Abstract Objective Toinvestigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisenseIGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using therecombinant adenovirus vector,GCV killing effeciency after combined gene transfer ofHSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCVand antisense IGF-1 and the survival rate of rats was observed.Results C6 cellstransfected with tk and antisense IGF-1 gene were more sensitive to GCV than thattransfected with tk gene alone.The survival of the combination gene therapy group wasprolonged significantly and large amounts of CD⁺ ₄ ,CD⁺ ₈ lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene mayenhance the tumor-killing effects of HSV-tk/GCV.
Key words HSV-tk;IGF-1;glioma;gene therapy
Chinese library classification R730.26405

Adenovirus-mediated HSV-tk/GCV system has beenextensively applied in the experimental investigation of glioma and it also has been putinto the clinical phase I treatment.Several studies reported HSV-tk/GCV system may makethe brain tumors regressed totally and the survival period of the tumor-bearing rats wasprolonged^[1] .But several other studies reported that the remaining tumorcells would prolify and tumor reoccurred^[2] .Antisense IGF-1 could extinctthe solid tumor of rat glioma and prevent it from reoccurring.We applied the combined genetherapy of HSV-tk/GCV and antisense IGF-1 to treat the rat glioma,and the results were asfollowed.

1 Materials and methods

1.1 Cell culture C6 cells wereobtained from the pathological department of the second military medical university.Humanembryonic renal 293 cells were kindly provided by Dr.Liu Guoqin (University of BritishColombia Canada).C6 cells and 293 cells were grown in DMEM medium (Gibco,Grand Island,NY)supplemented with 10% fetal calf serum,penicillin (100u/ml) and streptomycin (100μg/ml).
1.2 Cloning of antisense IGF-1 gene and construction of the recombinantadenovirus IGF-1 vector(pAd-IGF-1A) Total RNA was isolated from fresh confluent C6 cellsusing the method of one-step RNA isolation.IGF-1 primers were put into the RNA solutiondegenerated at 70℃ for 10 minutes,and AMV reverse transcriptase (Biolabs company) wasadded at 0℃,incubated at 37℃ for 1 hour,at 40℃ for 30 minutes,at 70℃ for 15minutes.The IGF-1 primers were as followed.Pl:5′-TTGCCTCATTATTCCTGC-3′,P2:5′-TCGCAGCCAGCATAGCCAGGTC-3′PCRwas performed using the above reaction liquid,degenerated at 95℃ for 5 minutes,annealedat 48℃ for 30 seconds,extended at 70℃ for 60 seconds.From the second cycle thedegeneration time was changed to 30 seconds.The PCR reaction was performed for 35cycles.The PCR product was analyzed with 1.8% agarose gel electrophoresis.The DNA fragmentwas retrieved and cloned into pGEM-T vector.The antisense clone was picked up and thesequence of the antisense clone was analyzed with the auto-sequencing meter.The antisenseIGF-1 cDNA fragment was subcloned into pBS、pCEP4 and pAd vector (pAd-IGF-1A).
1.3 Recombinant adenoviruses construction 293 cells werecotransfected with the recombinant adenovirus plasmide:pAdHCMV-lacZ、pAdHCMV-tk (providedby Fudan University)、pAd-IGF-1A and adenovirus fragment pJM17 using the method ofcalcium phosphate precipitation.The adenovirus mono-plaque was picked up and analyzed.Therecombinant adenovirus (Ad-lacZ、Ad-tk and Ad-IGF-1A) were amplified、purified and thetiters of the adenoviruses were assayed.
1.4 Infection efficiency of recombinant adenovirus C6 cells wereinfected with different M.O.I recombinant adenovirus Ad-1acZ.X-gal staining was performed48 hours later.The rate of the cells stained with blue dye was assayed.
1.5 Sensibility of C6 cells infected with different recombinantadenoviruses to GCV C6 cells were cultured in 96-pore culture plate in the concentrationof 1×10⁴ /pore.Twenty four hours later the different adenovirus Ad-tk、Ad-IGF-1Aand Ad-tk+Ad-IGF-1A (M.O.I.=200) were added.The different concentrations of GCV (1～5μmol/L)were added to the medium 3 hours later.GCV was purchased from Roche-Syntax Company.Themedium was changed in every 48 hours in the original concentrations.Five days later thevital cells were determined with the MTT method.The OD values (570nm wave-length) wereread in the enzyme-marking meter.
1.6 Establishment of rat C6 brain glioma model and treatment withdifferent recombinant adenoviruses in vivo The experimental rats were SD rats purchasedfrom the Second Military Medical University.The average weight was between 200～250g.Onverification of adequate anesthesia (absence of withdraw and blink reflexes) with sodiumpentobarbital,rats were placed in a rodent stereotactic frame (Jiangwan1-model,Shanghai).The head was prepared with 95% ethanol,and strict aseptic technique wasobserved thereafter.A 1cm incision was made in the anteroposterior direction over theright frontal region.A craniotomy was made with a 2mm bit round,the dura was puncturedwith a 25-gauge needle,and 10⁶ C6 cells in 10μl of normal saline solution wereinjected with syringe into the right frontal lobe at a depth of 3mm.The craniotomy defectwas filled with bone wax and the incision was sutured.Eight days after implantation,ratswere re-fixed in the stereotactic frame and 10μl recombinant adenoviruses were injectedinto the same place in aperiod of 15 minutes.Five groups were divided with every group often rats.(1) Ad-IGF-1A treatment group:The injected adenovirus Ad-IGF-1A is 6×10⁸ pfu/10μl.(2)Ad-tk treatment group:The injected adenovirus Ad-tk was 5×10⁸ pfu/10μl.(3)Ad-tk+Ad-IGF-1A group:The injected adenoviruses Ad-tk and Ad-IGF-1A were 5×10⁸ pfu/10μland 6×10⁸ pfu/10μl.(4)Saline solution group.(5)Ad-IGF-1A/GCV group:Theinjected adenovirus Ad-IGF-1A was 6×10⁸ pfu/10μl.In the group 2～5，12 hoursafter injection of the adenovirus GCV 50mg/kg^. 24 hours was givenintra-abdominally in the consecutive 10 days.
1.7 Study of the re-implantation of C6 cells into the rats Thelong-term survival rats were re-implanted 10⁶ C6 cells and the period ofsurvival was observed.

