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First published online 13 June 2007
doi: 10.1242/jcs.002345

Journal of Cell Science 120, 2284-2294 (2007)
Published by The Company of Biologists 2007
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Role of p53 in antioxidant defense of HPV-positive cervical carcinoma cells following H2O2 exposure

Boxiao Ding1,2, Sung Gil Chi3, Se Heon Kim4, Suki Kang1, Jae Ho Cho5, Dong Su Kim6 and Nam Hoon Cho1,2,*

1 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
2 Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
3 School of Life Sciences and Biotechnology, Korea University, Seoul, Korea
4 Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Korea
5 Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Korea
6 Genomine Research Division, Genomine Inc., Pohang, Korea


Figure 1
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  		Fig. 1. Expression of p53 and antioxidant enzymes in human cervical carcinoma cell lines and in HFKs. (A) Western blot analysis for p53, GPX1, SESN2 (Hi95), SOD2 and SOD1. GAPDH was used as a loading control. (B) RT-PCR for SESN1 and ACTB (β-actin).

 

Figure 2
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  		Fig. 2. Cell viability of HFK, SiHa, CaSki, HeLa, ME180 and C33A cells after H2O2 treatment as detected by Trypan Blue assay. The data represent the means of pooled results from five independent experiments.

 

Figure 3
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  		Fig. 3. Reduction of p53 levels in SiHa cells transfected with p53 siRNA or nonspecific siRNA. (A) Schematic illustration of the U6 promoter-driven shRNA constructs containing the sequence targeting p53 and the scrambled sequence. (B) Upper panel: Expression levels of p53, GPX1, SESN2, SOD2 and SOD1 in control or in si-p53-expressing cells, as detected by western blot analysis. GAPDH was used as a loading control. Middle panel: Expression of SESN1 (T2) and ACTB as detected by RT-PCR. Lower panel: Quantification of p53 in western blots above normalized to GAPDH expression.

 

Figure 4
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  		Fig. 4. Effect of deficiency in p53 on intracellular ROS level in the SiHa cell line. (A) Intracellular ROS levels in SiHa cells measured by CM-H2DCFDA staining and FACS analysis at different time intervals after transfection with p53 siRNA or nonspecific siRNA. (B) ROS levels expressed as the mean ± s.d. intensity of cell fluorescence. *P<0.001 compared with the corresponding negative control by Student's t-test. (C) FACS analysis of CM-H2DCFCA-stained cells treated with NAC (5 mM, 6 hours) 48 hours after transfection with p53 siRNA or nonspecific siRNA. *P<0.01 compared with the corresponding untreated group by Student's t-test. (D) Cytosolic superoxide levels in SiHa cells transfected with p53 siRNA or nonspecific siRNA measured by DHE staining and FACS analysis. (E) Mitochondrial superoxide levels in SiHa cells transfected with p53 siRNA or nonspecific siRNA measured by MitoSOX RED staining and FACS-analysis.

 

Figure 5
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  		Fig. 5. Characterization of SiHa cells with inhibited expression of p53. (A) Growth curves of SiHa/N and SiHa/sip53 cells untreated or treated with 5 mM NAC. *P>0.98 by Student's t-test. (B) Cell cycle distribution of SiHa/N and SiHa/sip53 was analyzed 48 hours after transfection.

 

Figure 6
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  		Fig. 6. ROS levels in HeLa (A), CaSki (B) and ME180 (C) cells 48 hours after inhibition of p53 with siRNA (sip53). For a negative control we used nonspecific siRNA. *P>0.95 compared with the corresponding cells with negative control vector (Student's t-test). (D) Expression levels of p53 mRNA in both control and si-p53-expressing cells 48 hours after transfection with corresponding plasmid, as detected by RT-PCR.

 

Figure 7
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  		Fig. 7. Effect of p53 deficiency on intracellular ROS level in SiHa cells undergoing oxidative stress. (A) ROS levels in untreated and H2O2-treated (12 hours) SiHa and SiHa cells with p53 inhibited with siRNA, as detected by FACS after CM-H2DCFCA-staining. (B) ROS levels were expressed as the mean ± s.d. intensity of cell fluorescence. *P<0.001 compared to the corresponding negative control by Student's t-test.

 

Figure 8
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  		Fig. 8. Inhibition of p53-sensitized SiHa cells to H2O2-induced oxidative damage. (A) 48 hours after transfection with negative control plasmid (SiHa/N) or p53 shRNA plasmid (SiHa/sip53), cells were treated with various concentrations of H2O2 for either 12 hours or 24 hours, after which time cell viability was measured with an XTT assay. Results shown are expressed as mean ± s.d. of triplicate microcultures. *P<0.01 compared with the corresponding negative control using Student's t-test. (B) Apoptosis and death levels of SiHa/N and SiHa/sip53 cells 12 hours after treatment with 0.1, 0.5 and 1 mM of H2O2 as detected by FACS after Annexin V/PI staining. Apoptotic (Annexin V+/PI–), dead (Annexin V+/PI+) and alive (Annexin V–/PI–) populations were readily identified. The rates of apoptotis and death are shown in upper right and lower right panels, respectively. *P<0.01 compared with the corresponding untreated cells (Student's t-test).

 

Figure 9
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  		Fig. 9. Reduction of p53 expression increased DNA oxidation in SiHa cells. (A) 8-oxo-dG level in SiHa cells expressing negative control plasmid (SiHa/N) or p53 shRNA plasmid (SiHa/sip53) 48 hours after transfection. (B) 48 hours after transfection with negative control plasmid or p53 shRNA plasmid, cells were treated with the indicated concentrations of H2O2 for 1 hour. Quantification of 8-oxo-dG staining was performed after FACScan analysis using CellQuest software. 8-oxo-dG levels are expressed as the mean ± s.d. intensity of cell fluorescence. *P<0.01 compared with the corresponding cells with negative control vector by Student's t-test.

 

Figure 10
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  		Fig. 10. p53 facilitated the removal of 8-oxo-dG residues from DNA in whole SiHa cells. (A) 48 hours after transfection with negative control plasmid (SiHa/N) or p53 shRNA plasmid (SiHa/sip53), cells were treated with 0.5 mM H2O2 for 1 hour. The cells were washed with fresh medium and then incubated in fresh medium for the indicated times. 8-oxo-dG levels were measured at each time point as indicated and expressed as fold of the control sample. (B) The percentage of remaining 8-oxo-dG residues in cellular DNA was plotted as a function of chase time. The t1/2 value reflects the estimated time for levels of 8-oxo-dG to decrease to 50% of its original level.

 

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© The Company of Biologists Ltd 2007