biomedical Sciences thesis and dissertation collection|
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