Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo.

AIM: To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS: We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS: The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice.

AIM: To investigate the inhibitory and anti-metastatic effect of mutant p27 gene (p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism. METHODS: Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intra-tumoral injection of Ad-p27mt in a pre-established transplantation model of human colorectal cancer in nude mice. Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor. Expression of MMP-9 in tumor tissue was detected by immunohistochemistry. RESULTS: The average sizes of transplantation tumors were 1.94 +/- 0.67 cm(3), 2.75 +/- 0.83 cm(3) and 3.01 +/- 0.76 cm(3) in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average proliferation rates were 37.34% +/- 1.45%, 53.16% +/- 3.27% and 54.48% +/- 2.43%, in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average apoptosis rates were 19.79% +/- 3.32%, 6.38% +/- 4.91% and 7.25% +/- 5.20% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). The average MMP-9 expression rates were 20%, 75% and 66.7% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). CONCLUSION: p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells, as well as decrease transpl-anted tumor metastasis.