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catty125699

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p53 activation can lead to apoptosis and/or cell cycle arrest
(29). To characterize the effects of ChREBP suppression on cell
cycle progression, control and ChREBP2 siRNA-transfected
HCT116 cells were assessed for propidium iodide (PI) staining
and BrdU incorporation. A 17% decrease in the number of cells
in S phase was observed, accompanied by an increase in the
percentage of cells in both G1 and G2/M phases in ChREBP2
siRNA-transfected cells compared to control cells (Fig. 3C).
p53 Contributes to the Metabolic and Growth Phenotype Induced by
ChREBP Knockdown in HCT116 Colorectal Cancer Cells. To test if p53
was required for the up-regulation of p21, MDM2, and TIGAR
mRNA in ChREBP-deficient cells, p53-/- HCT116 cells were
analyzed. The basal ChREBP level was higher in p53-/-
HCT116 cells compared with wild-type counterparts (Fig. 4B).
Transient transfection of ChREBP2 siRNA led to effective
reduction in ChREBP protein level in both p53+/+ and p53-/-
HCT116 cells (Fig. 4B). The basal mRNA level of p21 and
MDM2 was lower in p53-/- cells compared with p53+/+cells
(Fig. 4A). Furthermore, the up-regulation of p21 and MDM2
mRNA after ChREBP2 siRNA transfection was blocked by loss
of p53 (Fig. 4A). The basal mRNA and protein level of TIGAR
was slightly higher in p53-/- cells compared with p53+/+ cells
(Fig. 4 A and B). The up-regulation of TIGAR mRNA and
protein in ChREBP2 siRNA transfected cells was partially
blocked by loss of p53 (Fig. 4 A and B).
Suppression of ChREBP resulted in G1 and G2/M arrest in
p53+/+ HCT116 cells (Fig. 3C). To assess the role of p53 in
ChREBP-dependent G1 and G2/M arrest, cell cycle analysis was
performed using PI and BrdU staining for p53-/-HCT116 cells
transfected with either control or ChREBP2 siRNA. In contrast
to their wild-type counterparts, p53-/- cells transfected with
ChREBP2 siRNA readily entered S phase (Fig. 4C). However,
a greater proportion of p53-/- ChREBP-deficient cells had
accumulated in G2/M in comparison to p53-/- control cells
Attenuation of ChREBP Can Reduce Tumor Growth in Vivo. To examine
the effects of stable ChREBP gene knockdown on the in vivo
tumorigenicity of HCT116 cells, stable clones of HCT116 with
short hairpin RNA (shRNA)-mediated suppression of ChREBP
were established. Two clones (ChREBP-1 and ChREBP-12)
stably transfected with pSM2C-ChREBP which displayed reduced
ChREBP levels were chosen for further study. To determine
their tumorigenicity in vivo, vector control (Ctrl-1) and
ChREBP knockdown clones (ChREBP-1 and ChREBP-12)
were injected s.c. into nude mice and examined for tumor
formation. Each mouse was injected with a vector control
inoculation in one flank and a ChREBP shRNA clone in the
other, so that tumor comparisons were controlled for each
mouse. The growth of tumors was monitored every 3 days, and
tumors were excised and weighed 18 days post-injection. The
results demonstrated that ChREBP knockdown cells formed
smaller tumors in vivo compared with control cells (Fig. 5A).
Western blot analysis of protein lysates from the excised tumors
confirmed maintenance of the ChREBP knockdown phenotype
by the tumor cells (Fig. 5D).
To determine if decreased tumor growth was due to loss of
ChREBP expression, we generated stable cells which overexpressed
either vector control or mutant ChREBP cDNA in the
ChREBP-1 knockdown clone. A nonsuppressible ChREBP
cDNA containing several mutations in the region targeted by the
double-stranded RNA was created. Introducing the nonsuppressible
ChREBP cDNA in ChREBP shRNA knockdown cells
increased the tumor growth and rescued the tumorigenicity of
ChREBP knockdown cells to levels similar to those of control
cells (Fig. 5B). This finding demonstrates that the phenotype of
reduced tumorigenicity in ChREBP knockdown HCT116 cells
was due to the suppression of the ChREBP gene and not as a
nonspecific effect of RNAi.

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