Construction Of Eukaryotic Cell Expression Vector For Green Fluorescence Protein Reportor Gene Fused With E1A And Study On Killing Effects On Human Colorectal Cancer

Background: Human colorectal carcinoma (HCC) is one of the mostlethal cancers in humans. The majority of patients died of local recurrencedespite aggressive medical intervention including surgery, radiotherapy,chemotherapy. In recent years, gene therapy of tumors has become the mostnoticeable research field. It's the fifth model followed surgical therapy,radiotherapy, chemotherapy and immunotherapy. The initiation andprogression of human colorectal carcinoma are involved in many oncogenesand tumor suppressor genes, the over expressions of HER-2/neu gene, theinactivation of function of p53 gene etc, which makes gene prevention andgene therapy possible .The Ad5E1A gene transcribes two mRNAs of 12S and13S, and codes for two major related proteins of 243 and 289 amino acids bydifferently splicing. The ElA proteins are nuclear phosphoproteins that aremultifunctional transcription factors. The ElA products are positive andnegative regulators for the transcription of many cellular genes. At present,human colorectal carcinoma is less as the subject to the study of ElA genethan other. The aim of study is to research the effect of Ad5ElA gene onproliferation and metastasis of the human colorectal carcinoma.Methods:1.Construction of the mammalian expression recombinant pEGFP-E1AA E1A gene recombinant expression vector plasmid was constructed bysubcloning a 1.77kb E1A gene into a mammalian expression vectorpEGFP-C1 at the sites of EcoRâ…  and BamHâ… ,and named pEGFP-E1A. Therecombinant was cleaved with appropriate endoneuclear and sequenced. Theresults showed that the orientation of the insert was found to be correct, whileno rearrangement was found.2.Integratuion and expression of EIA gene in Human colorectalcarcinoma cell line (LoVo)The recombinant pEGFP-E1A was introduced LoVo by LipofectinTMReagent. After the cells resistant to G418, we obtained positive cell clones(LoVo-EIA). Neo and E1A gene PCR assay, E1A gene mRNA RT-PCR assay,LoVo-E1A cells SDS-PAGE and western-blot assay, immunocytochemistryassay showed that EIA gene has been integrated into LoVo-EIA cells andstably expressed.3.Effects of adenovirus type 5 EIA gene to human colorectal carcinomacells in virtoThe characteristics of the EIA expressing LoVo-EIA cells were studiedThe expressing change of HER-2/neu gene was investigated, too. Comparedwith the vector-transfected cells (LoVo -vect cells) and LoVo cells,theLoVo-EIA cells showed that(1)the growth rate of LoVo-EIA cells wasdiminished and their doubling-time increased;(2)the colony-formingefficiency of LoVo-EIA cells was inhibited significantly;(3)The cell cycle wasdramatically arrested at G2/M phase in the LoVo-EIA cells, and S phasedramatically decrease.(4)LoVo-EIA cells showed distinct sensitivity to theanticancer drugs. According to the IC50 value, the sensitivity of LoVo-EIAcellsincreased approximately 8-fold to cisplatin, 20-fold to bleomycin, 10-foldtopaclitaxel, 1-fold to irradiation compared with LoVo -vect cells. However,the sensitivity to 5-FU did not change. (5)Immunocytochemistry analysis alsoshowed that the products of HER-2/neu gene have decreased inEIA-expressing cells.4.Effects of adenovirus type 5 EIA gene to human colorectal carcinomacells in vivoTo study the effects of EIA gene on the growth and chemosensitivity oftransplantation tumor of nude mice of human colorectal carcinoma cellline(LoVo). LoVo, LoVo-vect and LoVo -EIA cells were transplanted intonude mice ,then the time of tumor formation,growth rates and weight of tumorwere observed. LoVo cells were injected into nude mice. After turmor formed,bleomycin, EIA gene and EIA gene+bleomycin were given respectively astherapy. The tumor volume was calculated,and growth curve was plotted.Representative histological sections were taken from mice bearingtransplantation tumor either treated or control groups, and expression ofHER-2/neu was detected. 1n nude mice, the expression of EIA genesignificantly suppressed the growth rates and elongated the time of tumorformation. Bleomycin or EIA gene and EIA gene+bleomycin can suppress thegrowth rates of transplantation tumor of nude mice, the suppressed rates oftumor growth was 50.2%,74.0% and 92.3%, respectively. The expression ofEIA gene can significantly suppressed the expression of HER-2/neu gene inLoVo-EIA cells EIA is able to significantly inhibit the growth rate oftransplantation tumor of nude mice of lymph node human colorectalcarcinoma cell line(LoVo), and dramatically enhance the sensitivity of thecellsto cytotoxic agents in vivo. Above of all functions of EIA gene may berelated to that it suppressed the expression of HER-2/neu gene.Conclusion: With these, we conclude that EIA gene is able tosignificantly suppress the malignant phenotye of human colorectal carcinomaand the expression of HER-2/neu gene. The above results showed that ElAgene possesses important effects on gene therapy of human colorectalcarcinoma. Our study provided data for gene therapy of tumor and possible ofusing E1A gene to improve tumor treatment.