Abstract

AIM In order to express suicide gene specifically incolorectal carcinoma cells for targeted gene therapy, we constructed the recombinantretroviral vector promoted by transcriptional regulatory sequences of carcinoembryonicantigen (CEA) gene.
METHODS A 1.3 kb EcoRⅠ/ BamHⅠ fragmentof the HSV-tk gene was isolated from pLNtk and inserted into pUC118, then the HindⅢ/ BamHⅠ fragmentfrom pCEA424/ 2CAT was isolated and subcloned into pUC118 to harvest HindⅢ/ EcoRⅠ CEATRS. These two fragments were ligated with the retroviral vector GINa.
RESULTS Restriction enzymeSal Ⅰwas used to identify a positive cloneB5. The recombinant retroviral vector, GICEAtkNa was constructed.
CONCLUSION Success inconstruction of GICEAtkNa provides a basis for colorectal carcinoma-specific genetherapy.INTRODUCTION
Herpes simplex virus thymidine kinase (HSV-tk) gene is the encoding gene of virusthymidine kinase. Catalyzed by herpes virus thymidine kinase, some avirulent nucleosideanalogi can be phosphorylated into drugs which are poisonous to mammallian cells and canlead to the death of cells by obstructing DNA synthesis. In order to avoid the death ofnormal cells, tk gene should be regulated to express specifically in tumor cells, with noexpression or at least low expression in normal cells. Using 5＇-transcriptional regulatory sequence (5＇TRS) of carcinoembryonic antigen (CEA) gene to promote HSV-tk genespecifically, we constructed retroviral eukaryotic vector, GICEAtkNa, which lays afoundation for further research of colorectal carcinoma tissue-specific gene therapy.

MATERIALS AND METHODS

Vectors, target genes andtranscriptional regulatory sequences
Retroviral vector, GINa was kindly provided by Professor Dusty Miller (Fred HuchisonCancer Center, USA), plasmid pLNtk by Dr. GE Kai (National Biochemistry Institute) and theplasmid pCEA424/ 2CAT containing 5＇TRSof CEA gene by Professor Zimmermann (Freiburg Albert-Ludwigs University, Germany). pUC118,host strain HB 101, JM 109 were kept at our laboratory.

Regents

All restriction endonucleases weremade by Shanghai Sino-American Biological Products Co.RNAase and T4 DNA ligase wereproducts of Shanghai Shisheng Co. PGE 8000 was purchased from Sigma Co.

Construction of colorectalcarcinoma tissue-specific retroviral vector

The amplified pCEA424/ 2CAT was digestedwith HindⅢand BamHⅠand purified in low melting point agarose to isolate a 420 bp 5＇TRS fragment of CEA gene which was subcloned into thepolyclone sites of pUC118. The recombinant was digested with HindⅢand EcoRⅠto harvestHindⅢ-EcoRⅠCEA fragment. The amplified pLNtk was digested with EcoRⅠand BamHⅠandpurified in low melting point agarose to isolate a 1.3kb EcoRⅠ-BamHⅠtk fragmentwhich was then subcloned into the polyclone sites of pUC118. The recombinant was digestedwith HindⅢand EcoRⅠto harvest EcoRⅠ-HindⅢtk fragment. GINa was digested with HindⅢ and dephosphorylated with calf intestinal alkalinephosphatase with T4 DNA ligase, the HindⅢ-EcoRⅠCEA fragment and the EcoRⅠBamHⅠtk fragmentwere ligated with the linearized and dephosphorylated GINa vector. and were employed totransfect the plasmids. A few plasmides were prepared using 20 clones, selected. SalⅠwas employed to define the orientation of the insertion.The positive clones which had been defined by the appearance of 1.7 kb band on agaroseelectrophoresis gel, were amplified. Finally, GICEAtkNa was constructed.

RESULTS
Acquirement of EcoRⅠ-HindⅢfragment and HindⅢ-EcoRⅠCEA TRSfragment

CEA TRS fragment and HSV-tk genefragment were inserted into the polyclone sites of pUC118 respectively (Figure 1). Therecombinant pUC118 was digested with the corresponding endonucleases to harvest HindⅢ-EcoRⅠCEATRS and EcoRⅠ-HindⅢ tk.

