J Korean Med Sci.  2004 Dec;19(6):834-841. 10.3346/jkms.2004.19.6.834.

In vivo Recombinant Adenovirus-mediated p53 Gene Therapy in a Syngeneic Rat Model for Colorectal Cancer

• Baek JH
• Agarwal ML
• Tubbs RR
• Vladisavljevic A
• Tomita H
• Bukowski R
• Milsom JW
• Kim JM
• Kwak JY

• KMID: 1778565
• DOI: http://doi.org/10.3346/jkms.2004.19.6.834

Abstract

The p53 gene has a significant role in controlling genomic stability of cancer. The purpose of this study was to evaluate the tumor response of allograft colorectal tumor treated with Ad5CMV-p53 in a syngeneic rat model. Two weeks after the inoculation of WB-2054-M5 tumor cells in the flank of rats, rats were randomly assigned by tumor size to one of three groups (n=18 in each): phosphate buffered saline (PBS), Ad5CMV, and Ad5CMV-p53. Recombinant adenovirus or PBS was administered through intratumoral injection at three divided doses every other day for 4 weeks. Apoptosis of the tumors was evaluated using TUNEL assay. After 2 and 4 weeks of treatment, the volume (cm3) of tumors in PBS, Ad5CMV, and Ad5CMV-p53 was as follows: 2 week: 1.66 +/-0.43, 1.40 +/-0.47, 0.75 +/-0.26 (p<0.001), 4 week: 4.41 +/-0.88, 3.93 +/-1.86, 2.33 +/-0.51 (p<0.001). Tumor growth showed no statistically significant difference between the PBS and Ad5CMV groups (6-week vol. p=0.32). The TUNEL assay results revealed more apparent apoptotic cells in Ad5CMV-p53-treated tumors than in other groups. Growth of allograft colorectal cancer in the syngeneic rat model was significantly suppressed by intratumoral Ad5CMV-p53 gene therapy. These results demonstrate that gene replacement therapy with p53 may provide a novel modality of treatment in conjunction with other present treatments for metastatic colorectal cancer.

Keyword

Colorectal Neoplasms; Gene Therapy; Protein p53; Recombinant Adenovirus; Transgenes

MeSH Terms

Adenocarcinoma/genetics/pathology/therapy
Adenoviridae/*genetics
Animals
Cell Line, Tumor
Cell Proliferation
Cell Survival/genetics
Colorectal Neoplasms/*genetics/pathology/*therapy
Disease Models, Animal
Female
Gene Therapy/*methods
Gene Transfer Techniques
Men
Protein p53/*genetics/*therapeutic use
Rats
Rats, Inbred WF
Recombinant Proteins/therapeutic use
Research Support, Non-U.S. Gov't
Transplantation, Isogeneic
Treatment Outcome

Figure

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  Fig. 1 The percentage of WB-2054-M5 cells transduced at varying amounts of viral particles. Cells were plated at a density of 200,000 cells/well and Ad5CMV-β-gal virus was used to infect the tumor cells at varying viral particle number. After 48 hr cells were stained with X-gal solution.Their transduction capability was assessed by determining the percentage of X-gal positive cells.

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  Fig. 2 Effect of Ad5CMV-p53 infection on the growth of WB-2054-M5 tumor cells by hexosaminidase assay. WB-2054-M5 cells were plated at a density of 100,000 cells/well and infected with Ad5CM-Vp53 (5×108 vp), Ad5CMV (5×108 vp) or PBS as a control. Cell growth in each treatment group was measured colorimetrically using the hexosaminidadse assay in duplicate wells daily for 5 days following infection. The absorbance of light waves at 405 nm showed a linear, direct proportion to the number of cells present and the time allowed for the reaction. WB-2054-M5 cell growth is significantly more inhibited in the Ad5CMV-p53-treated group, compared to the Ad5CMV and PBS groups (p<0.001).

• 

  Fig. 3 Effect of Ad5CMV-p53 infection on the growth of WB-2054-M5 tumor cells in the cell growth assay. WB-2054-M5 cells were plated at a density of 100,000 cells/well and were infected with Ad5CMV-p53 (5×108 vp), Ad5CMV (5×108 vp), or PBS as a control. Cell growth in each treatment group was measured by counting cells daily in quadruplicate. Growth of WB-2054-M5 cells is significantly inhibited by Ad5CMV-p53 infection (p<0.001).

• 

  Fig. 4 Comparison of mean G1/S ratio in cells infected with Ad5CMV-p53, Ad5CMV, or PBS. WB-2054-M5 cells were plated at a density of 200,000 cells in 60-mm cell plates and infected with Ad5CMV-p53 (1×109 vp), Ad5CMV (1×109 vp), or PBS as a control. Cell cycle distribution was assessed at days 1, 2, and 4 of incubation by analyzing DNA content using a FACScan, Cycletest kit and Cell Fit Software. Dead cells were gated out by pulse processing. The G1/S ratio is significantly increased in the cells infected with Ad5CMV-p53 (p<0.001).

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  Fig. 5 Comparison of the percentage of apoptotic cells in the cell groups infected with Ad5CMV-p53, Ad5CMV, or PBS. WB-2054-M5 cells were plated at a density of 200,000 cells in 60-mm plates and infected with Ad5CMV-p53 (1×109 vp), Ad5CMV (1×109 vp), or PBS as a control. Cells were assessed for apoptosis at days 1, 2, and 4 of incubation by exposure to fluorescent DNA binding dyes and by fluorescence microscopy to count cells with aberrant chromatin. An apoptotic index was developed: apoptotic cells/total cells ×100.

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  Fig. 6 The effect of intratumoral injection of AdCMV-p53, AdCMV, or PBS on the volume of allograft colorectal tumor. Allograft tumor cells were injected subcutaneously into the flank of F1 hybrid rats. Two weeks after inoculation, rats were randomly assigned to PBS, Ad5CMV, or Ad5CMV-p53. Intratumor injection of recombinant adenovirus (2×1011 vp) or PBS was done at 3 divided doses every other day. Then, tumor volume was measured. The outcome of the Ad5CMV-p53 group is significantly different from the other two groups (2 week: p<0.87, 3-6 Week: p<0.001), but there is no statistically significant difference between the outcomes of the PBS and Ad5CMV groups (6-week vol. p=0.32).

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  Fig. 7 The H-E staining & TUNEL staining of WB-2054-M5 allograft tumors. Intratumoral injections of recombinant adenovirus (2×1011 vp) or PBS was done at three divided doses every other day. The tumors were harvested 24 hours after the final treatment and assessed using the TUNEL assay. Apoptotic cells were more apparent in the Ad5CMV-p53-treated group than two other groups. (A) the H-E staining (poorly differentiated adenocarcinoma), (B) PBS-treated tumors, (C) Ad5CMV-treated tumors, (D) Ad5CMV-p53-treated tumors. Original magnification, (A) ×200, (B-D) ×400.

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