A p53-independent apoptotic mechanism of adenoviral mutant E1A was involved in its selective antitumor activity for human cancer

Abstract

The conserved regions (CR) of adenoviral E1A had been shown to be necessary for disruption of pRb-E2F transcription factor complexes and induction of the S phase. Here we constructed a mutant adenoviral E1A with Rb-binding ability absent (E1A 30-60aa and 120-127aa deletion, mE1A) and investigated its antitumor capacities in vitro and in vivo. The mE1A suppressed the viability of tumor cells as efficiently as the wild type E1A, and there was no cytotoxic effect on normal cells. Although the mE1A arrested tumor cell cycle with the same manner as E1A, the former played a different role on cell cycle regulation compared with E1A in normal cells, which might contribute to its selective antitumor activity. E1A and mE1A had accumulated inactive p53, decreased the expression of mdm2, Cdkn1a (also named p21), increased p21's nuclear distribution and induced tumor cell apoptosis in a p53-indenpent manner. Further, E1A or mE1A significantly suppressed tumor growth in subcutaneous hepatocellular carcinoma xenograft models. Especially, tumor-bearing mice treated with mE1A had higher survival rate than those treated with E1A. Our data demonstrated that mutant adenoviral E1A significantly induced tumor cell apoptosis in a p53-indenpednt manner and had selective tumor suppressing ability. The observations of adenoviral E1A mutant had provided a novel mechanism for E1A's complex activities during infection.

Keywords: Rb; adenoviral E1A; cell apoptosis; hepatocellular carcinoma; p53.

Figure 1. The mutant adenoviral E1A lost the ability to bind to cellular Rb protein

A. Schematic representation of adenoviral E1A 13s and the deletion mutant E1A structures. mE1A: mutant E1A with deletion 30-60aa and 120-127aa. B. Schematic diagrams of Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A and Ad-DC315-EGFP structures. C. Hela, HepG2, HK-2, H1299, U2OS, HCT116 p53+/+ and HCT116 p53−/− cells were lysed and lysates were to detect Rb and p53 expression by western blotting. GAPDH served as the protein loading control. D. HepG2 cells were infected with Ad-DC315, Ad-DC315-EGFP, Ad-DC315-E1A and Ad-DC315-mE1A at MOI=20. Cells were lysed at 48 hours post infection and subjected to immunoprecipitation with anti-E1A antibody. Protein Rb was detected by immunoblotting analysis (IB).

Figure 2. Ad-DC315-mE1A has antitumor activity against human cancer cells but not normal cells

H1299, Hela, HepG2, U2OS, HK2 cells were infected with Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A or Ad-DC315-EGFP at the indicated dose (0.1, 1, 10,100) for 4 days or for the indicated days (1-5 days) at MOI=10. Cell viability was measured by MTT assay. The value of MOI=0 or day 1 was set at 1. Cell viability data was expressed as mean values ±SD (n=6). Statistical significance was determined using Student's t test. *P<0.05, ** P<0.01.

Figure 3. Ad-DC315-mE1A or Ad-DC315-E1A affected cell cycle distribution in different manners

H1299, Hela, HepG2 and U2OS cells were infected with Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A or Ad-DC315-EGFP at MOI=20 for 72h. The cell cycle was assessed using flow cytometry analysis. Three independent trials were performed and the mean value was shown on the right.

Figure 4. Ad-DC315-mE1A or Ad-DC315-E1A induced apoptosis in a p53-independent manner

HCT116 p53+/+, HCT116 p53−/−, H1299, Hela, HepG2 and U2OS cells were infected with Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A or Ad-DC315-EGFP at MOI=20 for 72h. The early and late stage apoptosis was quantified using flow cytometry analysis. Representive data was shown. Statistical significance was determined using Student's t test. *P<0.05, ** P<0.01.

Figure 5. Ad-DC315-mE1A or Ad-DC315-E1A induced inactive p53 expression

HCT116 p53+/+, HCT116 p53−/−, H1299, Hela, HepG2 and U2OS cells were infected with Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A or Ad-DC315-EGFP at MOI=20 for 48h. The level of p53, p21, mdm2, Bax, Bcl-2, PARP and E1A proteins were analysed by western blotting. Beta-actin was assayed as a loading control.

Figure 6. Ad-DC315-mE1A or Ad-DC315-E1A affected cellular p21 localization

A. H1299, HeLa, U2OS and HepG2 cells were infected with Ad-DC315-E1A or Ad-DC315-mE1A at indicated MOIs for 72h. p21 and E1A levels were determined by immunoblot analysis. Beta-actin was used as a loading control. B. H1299, HeLa, U2OS and HepG2 cells were treated with adenoviruses at a MOI=20 for 48h. Cellular fractionation was performed. The p21 was detected in cytoplasmic and nuclear fraction (N, nuclear fraction; C, cytoplasmic fraction; T, total cell lysates.). H1 was used as a nuclear marker and btea-actin was a cytoplasm marker.

Figure 7. Protein p21expression was involved in E1A or mE1A mediated apoptosis

A. 3 siRNA (1120nt, 887nt and 376nt) targeted for p21was transfected into U2OS and Hela cells, respectively. Src, scrambled siRNA. GAPDH, a siRNA construct targeted for cellular GAPDH gene, was used as a positive control for detecting siRNA efficiency. Ad-DC315-E1A was added into cells tranfected with p21or control siRNAs post 24h. E1A, p21 or GAPDH were detected by western blotting. The adenoviral infectious virion production was assessed 48h post infection by TCID50 (upper, right). Cell survival was analyzed 96h later by MTT assay in U2OS and Hela cells (bottom, right). B. The p21stablely-overexpressed U2OS and Hela cells were infected with Ad-DC315-E1A for 48h. The p21 and E1A proteins were detected. The adenoviral infectious virion production on p21-overexpressed cells was assessed 48h post infection by TCID50 (right).

Figure 8. The antitumor efficacy of Ad-DC315-E1A and Ad-DC315-mE1A in tumor xenograft nude mice

A. HepG2 cells (5 × 10⁶ cells per site) were inoculated into the flank of 5-week-old female BALB/c nu/nu mice. When the tumors reached 3-5 mm in diameter, Ad-DC315, Ad-DC315-E1A, Ad-DC315-mE1A, Ad-DC315-EGFP or PBS was intratumorally injected on days 0, 2 and 4. Tumour growth is expressed as the mean tumor volume ± SD in each group (n = 6). Statistical significance was determined using Student's t test. *P<0.05, ** P<0.01. B. Survival rate in each group of HepG2 tumors-bearing mice (n = 6) was shown using the Kaplan–Meier method. Statistical significance was determined using log-rank test. *P<0.05, ** P<0.01. C. The tumor mass removed from survival mice was shown.