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First published online 1 August 2006
doi: 10.1242/jcs.03069

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Casein kinase 2 inhibition decreases hypoxia-inducible factor-1 activity under hypoxia through elevated p53 protein level

Laboratory of Biochemistry and Cellular Biology, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium

View larger version (48K): [in a new window]   Fig. 1. Effect of CK2 inhibitors on HIF-1 and p53 protein level in HepG2 and HeLa cells. Cells were incubated for 5 hours under normoxia or hypoxia in the presence or the absence of different concentrations (µM) of DRB (A,C), apigenin (A,C) or of 10 µM TBB (B). Total cell lysates were tested for HIF-1, p53, phospho-Ser392 p53 and -tubulin by western blotting.

View larger version (19K): [in a new window]   Fig. 2. Effect of CK2 inhibitors on HIF-1 and p53 DNA binding activity in HeLa cells. Cells were incubated for 5 hours under normoxia or hypoxia in the presence or absence of different concentrations (µM) of DRB or apigenin (API). Nuclear extracts were tested for HIF-1 (A) or p53 (B) binding to a specific probe. Results are expressed in absorbance as a percentage of normoxic control (means ± 1 s.d.; A, n=3; B, n=6). *P<0.05, **P<0.01, ***P<0.001, NS, not significant vs. normoxia; (*)P<0.05, (**)P<0.01, (NS) not significant vs. hypoxia.

View larger version (18K): [in a new window]   Fig. 3. Effect of CK2 inhibitors on HIF-1 transcriptional activity in HepG2 cells. Cells were incubated for 16 hours under normoxia or hypoxia in the presence of different concentrations (µM) of DRB or apigenin (API). (A,B) Cells were cotransfected with pGL3-6HRE and pRL plasmids. HIF-1 activity is expressed as the ratio of native firefly luciferase to synthetic Renilla luciferase activity (means ± 1 s.d.; n=3). (C) Real-time PCR analysis of the aldolase mRNA level. Relative aldolase mRNA levels were calculated as the ratio of aldolase expression in hypoxia-incubated cells to these in control normoxic cells, both normalised to -tubulin levels. *P<0.05, **P<0.01, ***P<0.001, NS, not significant vs. normoxia; (*)P<0.05, (**)P<0.01, (***)P<0.001, (NS), not significant vs. hypoxia.

View larger version (24K): [in a new window]   Fig. 4. Effect of CK2 inhibitors on p53 transcriptional activity in HepG2 cells. Cells were incubated for 16 hours under normoxia or hypoxia in the presence or the absence of different concentrations of DRB (µM), 20 µM apigenin (API) or 10 µM TBB. (A) Cells were cotransfected with pG13-Luc and pRL plasmids. p53 activity is expressed as the ratio of native firefly luciferase to synthetic Renilla luciferase activity (means ± 1 s.d.; n=3). (B) Real-time PCR analysis of the p21 mRNA level. Relative p21 mRNA levels were calculated as the ratio of p21 expression in hypoxia-incubated cells to these in control normoxic cells, both normalised to -tubulin levels. (C) Total cell lysates were tested for p21 and -tubulin protein expression by western blotting.

View larger version (27K): [in a new window]   Fig. 5. Effect of CK2 siRNA on HIF-1 transcriptional activity in HepG2 cells. Cells were transfected with CK2 siRNA. (A) 42 hours post-transfection, cells were incubated for 6 hours under normoxia or hypoxia in the absence or in the presence of 20 µM apigenin (API). Total cell lysates were tested for CK2, HIF-1, p53 and -tubulin proteins by western blotting. (B) 24 hours post-transfection, cells were cotransfected with pGL3-6HRE and pRL plasmids and then incubated for 16 hours under normoxia or hypoxia. HIF-1 activity is expressed as the ratio of firefly luciferase to Renilla luciferase activity (means ± 1 s.d., n=3). Xtreme, cells exposed to the transfection reagent alone; neg si CTL, cells transfected with the negative control siRNA; siRNA, cells transfected with CK2 siRNA. *P<0.05 or NS, not significant vs. normoxia; (*)P<0.05 and (NS), not significant vs. hypoxia.