Construction of humancolorectal carcinoma-specific retroviral vector

The recombinant retroviral vector,GICEAtkNa, was constructed by inserting CEA TRS fragment and HSV-tk fragment into thepolyclone sites of GINa simultaneously (Figure 2). The illustrated construction ofG1CEAtkNa is shown in Figure 3. It contained two transcriptional regulatory components: 5＇LTR and CEA424/ 2. 5＇LTRregulated the expression of selecting gene, and Neo, CEA424/ 2 regulated theexpression of target gene, HSV-tk.

Figure 1 Identification of the positive clones resulting from separate insertion of tk gene fragment and CEA gene fragment into pUC118.
A:Lanes 1,2:1.3 kb fragment resulting from digestion of recombinant pUC118 tk with EcoRⅠ and BamHⅠ; Lane 3:λDNA HindⅢ marker B:Lane 1:420 bp fragment resulting from digestion of recombinant pUC118 CEA with HindⅢand BamHⅠ;Lane 3:λDNA HindⅢ marker
Figure 2 Identification of the positive clones resulting from simultaneous insertion of tk gene fragment and CEA TRS into GINa with SalⅠ.
Lanes 1,2:1.7 kb fragment resulting from digestion of GICEAtkNa with SalⅠ; Lanes 3: λDNA HindⅢ marker.

Figure 3 Scheme of construction of G1CEAtkNa.

DISCUSSION

Recently, the incidence ofcolorectal carcinoma has been increasing gradually. Although many surgeries andsupplementary therapies have been employed clinically, its 5-year survival rate has notbeen improved obviously^[1] . The quantity of chemotherapeutic drugs are limitedgreatly because of their side effects on normal tissues. Currently, virus-directed enzyme/ prodrug therapy(VDEPT) for tumors has being paid great attention to^[2] . Accordingto VDEPT, the transcriptional difference of CEA gene between tumor cells and normal cellsmainly explains the specific and selective killing effect on the tumor cells. In otherwords, suicide genes, such as tk, are transfected into human tumor cells and are expressedspecifically by retroviral vector, and then the prodrugs which are not poisonous to normalcells, such as GCV, are employed to kill tumor cells selectively. Nowadays, remarkableachievements in HSV-tk gene therapy for malignant brain tumors have been obtained^[3] . The key ofgene therapy for tumors is targeted expression. Targeting suicide gene into tumor cellscan not only increase the efficiency of gene transference, but also protect the normalcells against the suicide gene. In this way, much more prodrugs can be used to kill tumorcells to maximum. CEA is a tumor-associated marker especially for advanced colorectalcarcinoma, so transcriptional regulatory sequences of CEA have been used to promotetissue-specific expression of suicide genes for tumor-specific gene therapy^[4,5] . Schrewe etal^[6] cloned CEA 5＇TRSfrom human genome, and considered that the transcriptional regulatory activity of pCEA424/ 2 CAT was higher thanthat of other sequences. Tadasi et al^[7] transduced pCEA424/ 2CAT intoCEA-producing human lung cancer cells, A549 (A549 cells expressing HSV-tk specifically arehighly sensitive to GCV), and injected GCV into nude mice bearing tumor resulting frompCEAtk-s conversed A549 cells, and obvious tumor-inhibitory effect was observed. TakingpCEA 424/ 2CATas colorectal carcinoma-specific promoter, we constructed pGICEAtkNa by cloning HSV-tkinto polyclone sites of GINa. Up to now, the incasement and transfection have beenfinished. The preliminary results indicate that LoVo cells transfected by colorectalcarcinoma-specific HSV-tk gene are more sensitive to CEA than cells transfected byordinary vector free of CEA transcriptional regulatory sequences. To improve colorectalcarcinoma tissue-specific gene therapy, we will carry out preclinical research on animals.

ACKNOWLEDGMENTS We thank Professor Zimmermann (Albert-ludwigs University,Germany) for providing pCEA 424/ 2CAT, Doctor GE Kai (National Biochemistry Institute) for providingpLNtk.
¹ Department of General Surgery, ChanghaiHospital, Second Military Medical University, Shanghai 200433, China
² Department of Microbiology, Institute of Basic Medical Sciences, SecondMilitary Medical University, Shanghai 200433, China
³ Department of General Surgery, Changzheng Hospital, Second Military MedicalUniversity, Shanghai 200004, China
CUI Long, MD, male, Chief of Department of General Surgery, Changhai Hospital, SecondMilitary Medical University, having 6 papers published.
Project supported by the National Natural Science Foundation of China,No.39600142.
Correspondence to: CUI Long, Department of General Surgery, ChanghaiHospital, Second Military Medical University, Shanghai 200433, China
Received 1997-07-12 Revised 1997-08-25

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