Targeted Antitumor Effect Induced By HTERT Promoter Mediated Ornithine Decarboxylase And S-adenosylmethionine Decarboxylase Antisense Adenovirus

Polyamines including spermine,spermidine,and putrescine are essential for normal cell growth and differentiation,but the abnormal polyamine metabolism plays an important role in the development of tumors.Decarboxylating ornithine to produce putrescine is the first rate-limiting step in polyamine biosynthesis,so ornithine decarboxylase(ODC) which catalyzes this response has been the major target for anticancer investigation.The second rate-limiting enzyme is S-adenosylmethionine decarboxylase(SAMDC).It is reported that irreversibly inactivating ODC/SAMDC activity can inhibit prostate cancer,and inhibition of ODC has been used in clinical chemoprevention trials for epithelial cancers,including colon,esophageal,breast, cutaneous,and prostate malignancies.Our recent work has proven that inhibition of ODC/SAMDC activity by recombinant antisense ODC/SAMDC adenovirus has had antitumor effects on human prostate cancer and colorectal cancers.Encouraging results of Gene therapy had emerged from the clinic,but questions are still being asked about its safety.Because the target gene therapeutic has an important aim as concentrating the target therapeutic gene expression into the specific target tissue.Telomerase is a ribonucleoprotein enzyme that specifically adds back telomere sequences lost during replication by using an intrinsic RNA component as a template for polymerization.Telomerase is activated in the majority of immortal cell lines in culture and in most but not all malignant tumor.Expression of hTERT appears to represent a rate-limiting step regulating overall telomerase activity.Unsurprising,hTERT promoter can minimize secondary effects and maximize the therapeutic index.Some researchers have applied the hTERT promoter into the adenovirus mediated targeted cancer gene therapy.The region of the hTERT promoter contains a potential Myc oncoprotein binding site,and the c-Myc proto-oncogene is an ubiquitous transcription factor involved in the control of cellular proliferation and differentiation.The hTERT promoter may allow the use of proapoptotic genes for cancer treatment without noticeable effects on progenitor cells.In the present study,we cloned the antisense ODC/SAMDC RNA which was driven by hTERT promoter into the adenovirus vector(rAd-hTERTp-ODC/SAMDC). Human cancer cell lines and normal cell lines were infected separately with rAd-hTERTp -ODC/SAMDC as well as with control vector(rAd-CMV-GFP).The results showed that rAd-hTERTp-ODC/SAMDC successfully targetly inhibited the growth of cancer cells through inhibition of ODC/SAMDC expression,and was safe for normal cells.Part1:Detection of telomerase activity and detection of hTERT promoter activity in cell Strains【Aim】To detect the telomerase activity and exogenous hTERT promoter activity in the five cell lines used in this research(A549,Bel-7402,HepG2,HELF and LO2) respectively.【Methods】Culture the cells in vitro.Telomerase protein was purified and telomerase activity was detected by TRAP method.The specific sequence of telomerase product was dyed after PAGE electrophoresis,the positive result has more than four bands.The recombinant plasmid containing the hTERT promoter sequence (pGL3-hTERT-luc) was transferred into each cell strain respectively.According to the expression level of the luc gene downstream hTERT promoter,we conclude the activity of hTERTp in cancer cells and normal cells.【Results】The TRAP results of A549,Bel-7402 and HepG2 cell indicated that more than four bands appear on the gels.The results of HELF and LO2 had no positive bands.The specific activity of hTERTp was detected by Dual-Lucy Assay,the results showed that:A549(4.5±0.25);Bel-7402(5.1±0.24);HepG2(4.9±0.3); HELF(0.16±0.02);LO2(0.11±0.01).