Dissertation > Excellent graduate degree dissertation topics show

Characterization of Oncolytic Mechanism and Entry into Impermissive Cells NIH3T3 of Recombinant HSV-1

Author: SuYue
Tutor: QiYiPeng
School: Wuhan University
Course: Microbiology
Keywords: recombinant HSV-1 (mtHSV) oncolytic mechanism Ras reticulon 3(RTN3) impermissive cell
CLC: R730.5
Type: PhD thesis
Year: 2006
Downloads: 149
Quote: 0
Read: Download Dissertation

Abstract

We previously reported that mtHSV, a mutant conditionally replicating, recombinant HSV-1, which was constructed by inserting E. coli beta-galactosidase gene into the loci of the apoptosis-inhibiting gene icp34.5, and the major determinant of HSV neurovirulence of HSV-1. It can kill selectively the cancer cells, but not the normal cells. Selectively oncolytic effects of mtHSV, on several tumor cells in vitro and its antitumor effects by the intratumoral (IT) route to nude mice body loaded the human hepatoma xenografts were explored,however, its oncolytic mechanism is under confirmation.Farassati F et al. proved that NIH3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. It suggests that HSV-1 specifically targets cells with an activated Ras signaling pathway. Our previous study indicated reticulon 3 (RTN3)(RTN3, also named HAP, ASYIP, homologous interacting protein of ASY protein)as a important member in RTN family is the target gene of ER stress in cells and overexpression of RTN3 could cause ER overload response. Our prevenient study also suggested one of significant characteristics of RTN3 protein is that it locate at the ER. However, nobody was aware that there were certain relations among the novel ER–targeted protein RTN3 and Ras as well as HeLa cells infected by mtHSV.In this research, we aimed to study selectively oncolytic molecular mechanism of mtHSV, that is, the relations between Ras/RTN3 and HeLa cells infected by mtHSV.The results showed that HeLa cells could be killed efficiently by mtHSV in vitro. In the flow cytometry and Western blot experiment, Ras protein was obviously down-regulated on plasma membrane (PM) while the whole Ras protein didn’t change along with up-regulation of reticulon 3 (RTN3) protein at endoplasmic reticulum(ER) at 48h post infection of mtHSV in HeLa cells . Expression of Ras protein on PM and whole Ras protein in HeLa cells was down-regulated by siFTa(inhibitor ofαsubunits of human farnesyltransferase with siRNA)and siRTN3(inhibitor of RTN3 with siRNA) respectively. HeLa cells could be killed effectively by siFTa and siRTN3 at 48h post transfection. siFTa and siRTN3 effectively inhibitted HeLa cells infected by mtHSV. Further experiments were made to study the relationship between Ras and RTN3 using confocal colocalization and co-immunoprecipitation.The results exhibited that Ras could interact with RTN3 at ER. The data put forward that Ras/RTN3 is an important factor to HeLa cells for mtHSV. The molecular interaction between Ras and RTN3 may further improve the understanding of the function of Ras and RTN3 during mtHSV infection.Furthermore,in order to find the relations among HeLa cells infected by mtHSV and P53 as well as cell apoptosis. The expression of P53 protein was detected by the flow cytometry and Western blot experiments respectively. P53 protein didn’t change in flow cytometry and western blot experiments in HeLa cells at 24h,48h post infection (p.i) respectively. DNA ladder analysis, hoechst33258 staining and Annexin V/PI analysis demonstrated that mtHSV couldn’t induce HeLa cells apoptosis at early stage. p53 gene sequence is normal in HeLa cells and Hpv-16 was transformed into HeLa cells, so P53 lost normal function in HeLa cells. In such instances, mtHSV couldn’t induce HeLa cells apoptosis at early stage. The results suggest that selectively oncolytic mutant of HSV-1 lyses HeLa cells via inducing P53-independent cell death. Therefore, it’s important to find a novel access for mtHSV entering into cell thoroughly understanding molecular mechanism for mtHSV targeting cells, mtHSV were used to screen a 15-mer phage display peptide library. After four rounds of screening and sequence analysis, A special segment of screened peptides was found. Its amino acid sequence APFSRLLFPDFRSFV was deduced and showed 84% homologue with RasGRP2. Recombinant plasmid AcBacmid-RasGRP2 and recombinant Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) vAc-RasGRP2 were constructed to express RasGRP2 in Sf9 (Spodoptera frugiperda) cells. The molecular weight of expressed RasGRP2 protein was identified approximately 68kD. Tansmembrane domain of RasGRP2 protein was predicted by biosoft“DAS”–Transmembrane prediction server. The results showed RasGRP2 protein is a transmembrane protein. The domain has continuous 18 hydrophobic amino acid from 297 to 314 (LGVHLKDLVALQLALPDW) is tansmembrane domain of RasGRP2 protein.To identify the function of RasGRP2 for mtHSV and HSV-1 entering into cells, A eukaryotic expression recombinant pAdtrackCMVRasGRP2 were constructed , which can express RasGRP2 gene, using gfp (green fluorescence protein gene) as reporter gene. pAdtrackCMVRasGRP2 was tranfected into mtHSV and HSV-1 impermissive cell line—NIH3T3 cells. After 48h post transfection, NIH3T3 cells was infected with mtHSV and HSV-1 respectively. The infection experiment showed that RasGRP2-expressing NIH3T3 cell could be infected by mtHSV and HSV-1. Ston JC et al. reported RasGRP2 lies upstream of Ras on Ras signal pathway. The results that impermissive cell line—NIH3T3 can be infected by mtHSV and HSV-1 via RasGRP2 imply RasGRP2 is a novel important entry for mtHSV and HSV-1 entering into cells and Ras signal pathway is an important access for mtHSV targeting cells. The results provide further theoretical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.

Characterization of Oncolytic Mechanism and Entry into Impermissive Cells NIH3T3 of Recombinant HSV-1

Abstract

Related Dissertations