p21WAF1/CIP1 is more effective than p53 in growth suppression of mouse renal carcinoma cell line Renca in vitro and in vivo

Abstract

Purpose: Although there are many controversial reports about the effect of p53 and p21(WAF1/CIP1) overexpression in different human tumor cells, the p53 gene is shown to be a more effective candidate for cancer gene therapy because of its more pronounced ability to induce apoptosis. In the present study, we present the effect of p53 and p21(WAF1/CIP1) overexpression on mouse renal carcinoma cells in vitro and in vivo.

Methods: p53 and p21(WAF1/CIP1) genes were introduced into Renca cells using adenoviral vectors (Ad5CMV-p53 and Ad5CMV-p21). The induction of apoptosis was measured using Annexin V assay and DNA fragmentation analysis. The expression of proteins was examined using immunocytochemistry and Western blot methods. The ability of adenoviral vectors to inhibit tumorigenicity of Renca cells, as well as the growth of pre-established tumors was measured.

Results: In vitro growth assays revealed higher growth suppression after Ad5CMV-p21 infection. Although both vectors induced apoptosis, Ad5CMV-p53 was slightly more efficient. In vivo studies in Balb/c mice, demonstrated that tumorigenicity was completely suppressed by Ad5CMV-p21. Besides this, Ad5CMV-p21 significantly inhibited the growth of established tumors, while Ad5CMV-p53 did not.

Conclusions: These data suggest that p21(WAF1/CIP1) is a more potent growth suppressor than p53 of mouse tumor cells Renca. The divergent responses of tumor cells to p21(WAF1/CIP1) overexpression could be due to various networks that differ between species.

Fig. 1A-C.

Time- and dose-response curves obtained after infection of Renca cells with A dl 312, B Ad5CMV-p21, and C Ad5CMV-p53 at different MOI values ranging from 20 PFU/cell to 100 PFU/cell. Cells were plated in triplicates on 24-well plates and the growth was determined by counting the cell numbers at different time points after infection. Results represent means±SD. The experiment was repeated three times, and the results were similar

Fig. 2A-E.

Immunocytochemical analysis of p53 and p21 protein expression in Renca and B16Bl6 cells. The cells were infected with Ad5CMV-p53 and Ad5CMV-p21 at MOIs 50 and 100 and compared to uninfected cells. Antibodies towards human and mouse p53 protein and towards human p21 protein were used. Percentages of A p53- and B p21-positive cells and immunocytochemical staining of C Renca control, D Ad5CMV-p53-infected and E Ad5CMV-p21-infected cells are shown

Fig. 3A,B.

Western blot analysis of A p53 and B p21 protein expression in Renca cells. The cells were infected with Ad5CMV-p53 or Ad5CMV-p21 at a MOI 100. After 24 h, cell lysates were prepared and subjected to Western blot analysis using monoclonal antibody towards human and mouse p53 protein and polyclonal p21 antibody towards mouse p21 protein. The right half of each panel represents the densitometric analysis of the bands

Fig. 4A,B. A

Percentage of apoptotic cells. The cells were mock infected or infected with dl 312, Ad5CMV-p53, and Ad5CMV-p21 at a MOI 100 and examined after different time points using Annexin V assay. Results represent means±SD. B Nucleosomal DNA fragmentation in Renca cells. Low-molecular-weight DNA was isolated 24 h after infection with Ad5CMV-p53 and Ad5CMV-p21 at a MOI 100. Lane 1 DNA from control cells; Lane 2 DNA from cells infected with Ad5CMV-p53; Lane 3 DNA from cells infected with Ad5CMV-p21

Fig. 5A,B.

Inhibition of tumor formation. Renca cells were exposed to MOI 100 of Ad5CMV-p53, Ad5CMV-p21 and dl 312, or mock-infected and inoculated (5×10⁵ cells/0.1 ml PBS) into recipient animals. The A presence of tumor and B tumor volume are shown. Results represent means±SD

Fig. 6A,B.

Inhibition of tumor growth. Renca cells (5×10⁵ cells/0.1 ml PBS) were injected into Balb/c mice and tumors formed. Effect of A a single intratumoral injection of 5×10⁸ PFU or B five consecutive injections of 1×10⁸ PFU of Ad5CMV-p53, Ad5CMV-p21, dl 312 or PBS is shown (n=6). Results represent means±SD