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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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zerohead
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catty125699
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