View larger version (14K): [in a new window]   Fig. 6. Effect of p53 overexpression on HIF-1 and p53 transcriptional activity in HepG2 cells. Cells were incubated for 16 hours under normoxia or hypoxia. Cells were cotransfected with pGL3-6HRE (A) or pG13-Luc (B), p53 expression plasmid or pCMV-myc and pRL. p53 activity is expressed as the ratio of firefly luciferase to Renilla luciferase activity (means ± 1 s.d.; n=3). *P<0.05 and ***P<0.001 vs. normoxia; (**)P<0.01 and (***)P<0.001 vs. hypoxia.

View larger version (18K): [in a new window]   Fig. 7. Effect of CK2 inhibitors on HIF-1 transcriptional activity in Hep3B cells. Cells were incubated for 16 hours under normoxia or hypoxia in the presence or the absence of different concentrations (µM) of DRB or apigenin (API). (A) Cells were cotransfected with pGL3-6HRE and pRL plasmids. HIF-1 activity is expressed as the ratio of firefly luciferase to Renilla luciferase activity (means ± 1 s.d.; n=3). (B) Real-time PCR analysis of the aldolase mRNA level. Relative aldolase mRNA levels were calculated as the ratio of aldolase expression in hypoxia-incubated cells to these in control normoxic cells, both normalised to -tubulin levels. **P<0.01, ***P<0.001 or NS, not significant vs. normoxia; (*)P<0.05 or (NS), not significant vs. hypoxia.

View larger version (35K): [in a new window]   Fig. 8. Effect of nutlin and TBB on MDM2, HIF-1 and p53 protein levels in HepG2 cells. Cells were incubated for 5 hours under normoxia or hypoxia in the presence or the absence of 10 µM TBB or of 5 µM nutlin. Total cell lysates were tested for HIF-1, p53, MDM2 and -tubulin protein expression by western blotting.

View larger version (28K): [in a new window]   Fig. 9. Effect of CK2 inhibitors on HIF-1-p53 interaction in HepG2 cells. Cells were incubated 5 hours under normoxia or hypoxia in the presence or the absence of 50 µM DRB or 20 µM apigenin (API). 50 µl of total cell lysates were tested for HIF-1, p53 and -tubulin by western blotting and the remaining 950 µl were immunoprecipitated for p53. The amount of p53 and HIF-1 protein in the immunoprecipitated samples was detected by western blot. CTL(-) are samples immunoprecipitated without anti-p53 antibodies.

View larger version (16K): [in a new window]   Fig. 10. Effect of wild-type and mutated p53 overexpression on HIF-1 transcriptional activity in HepG2 cells. Cells were incubated for 16 hours under normoxia or hypoxia. Cells were cotransfected with pGL3-6HRE (A) or pG13-Luc (B), p53 expression plasmid or pCMV-myc and pRL. HIF-1 or p53 activity is expressed as the ratio of firefly luciferase to Renilla luciferase activity (means ± 1 s.d., n=3). *P<0.05, **P<0.01 or ***P<0.001 vs. normoxia; (***)P<0.001 or (NS), not significant vs. hypoxia.

View larger version (22K): [in a new window]   Fig. 11. Effect of pifithrin- on HIF-1 and p53 protein level and transcriptional activity in HepG2 cells. (A,B) Cells were incubated for 5 hours under normoxia or hypoxia in the presence of different concentrations (µM) of pifithrin- or 50 µM DRB. Total cell lysates were tested for HIF-1 (A), total p53 (B) and -tubulin by western blotting. (C,D) Cells were incubated for 6 hours under normoxia or hypoxia in the presence of 25 µM pifithrin and/or 50 µM DRB. Real-time PCR analysis of the aldolase (C) and p21 (D) mRNA level was performed. Relative aldolase or p21 mRNA levels were calculated as the ratio of aldolase or p21 expression in hypoxia-incubated cells to these in control normoxic cells, both normalized to -tubulin levels.

View larger version (16K): [in a new window]   Fig. 12. Schematic representation of the possible role of CK2 in p53-dependent regulation of HIF-1 activity. Under hypoxia, elevated CK2 activity triggers p53 degradation, thus relieving p53 inhibition on HIF-1, leading to active HIF-1. When CK2 is inhibited, p53 is stabilized, competes with HIF-1 for p300, lowering HIF-1 activity.

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