The Study on Potential of cdc25-Fas Chimeric Expression Vector in Inducing Apoptosis of Tca8113 Cell Line In Vitro
Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong
BACKGROUND & OBJECTIVE: Tumorigenesis is associated with not only abnormal cellular proliferation and differentiation, but also apoptosis involving many apoptotic signals. This study was designed to explore the effect of transfer of cdc25A-Fas chimeric gene on human oral squamous cell carcinoma cell line Tca8113(OSSC Tca8113). METHODS: The two recombinant pAdTrack-CMV-cdc25A-Fas(pCCF), pAdTrack-cdc25A-Fas(pCF), which were designed and constructed by our group, and the two control plasmids, pAdTrack-CMV, pAdTrack were transfected into Tca8113 cells with lipofecting technique, respectively. The transfection efficiency was determined with the expression of the report gene, GFP. The RNA and protein levels of Fas were detected by Northern blot, RT-PCR, Western blot and Immunohistochemistry . The apoptosis of transfected Tca8113 cells was analyzed through DNA agarose gel electrophoresis, TUNEL, Annexin V and FCM. RESULTS: ① The target gene, Fas, and report gene GFP link-expressed. The maxim transfection presented by GFP ‘s expression after tranfected 5-7d was 15%.② After 5 d, Up-regulation of Fas expression were found in pCCF and pCF transfected Tca8113 cells comparing with the control groups at the same time.③ Fas protein expressions were detected positively at 3,5,7d and located at cell membrane and plasma. ④After 2.5d(60hr), the increasing apoptosis of Tca8113 cells, which were transfected with pCCF and pCF were observed through AnnexinV, DNA ladder, TUNEL, and morphological observation. About 25% experimental Tca8113 cells was detected appearing apoptosis. FCM investigation shows the GFP expression cell peak and the apoptotic cell peak. ⑤ The increased apoptosis index (AI) among the three groups of Tca8113 cells after 72hr of transfection showed the significant difference among pCCF, pCF transfected groups and control cells groups (p<0.05), whereas no significant difference between pAdTrack-CMV-cdc25A-Fas group and pAdTrack-cdc25A-Fas transfected group. (p>0.05). The proliferation index (PI) among these three groups of Tca8113 cells was no significant difference (p>0.05). CONCLUSION: The eukaryotic expression plasmids pCCF and pCF were transfected into human OSSC cell line Tca8113, and the up-regulation of Fas expression was observed. The cdc25A-Fas chimeric gene induced/accelerated the apoptosis of the human OSSC cell line Tca8113 cells. It has potential for inducing apoptosis of human oral squamous cell carcinoma. The experiment primarily confirmed that the cell proliferation signal, C-Myc/Max, should act on cis-regulation factor, the 27bp cdc25A fagment to up-regulation the expression of Fas.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 994 (Signaling pathways - Part II).