2 Results

2.1 Cloning of antisense IGF-1 gene 300bpIGF-1 cDNA fragment was obtained from C6 cells using RT-PCR,among which contained the BglⅡenzyme-cutting site.The antisense IGF-1 pGEM-T plasmid was obtained.The sequence of theantisense clone was correct.
2.2 Recombinant adenoviruses construction 293 cells werecotransfected with the recombinant adenovirus plasmids pAdHCMV-lacZ、pAdHCMV-tk、pAd-IGF-1Aand adenovirus fragment pJM17.The adenoviruses were analyzed with PCR and were confirmedto contain the target gene.The titers of the purified adenoviruses Ad-IGF-1A、Ad-tk and Ad-lacZ were 6.5×10¹⁰ pfu/ml、5×10¹⁰ pfu/ml and 2×10¹⁰ pfu/ml.
2.3 Infection efficiency of recombinant adenovirus The infectionefficiency of Ad-lacZ in the M.O.I.of 50、100、200、400 was 75%、95%、100% and100%.C6 cells were infected 100 percent by adenovirus in the M.O.I.of 200. This impliedthat recombinant adenovirus might mediate high efficiency gene transfer and expression.
2.4 The sensibility of C6 cells infected by different recombinantadenoviruses to GCV C6 cells infected by Ad-tk in the M.O.I.=200 were killed totally byGCV in the concentration of 5μmol/L.C6 cells infected by Ad-tk and Ad-IGF-1A were killedtotally by GCV in the concentration of 2μmol/L.This implied that the C6 cells infected byAd-IGF-1A and Ad-tk were more sensitive to GCV than that the cells infected by Ad-tk.TheC6 cells and the C6 cells infected by Ad-IGF-1A or Ad-tk were not sensitive to GCV.(Figure1)

