[Potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of Tca8113 cells in vitro].
OBJECTIVE
Previous studies have revealed the close relationship between Fas/FasL pathway and carcinogenesis of oral squamous cell carcinoma (OSCC). This study was designed to explore the potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of human OSCC cell line Tca8113.


METHODS
The 2 chimeric expression vector pAdTrack-CMV-cdc25A-Fas (pCCF), and pAdTrack-cdc25A-Fas (pCF) were constructed by gene engineering, pCCF, pCF, and the control plasmid pAdTrack-CMV were transfected into Tca8113 cells by liposome, respectively. The transfection efficiency was presented by the expression of the report gene, green fluorescence protein (GFP). The mRNA and protein levels of Fas were determined by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry methods. The apoptosis of transfected Tca8113 cells was analyzed by techniques of DNA agarose gel electrophoresis, TUNEL, Annexin V label, and flow cytometry (FCM).


RESULTS
(1) The chimeric expression vectors pCCF, and pCF were successfully transfected into Tca8113 cells and the maximum transfection (15%) was observed at the 5th-7th day after transfection. (2) Up-regulation of Fas expression was detected at the 3rd day after transfection in pCCF, and pCF transfection groups. At 3rd, 5th, and 7th day after transfection, Fas protein was found to express on membrane and in plasma of transfected Tca8113 cells with distinct morphology of partial apoptosis. (3) From 2.5 days after transfection, early apoptosis had been observed in pCCF, and pCF transfection groups. At 3rd day, the increased apoptosis index (AI,=25%) of pCCF, and pCF transfection groups was significant higher than that of control group (P< 0.05), whereas there was no significant difference in AI between pCCF transfection group and pCF transfection group (P >0.05). (4) FCM analysis showed that the peak of GFP-expression cells was identical to that of the apoptotic cells.


CONCLUSIONS
The cdc25A-Fas chimeric expression vector was able to initiate the apoptosis of Tca8113 cells by up-regulating Fas expression. This result indicated that the 27 bp cdc25A fragment can be designed as a cis-regulatory element to modulate the effects of C-Myc/Max in cellular proliferation and apoptosis.
Feng
Chong-Jin
CJ
Department of Stomatology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510080, PR China.
Li
Chun-Yang
CY
Hong
Yun
Y
Yang
Ling-Lan
LL
Ye
Ping
P
Cheng
Bin
B
D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation; D005822:Genetic Vectors; D006801:Humans; D009062:Mouth Neoplasms / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D020687:cdc25 Phosphatases / Q000096:biosynthesis* / Q000235:genetics; D019014:fas Receptor / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases