Study Of Interaction Of Protein Annexin A3and Protein TAp73in Platinum-resistant Ovarian Cancer Cell Line

Background and PurposeThe morbidity of ovarian caner in female reproductive system accounts for the third place.But it is the first lethal disease in female genital malignant tumor. Clinicia ns have designated ovarian cancer a "silent killer".Most cases has already been late stage when diagnosed,the5-year survival rate is below45%. The most important treatment strategy for the primary cancer when diagnosed is given first line with cytoreductive surgery followed by chemotherapy doublets consisting of a Platinum-based compound combined with a Taxane. But more than80%of the patients experience an early relapse because of drug resistance which is the main obstacle of ovarian cancer treatment. The mechanism of drug resistance have multi-factors, multi-channel, multi-level characteristics,there have no consensus currently. Genomics research has opened up a new chapter in proteomics, ordered of interactions between the proteins involved in the regulation of intercellular and intracellular signal transduction. One of functional proteomics technologies, Yeast two-hybrid system is carried on eukaryotic model organism yeast, studying on interaction of proteins in live cells.AnnexinA3is the only enzyme in the Annexin family members, have functions of Inhibiting phospholipase A2and anticoagulant, also play a role in controlling cell proliferation. New study finds expressing of AnnexinA3protein in Platinum-resistant ovarian cancer cell lines clearly increased.It was also confirmed at both the mRNA and protein expression levels. Further validation testing clinical specimens found expression of AnnexinA3in tissue of Platinum-resistant clinical patients with ovarian cancer elevated clearly, the disease-free survival period of high expression of AnnexinA3groups was significantly reduced. In addition, drug resistance of Platinum of high expression of AnnexinA3cells had been increased obviously. But there is no cross tolerance with drugs such as paclitaxel and epirubicin,pointing out AnnexinA3is likely to be ovarian tumor specificity of platinum resistance-related protein.P73gene was found in a false positive cDNA clones accidentally by Kaghad et al in1997when going on hybridization in the cDNA library of COS cells.It expresses two functional antagonistic interaction of various isomers:one kind is the carboxyl-terminal isoforms (TAp73); another kind is the amino-terminal isoforms (ANp73). TAp73protein has transcriptional activation ability,capable of up-regulating target of p53gene, causing cell growth and cell proliferation inhibition,inducing apoptosis, increasing sensitivity of tumor cells to chemotherapy and radiotherapy. While terminal expression of isoforms (ANp73) can result in drug resistance of tumor cells to chemotherapy.In our study we used yeast two-hybrid system in Platinum-resistant ovarian cancer cell line COC1/DDP on the detection of TAp73protein and AnnexinA3protein interaction.MethodsWe used RT-PCR method to amplify AnnexinA3and TAp73CDS gene coding sequence in Platinum-drug resistance ovarian cancer cell line COC1/DDP. After Blue-white selection and sequence without error, then connected to the pGBKT7and pGADT7plasmid, we built pGBKT7-AnnexinA3and pGADT7-TAp73recombinant vector.Then we transfected them into Y2HGold and Y187yeast strains respectively. With fusion proteins detected of no toxicity and auto-activation to yeast strains, we made sex fuse of the two types of transformed yeast strains. By SD/-Ade/-His/-Leu/-Trp/X-a-gal/Aba screening, we gained positive pGBKT7-AnnexinA3and pGADT7-TAp73clones with identification of positive interaction between recombinant plasmid with both pGBKT7-AnnexinA3and pGADT7-TAp73each other.ResultsWe amplified AnnexinA3and TAp73CDS gene coding sequence in Platinum-drug resistance ovarian cancer cell line COC1/DDP. After Blue-white selection and sequence without error,we built pGBKT7-AnnexinA3and pGADT7-TAp73recombinant vector and transfected them into Y2HGold and Y187yeast strains respectively. With fusion proteins detected of no toxicity and auto-activation to yeast strains, we made sex fuse of the two types of transformed yeast strains. By SD/-Ade/-His/-Leu/-Trp/X-a-gal/Aba screening, we gained positive pGBKT7-AnnexinA3and pGADT7-TAp73clones with identification of positive interaction between recombinant plasmid with both pGBKT7-AnnexinA3and pGADT7-TAp73each other.ConclusionThis research successfully built pGBKT7-AnnexinA3and pGADT7-TAp73recombinant vector and stable expression of pGBKT7-AnnexinA3and pGADT7-TAp73fusion protein in yeast strains. By yeast two-hybrid technology we identified the positive interaction between AnnexinA3and TAp73proteins.