【Conclusion】The three malignancy cell Strains(A549,Bel-7402 and HepG2)which were used in this research all had the positive telomerase activity.In the normal cell Strains(HELF and LO2),the telomerase activity were negative.Dual-Lucy Assay showed that:hTERTp has high activity in the cancer cells but has no activity in the normal cells.Part2:Construction of an antisense recombinant adenovirus vector of ODC and SAMDC mediated by hTERT promoter【Aim】To construct the antisense recombinant adenovirus vector of ODC and SAMD respectively mediated by the hTERT promter. 【Methods】The 120bp and 205bp cDNA around start codon in ODC mRNA and SAMDC mRNA was amplified by PCR.The antisense sequence of ODC and SAMDC took the place of luc gene in the plasmid(pGL3-hTERT-luc),then we got the recombinant plasmid(pGL3-hTERT-ODC and pGL3-hTERT-SAMDC). Plasmids(pGL3-hTERT-ODC and pGL3-hTERT-SAMDC) were digested by KpnI/SalI;the short sequence(-hTERT-ODC- and -hTERT-SAMDC-) were purified and inserted into shuttle vector pAdTrack.The resulted vectors pAdTrack-hTERT-ODC and pAdTrack-hTERT-SAMDC were then linealized by PmeI and transformed into adeasy-1 cells.The positive recombinant vectors pAdeasy-hTERT-ODC and pAdeasy-hTERT-SAMDC were digested with PacI and subsequently transfected into HEK293 cells to pack and amplify recombinant adenovirus particles Ad-hTERT-ODC and Ad-hTERT-SAMDC.The transfection and viral production were monitored and proved by fluorescent microscopy and PCR technique.【Results】A 120bp and a 205bp cDNA of ODC and SAMDC successfully amplified by PCR and successfully took the place of luc gene,the hTERTp gene sequence combining the target gene seuence were ligated into the shuttle vector successfully.The sequence and derection of inserted genes were confirmed by sequencing.Several positive recombinant clones were identified after transformation of Adeasy-1 cells with linearized pAdTrack-hTERT-ODC and pAdTrack-hTERT-SAMDC.During the processure of viral package and amplification in 293 cells,lots of obvious viral plagues and GFP expression were detected with the light microscopy and fluorescent microscopy.The appearance of target gene in adenovirus genome was confirmed by PCR technique.【Conclusion】The antisense recombinant adenoviruses mediated by hTERT promoter Ad-hTERT -ODC and Ad-hTERT-SAMDC were successfully constructed and amplified.The recombinant adenovirus vector can support an possible vector to block the polyamine synthesis targeted in the cancer cells.Part3:Targeted inhibitory effects of ODC and SAMDC virus mediated by hTERTp on cancer cell growth and invasion in vitro【Aim】To study the Targeted inhibitory effects of Ad-hTERT-ODC and Ad-hTERT-SAMDC on cancer cell proliferation and invasion in vitro.【Methods】MTS assay was used to analyze Adenovirus-mediated gene transduction efficiency of different MOI in A549;Bel-7402;HepG2;HELF and LO2 cell strains.Western Blot was used to detect ODC and SAMDC expression.MTS assay was used to analyze the effect on the growth of cells.Cell cycle progression was detected by flow cytometry analysis.The invasion activity of cancer cell in vitro was detected with Matrigel invasion assay.【Results】MTS assay results showed that adenovirus could significantly inhibit the growth of A549;Bel-7402 and HepG2 cells about 65%;62%and 55% respectively,by 50MOI,25MOI and 25MOI without cell toxicity. Western blotting results demonstrated that Ad-hTERT-ODC and Ad-hTERT-SAMDC could decrease the expression of ODC and SAMDC about 50%,60%,50%and 40%,50%,40%in cancer cells(A549;Bel-7402 and HepG2)respectively,and had no obviously effect in the normal cells(HELF and LO2).Cell flow cytometry analysis showed that Ad-hTERT-ODC and Ad-hTERT-SAMDC could lead the infected cancer cells to arrest at G₀/G₁ phase. Matrigel invasion assay showed the recombinant virus decreased invasiveness of cancer cell obviously.【Conclusions】Ad-hTERT-ODC and Ad-hTERT-SAMDC had significant inhibitory effects on cancer cell proliferation and invasion and had no obvious effect on normal cells,so the recombinant virus bears target-therapeutic potential for the treatment of cancer.