Figure 1 Sensibility of C6 cells infected by different recombinantadenoviruses of to GCV
-◆-Ad-tk+Ad-IGF-1A -▲-Ad-IGF-1A
Ad-tk Control

2.5 Treatment with different recombinantadenoviruses in vivo Eight days after C6 cells implantation,in the group of the Ad-tk/GCV treatment there was 50% of the rats surviving till 90 days and the averagesurvival period was 56.5±4.9 days.In the group of the Ad-IGF-1A treatment alone there was38% of the rats surviving till 90 days and the average survival period was 47.3±1.2days.In the group of combination treatment the average survival period was 90 days (P<0.01).Thisimplied that combining application of Ad-tk/GCV and Ad-IGF-1A might prolong the survivalrate of the tumor-bearing rats.The average survival period of the control group was 15.7±1.6days.(Figure 2)

Figure2 The survival rate of every group eight daysafter C6 cells implantation
-●-Ad-tk/GCV+Ad-IGF-1A Ad-IGF-1A
-▲-Ad-IGF-1A/GCV Ad-tl/GCV -◆-Control

2.6 Study of the re-implantation of C6 cells In thegroup of the HSV-tk/GCV treatment the average survival period was 26.1±2.5 days when therats surviving till 90 days were re-implanted C6 cells.In the group of the combiningtreatment the average survival period was 40.2±2.8 days when re-implanted.
2.7 Histological examination There were a lot of the CD⁺ ₄ and CD⁺ ₈ lymphocytes in the tumor tissue in the group of thecombining treatment.There were a little CD⁺ ₄ and CD⁺ ₈ lymphocytes in the tumor tissue in the group of the Ad-tk/GCV treatment.There were more CD⁺ ₄ and CD⁺ ₈ lymphocytes in the group of the Ad-IGF-1A than that of Ad-tk/GCV treatment.(Figure 3 and 4)

Figure3 Immunocytochemical analysis of the tumor tissuein the group of the Ad-tk/GCV treatment using
CD4 adn CD8 antibodies.(A)a little CD4+lymphocyted(the right),(B)a littleCD8+lymphocytes(the left)

Figure4 Immunocytochemical analysis of the tumor tissuein the group of the Ad-tk/GCV+AdIGF-1A
treatment.(A)large amout of CD4+lymphocytes(the right),(B)large amout ofCD8+lymphocytes(the left)

