[Kiss-1 gene expression after radiation and its association with proliferation and apoptosis in colorectal cancer cells].

OBJECTIVE: To investigate the change of expression level of metastasis suppressor gene Kiss-1 in the colorectal cancer cell line SW480 after radiation, and to determine its association with the proliferation and apoptosis of SW480 cells. METHODS: SW480 cells were divided into control group (0 Gy) and study groups (2, 4, 6, 8 Gy). Cells in the study groups were irradiated by 6-MV X-ray radiation for 48 hours. Immunohistochemistry and real-time PCR methods were used to investigate the influence of radiation on Kiss-1 gene expression of SW480. Colony formation assay was used to detect the proliferation of SW480. Flow cytometry-Annexin- V/PI assay was used to observe the change of the apoptosis rate. RESULTS: Compared with the control group, Kiss-1 protein expression increased after radiation of 6, 8 Gy (P<0.05), but no significant changes were observed after radiation of 2, 4 Gy(P>0.05). Kiss-1 gene mRNA level increased after radiation of 2, 4, 6 Gy, while no obvious change was observed for 8 Gy radiation. The apoptosis rates increased for 4, 6, 8 Gy radiation(P<0.05), however, there was no significant difference for 2 Gy radiation (P<0.05). CONCLUSION: Radiation may increase Kiss-1 gene expression in SW480 cells, which results in decreases proliferation and increases apoptosis in residual surviving cells.

Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein.

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogenes could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.