An Experimental Study On The Construction Of PIRES-CD And The Suicide Gene Therapy Of Adenoid Cystic Carcinoma

Objective Adenoid cystic carcinoma, which is also called cylindroma,was first reported by Billroth.Most scholar considered that it comes from salivary ducts,or probably the oral squamous basal cells.It characterizes by encroaching the nerve and far matastasis.The treatment of adenoid cystic carcinoma gives priority to operation combined with post operation irradiation and chemistry therapy,but the curative effect is still dissatisfactory.Therefore,it makes sense to explore new therapy.Recently, the gene therapy of malignat tumor is developed rapidly and there are many methods about the gene therapy that include: suicide gene therapy, immunologic gene therapy, drug resistance gene therapy, angiostatin gene therapy and so on.The sucide gene therapy is one of the most potential approach of antitumor. Suicide gene therapy which is also named enzyme-prodrug therapy, encode enzyme that convert nontoxic prodrugs into highly toxic metablites .Cells transfected with suicide genes are targeted for specific negative selection,witch can be induced by administrtion of the corresponding produg. Among the enzyme/produg combinations,two of the best characterized system are herpes simplex virus thymidine kinase(TK)/ganciclovir(GCV) and Escherichia Foli cytosine deaminase(CD)/5-flourocytosine(5-FC).The formor can convert the antiviral nucleoside analogs acyclovir(ACV),gancielovir(GCV) to their nucleoside monophosphate derivatives,the monophosphate forms are subsequently phosphorylated by endogenuscellular kinases to triphosphates, these molecules are potent inhibitors of DNA synthesis ; The later can convert 5-FC into 5-FU, 5-FU is metabolized to 5-fluorourikine 5-triphosphate(5-FUTP) and 5-fluoro-2-deoxyuridine-5- monophosphate(5-FdUMP) , 5-FUTP can be incorporated into RNA in place of UTP resulting in the inhibition of nuclear processing of rRNA and mRNAs, 5-FdUMP irreversibly inhibits TS, which subsequentlyprevents DNA synthesis and induces cellular apoptosis.In this study,we constructed eukaryotic expression plasmids pIRES-CD, and transfected it into ACC-2 cells by electroporation.The prodrug 5-FC was used in order to observe the cytotoxicity effects and the bystander effects of the suicide gene on ACC-2 cells and find more effective suicide gene therapy strategy.Methods The primer of CD gene was designed and composed at the base of CD's DNA sequence.The gene extended productions were inserted into clone vector pMD18-T,then the recombination plasmid pMD18-T-CD was obtained,which was identified by PCR and enzyme digestion.The fragements of CD were inserted into the eukaryotic expression plasmid pIRES,which were digested by Xbal and NotI.The recombination expression plasmids pIRES-CD,which were also identified by PCR and enzyme digestion,were obtained. The pIRES-CD was transferred into ACC-2 cells through electroporation, then stable virus-producing cell ACC-2/CD was established after geneticin(G418) selection.RT-PCR was resorted to demonstrate the successful transduction and transcription of CD gene. ACC-2/CD cells in culture were treated respectively at varying concentration of 5-FC(10 to 320 ug/ml),the cytotoxicity effects of prodrug (5-FC ) on ACC-2/CD and ACC-2 cells were measured by microculture tetrajolium test (MTT) method and the growth inhibition rate(GIR) of cells and the 50% inhibitory concentration(IC₅₀) of prodrug were counted. In the meantime, bystander effect of 5-FC on ACC-2/CD and ACC-2 cells mixture in culture at varying ratio were observed.Results We successfully cloned the Escherichia Foli CD gene and analyzed the CD gene sequence,which basically accorded with the gene bank.Later,we inserted the cloned CD gene into clone vector pMD18-T. The fragements of CD gene were inserted into the eukaryotic expression plasmid pIRES,which was digested by XbaI and NotI.We successfully constructed the recombination expression plasmids pIRES-CD,which were identified by PCR and enzyme digestion. The pIRES-CD was transferred into ACC-2 cells through electroporation,stable virus-producing cell ACC-2/CD were established after geneticin(G418) selection.228bp positive band were seen through RT-PCR,which showed that suicide gene could be transcribed and expressed in gene-transferred ACC-2 cells.When the concentration of 5-FC was larger than 20ug/ml,the growth of ACC-2/CD-TK cells were inhibited. IC₅₀ was 55.42ug/ml,whereas IC50 for ACC-2 cell was 265.56ug/ml.There was significant difference between them (P<0.01).Conclusions 1. We successfully cloned the Escherichia Foli CD gene,constructed the clone vector pMD18-T-CD,and analyzed the CD gene sequence,which basically accorded with the gene bank.2.At the basis of pMD18-T-CD we successfully constructed the recombination eukaryotic expression plasmid pIRES-CD,which was identified by PCR and enzyme digestion.3. The pIRES-CD were transferred into ACC-2 cells through electroporation. 228bp positive band was seen by RT-PCR.4. CD/5-FC system could make strong cytotoxic efect on ACC-2/CD; CD/5-FC system also had significant bystander effect.