Cancer Res Treat.  2006 Jun;38(3):168-177.

Cell Cycle Regulatory Protein Expression Profiles by Adenovirus p53 Infection in Human Papilloma Virus-associated Cervical Cancer Cells

• Lee YS
• Bae SM
• Kwak SY
• Park DC
• Kim YW
• Hur SY
• Park EK
• Han BD
• Lee YJ
• Kim CK
• Kim DK
• Ahn WS

Abstract

PURPOSE: The tumor suppressor gene, p53, has been established as an essential component for the suppression of tumor cell growth. In this study, we investigated the time-course anticancer effects of adenoviral p53 (Adp53) infection on human ovarian cancer cells to provide insight into the molecular-level understanding of the growth suppression mechanisms involved in Adp53-mediated apoptosis and cell cycle arrest.
MATERIALS AND METHODS
Three human cervical cancer cell lines (SiHa, CaSki, HeLa and HT3) were used. The effect of Adp53 infection was studied via cell count assay, cell cycle analysis, FACS, Western blot and macroarray assay.
RESULTS
Adp53 exerts a significant role in suppressing cervical cancer cell growth. Adp53 also showed growth inhibitory effects in each cell line, and it induced apoptosis and cell cycle arrest. Adp53 differentially regulated the expression of genes and proteins, and the gene expression profiles in the SiHa cells revealed that the p21, p53 and mdm2 expressions were significantly up-regulated at 24 and 48 hr. Western blot shows that the p21 and p53 expressionlevels were significantly increased after Adp53 infection. In addition, in all cell lines, both the CDK4 and PCNA protein expression levels were decreased 48 h after Adp53 infection. Cell cycle arrest at the G1 phase was induced only in the SiHa and HeLa cells, suggesting that exogenous infection of Adp53 in cancer cells was significantly different from the other HPV-associated cervical cancer cells.
CONCLUSION
Adp53 can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest, as well as through the regulation of the cell cycle-related proteins. The Adp53-mediated apoptosis can be employed as an advanced strategy for developing preferential tumor cell-specific delivery.

Keyword

Adenoviral p53; Cervical cancer; Growth arrest; Apoptosis; Gene therapy

MeSH Terms

Adenoviridae*
Apoptosis
Blotting, Western
Cell Count
Cell Cycle Checkpoints
Cell Cycle*
Cell Line
G1 Phase
Genes, Tumor Suppressor
Genetic Therapy
HeLa Cells
Humans*
Ovarian Neoplasms
Papilloma*
Proliferating Cell Nuclear Antigen
Transcriptome
Uterine Cervical Neoplasms*
Proliferating Cell Nuclear Antigen

Figure

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  Fig. 1 (A) Time course of the p53 protein expression after Adp53 (50 MOI) infection in the SiHa cell line. The blots are shown with the number of days after infection being indicated above the lanes; 0 days (lane 1), 2 days (lane 2), 4 days (lane 3) and 6 days (lane 4). The cells were harvested at the indicated days to prepare the crude cell extracts. Ten µg of proteins were subjected to 10% SDS-PAGE, and then they were transferred to a membrane and probed with antibodies recognizing p53. (B) Growth inhibition of the SiHa cells following Adp53 infection, as determined by cell count assay. The SiHa cells were inoculated at a density of 105 cells/well in 12-well plates in triplicate at 24 h before Adp53 and LacZ infection. After infection, the SiHa cells were cultured for the indicated periods, and then the cell lysates were prepared for counting under a microscope. The mean cell counts for triplicate wells were plotted. ♦Control, ▪AdLacZ infected, ▴Adp53-infected.

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  Fig. 2 Gene expression profiles of the SiHa cell at 24 h (B) and 48 h (C) after Adp53 infection. The controls (A) were not infected by Adp53. The total RNA was obtained from SiHa cells after Adp 53 infection via the apoptosis pathway GEArray Q Series Human Cell Cycle Gene Array. Selected hybridization signals refer to controls or examples of the differentially expressed genes, namely: 1. human housekeeping genes (GAPD); 2. human housekeeping genes (ACTB); 3. blank; 4. negative control (PUC18); 5. positive control (PPIA); 6. positive control (RPL13A); 7. TP53; 8. ATM; 9. CCNF; 10. MDM2; 11. FOXM1; 12. CCNG2; 13. E2F2 and 14. CCNE1. (D) The protein expression on western blot analysis in the SiHa cells after infection with the controls, AdLacZ and Adp53 for 24 and 48 hr.

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  Fig. 3 Protein expression in the CaSki, HeLa and HT3 cancer cells after infection with the control, AdLacZ and Adp53 for 24 and 48 hr.

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  Fig. 4 Cell cycle arrest in the G0/G1, S and G2/M phase by the control, AdLacZ and Adp53 in SiHa, CaSki, HeLa and HT3 cells. The cells (105 cells/well) were cultured in a 12-well plate overnight in triplicate, and then the control cells (▪), LacZ (▒) and infected with Adp53 (□) were cultured at 50 MOI for 2 days. The cell cycle distribution was determined by propidium iodide staining. The mean value of three experiments±standard deviation is shown.

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  Fig. 5 Effects of Adp53 on apoptosis. The SiHa, CaSki, HeLa and HT3 cells (105 cells/well) were cultured in 12-well plates overnight in triplicate, and then the control cells or the cells infected with adp53 or LacZ were cultured at 50 MOI for 2 days. For the apoptosis analysis, the cells were stained with annexin V. The stained cells were analyzed via FACS. The thin line represents the negative control, the dotted line represents AdLacZ and the thick line represents Adp53.

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