Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells.
OBJECTIVE
To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis.


METHODS
PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow- cytometry.


RESULTS
PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population- doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the G0-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells.


CONCLUSIONS
PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620 cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.
Xiao
De-Sheng
DS
Department of Pathology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan Province, China.
Wen
Ji-Fang
JF
Li
Jing-He
JH
Hu
Zhong-Liang
ZL
Zheng
Hui
H
Fu
Chun-Yan
CY
D017209:Apoptosis; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D002455:Cell Division; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators