IRES Dependent Translation Improved The Tumor Therapeutic Effect By A Vector Expressing Double Tumor Suppressor Genes

There are two modes of translation initiation in eukaryotes cells.The overwhelming majority of eukaryotic m RNAs initiates the synthesis of the corresponding proteins by the cap-dependent model.In this mechanism,the interaction of the cap structure with factor e IF4 E plays a critical role in the recruitment of the translation initiation factor proteins(e IFs)to the 5’-terminal of m RNA.Initiation of protein translation from an internal ribosome entry site(IRES)is an alternative type of protein translation in which ribosomes are recruited directly to an m RNA by virtue of its secondary structure,independently of the 5’-terminal cap structure.IRES often eliminates the involvement of e IFs,activated by binding to specific sets of proteins,referred to as IRES trans-acting factors(ITAFs).IRES-dependent translation initiation was first described in poliovirus RNA and encephalomyocarditis virus(EMCV)RNA,and is now thought to contribute to the translation of certain cellular RNAs as well,particularly under abnormal cellular states triggered by hypoxia or cellular stress.IRES have attracted interest in cancer gene therapy because they can be used in the design of gene transfer vectors that provide bicistronic co-expression of two transgene products under the control of a single promoter.The p53 tumor suppressor controls a key cell cycle arrest and apoptosis pathway that is central to tumor suppression.The pathway is triggered through stabilization of the p53 protein and activation of its transcriptional transactivation properties in response to a variety of cellular abnormalities commonly found in cancer,including oncogene expression and DNA damage.Abrogation of the p53 pathway is probably common to most cancers and may therefore be an essential step in tumor development,with loss or mutation of the p53 gene occurring in some 50%-60% of cancers overall.For this reason,the p53 pathway has attracted attention not only for its importance tocancer cell biology but also for its potential for cancer treatment.The fact that p53 is regulated by activation signals,such as DNA damage,and regulatory proteins,such as p14 ARF and mdm2,may explain why permanent cell cycle arrest or apoptosis is not always achieved in tumor cells by ectopic overexpression of p53 alone.Deletion mutations of p14 ARF as a newly found tumor suppressor gene have been reported in varieties of human tumors.The p14 ARF tumor suppressor has played a pivotal role in the field of cancer biology via interacting with binding partners.One of the most well-defined function of p14 ARF is to suppress aberrant cell growth in response to oncogene activation,by activating the p53.p14 ARF is thought to stabilize and stimulate p53 activity by neutralizing the inhibitory effects of mdm2.In addition,p14 ARF has also the ability to restrain cell growth independently of p53.p14 ARF is also a potential target for gene therapy.We constructed Adp14 and Adp14/p53 adenoviruses to compare the inhibitory effect of a combination of Adp14 plus Adp53 with Adp14/p53 alone on tumor cells,and to find out which is more effective treatment.And then we compare the effects of the p14 ARF tumor suppressor on cap-dependent translation versus cap-independent translation.Methods:(1)Adp14 and Adp14/p53 adenovirus were constructed by Ad Easy TM adenovirus vector system;(2)Human colon cancer cell line DLD-1 viability was measured by MTT assay;(3)The m RNA levels of p14 ARF and p53 were evaluated by real-time PCR;(3)Total protein were extracted,and the expression of p14 ARF,p53 and mdm2 were analyed by Western blot;(4)p53 translational rates was detected using polysomal analysis;(5)DLD-1 cells were treatmented with si RNA to downregulated mdm2 protein levels;(6)Retrovirus carrying GFP or IRES-GFP were constructed,and infected human esophageal cancer cell OE33 to establish stable cell lines OE33-GFP and OE33-IRES-GFP;(7)The rate of GFP protein synthesis was monitored by pulsing OE33-GFP and OE33-IRES-GFP cells with [35S]-methionine,followed by SDS-PAGE and fluorography of immunoprecipitated GFP.Results:(1)Compared with Adp14 or Adp53 group,Adp14 combined with Adp53 could reduce cell viability of DLD-1.But the protein levels of p53 in DLD-1 treated with Adp14 combined with Adp53 was very low;(2)Polysomal analysis indicated that the translation rate of p53 in DLD-1 cells treated with Adp14 was reduced.It was indicated that overexpression of p14 ARF could inhibit the translation of p53.p53 message was efficiently translated in DLD-1cells treated with Adp14/p53;(3)si RNA treatment to downregulate mdm2 protein levels in Adp14/p53-treated DLD-1 cells had no effect on the translation rates of p53.It rued out a possible role of the mdm2 protein in maintaining p53 translation rates in Adp14/p53-treated DLD-1cells;(4)By comparing Adp14 and Adp53 combined group and Adp14/p53 group it found that p53 was translated in different manners.p53 was translated in cap-depandent manner in Adp14 and Adp53 combined group,in IRES-dependent manner in Adp14/p53 group.Adp14 and Adp53 were co-transfected with the Adp14 /p53 group by IRES-dependent translation.Polysomal analysis indicated that p53 message was less efficiently translated in Adp14 combined with Adp53 treated DN-MEFs,and p53 message was efficiently translated in Adp14/p53 treated DN-MEFs.Thus,cap-dependent translation of p53 was less efficient in the presence of overexpressed p14 ARF,and IRES-mediated translation of p53 was efficient even in the presence of overexpressed p14ARF;(5)OE33-GFP and OE33-IRES-GFP stable cell lines were constructed.The rate of GFP protein synthesis was monitored by pulsing cells with [35S]-methionine.The results shown that cap-dependent translation of GFP in OE33-GFP cells was suppressed on the average by some 90% in Adp14 treated cells compared to Ad Luc-treated cells,while IRES-dependent translation of GFP in OE33-IRES-GFPcells shown only weak suppression by Adp14.The ability of IRES-mediated protein translation to escape suppression by p14 ARF appeared therefore to be a general phenomenon with potential relevance to any gene transfer protocol involving p14 ARF.Conclusion:(1)A combination of Adp14 plus Adp53 can inhibit tumor growth,as well as Adp14/p53,but inhibiting effect of Adp14/p53 is more significant.(2)Cap-dependent translation of proteins can be inhibited by p14 ARF.In contrast,IRES-mediated translation of proteins could overcome suppression by p14 ARF.In summary,p14 ARF is not only a tumor suppressor protein,but also has the function of inhibiting cap-dependent translation initiation.This suggests that transgenes placed downstream of an IRES element will retain efficient translation of their gene products in the presence of high levels of ectopic or endogenous p14 ARF,a finding that could be particularly relevant to therapeutic gene therapy strategies for cancer.