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Fig. 6. mTOR inhibition causes p53-dependent cell death by affecting Mdm2
protein levels in developing livers. (A) Analysis of Mdm2 (top) and
mTOR (bottom) inhibition in E12.5 wild-type (+/+) and
p53-/- mutant livers. Panels show TUNEL-positive cells in
representative liver sections treated with Nutlin-3 or rapamycin. Cell death
for each genotype is shown (right) as a percentage increase in the number of
apoptotic cells compared with non-injected mice
(*P<0.01; **P<0.001; Student's
t-tests). Basal levels of TUNEL labeling were indistinguishable in
non-injected wild-type and p53-/- mutant embryos. Analysis
on p53 mutants indicated that death of embryonic hepatocytes is also
controlled by unknown p53-independent pathways. (B) Western blot
analysis of total lysates of E12.5 livers of wild-type embryos treated or not
with rapamycin. Rapamycin treatment causes a reduction in the levels of Mdm2
(top), which correlates with an increased phosphorylation of p53 on the
S18 residue (pS18-p53; middle), whereas tubulin levels
are unchanged. Levels of Mdm2 protein and p53 phosphorylation were quantified
(right) after normalization with tubulin. *P<0.01;
**P<0.001; Student's t-tests. (C)
Immunohistochemical analysis of p53 phosphorylation on the S18
residue of untreated (control), wild-type and p53-/- mice injected
with rapamycin. Quantitative analysis (right) is reported as the percentage
increase of cells positive for p53 phosphorylation in rapamycin-injected
wild-type mice over the number of positive cells in non-injected mice.
*P<0.01; Student's t-tests. The numbers of
embryos analyzed in these studies are indicated (n).
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