3 Discussion

As apromising gene therapy method,adenovirus mediated Ad-tk/GCV system has been extensivelyapplied in the experimental study of the treatment of brain tumors and the results wereinspiring.But in the recent reports the Ad-tk/GCV system may not cure the malignant braintumor and kill all the tumor cells.When the treatment ceased,the remained tumor cells maymultiply again and tumor reoccurred.Although the efficacy of Ad-tk/GCV system killingglioma cells was strong,the immunologic reaction in vivo was less effective and the bloodbrain barrier may hider GCV from entering the tumor.The bystander effect in vivo wasrelated to the immunologic reaction.Therefore various methods were used to strengthen theefficiency of the suicide gene,the more strategies adopted were various kinds of cytokinescombined with HSV-tk/GCV and some favorable effects were reported.We conducted thecombining gene therapy of Ad-tk/GCV and antisense IGF-1 for glioma,there was no reportabout this area,and we found the tumor-bearing rats underwent the combining gene therapywould have long survival period than that treated by Ad-tk/GCV or antisense IGF-1 alone.
In 1993,Trojan^[3] employed antisense IGF-1 to treat glioma and foundthe antisense IGF-1 may extinct the solid tumor of glioma and prevent it fromreoccurring.The possible mechanism may lie in two aspects,the one was that antisense IGF-1may block the tumor cells autocrine of IGF-1 and inhibit the tumor growth and the otherwas that antisense IGF-1 may change the phenotype of the tumor cells and lead the body todevelop the specific anti-tumor immunity.Shevelev^[4] found that theexpression of MHC-1 antigen was higher in C6 cells transfected with antisenseIGF-1.Lafarge-Frayssinet^[5] also found that the expression of MHC-1 and B7antigen was increased in tumor cells transfected with antisense IGF-1.And in the treatmentin vivo there were a lot of CD⁺ ₄ and CD⁺ ₈ lymphocytes in the tumor tissue.We have conducted adenovirus-mediated antisense IGF-1 genetherapy for glioma and found that 3 days and 5 days after inoculation the rats receivingthe antisense IGF-1 would survive in long-term.
Bystander effect was the major mechanism in the suicide gene treatment of thetumors.The immunity reaction was related to the bystander effect in vivo.The immunityreaction leaded by Ad-tk/GCV was less strong in treating the tumor.So Freeman^[6] pointed out that to strengthen the bystander effect of the immunity reaction might be aneffective method.Benedetti^[2] conducted the combining gene therapy forglioma with HSV-tk and IL-4.He found that the rate of mouse survival in 5 months was80%,but the group of HSV-tk/GCV or IL-4 was 10% or 23%.Chen^[7,8] conductedthe combining gene therapy for hepatocarcinoma with HSV-tk and IL-2.He found that resultof the combination treatment was more effective than any gene therapy alone.Hayashi^[9] conducted the combining gene therapy for rectal cancer with HSV-tk and GM-CSF and alsofound the tumor-bearing rats of the combination treatment group survived in long-term.Theresult of our experiment displayed that combining gene therapy with antisense IGF-1 and HSV-tk/GCV would made the larger tumor disappear and activate the body immunity totumor.The rats receiving the treatment may acquire the specific anti-tumor immunity.Therewere a lot of CD⁺ ₄ and CD⁺ ₈ lymphocytes in thetumor tissue.This implied that antisense IGF-1 might strengthen the tumor-killing effectof HSV-tk/GCV and prevent the tumor from re-occurring.Therefore combining gene therapywith antisense and HSV-tk may be an effective method of treating the glioma.

References

1，Cool V,Pirohe B,Gerard C,etal.Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with9L gliosarcoma.Human Gene Ther,1996,7∶627
2，Benedetti S,Dimeco F,Pollo B,et al.Limited efficacy of the HSV-tk/GCV system for genetherapy of malignant gliomas and perspectives for the combined transduction of the IL-4gene.Human Gene Ther,1997,8∶1345
3，Trojan J,Johnonson TR,Rudin SD,et al.Treatment and prevention of rat glioblastoma byC6 cells expressing antisense IGF-1 RNA.Science,1993,259∶94
4，Shevelev A,Befriend AP,Schoolgirl E,et al.Potential triple helix-mediated inhibitionof IGF-1 gene expression significantly reduces tumorigenicity of glioblastoma in an animalmodel.Cancer Gene Ther,1997,4∶105
5，Lafarge-Frayssinet C,Duc HT,Frayssinet C,et al.Antisense insulin-like growth factors-1transferred into hepatoma cell line inhibits tumorigenesis by modulating majorhistocompatibility major l cell surface expression.Cancer Gene Ther,1997,4∶276
6，Freeman SW,Ramesh R,Marrogi AJ,et al.Immune system in suicide genetherapy.Lancet,1997,349∶2
7，Chen SH,Chen XHL,Wany Y,et al.Combination gene therapy for liver metastasis of coloncarcinoma in vivo.Proc Natal Acad Sci,1995,92∶2577
8，Chen SH,Kosai KI,Xu BS,et al.Combination suicide and cytokine gene therapy for hepaticmetastasis of colon carcinoma:sustained antitumor immunity prolongs animal survival.CancerRes,1996,56∶3758
9，Shuji Hayashi,Nobwhiko E,Itsuo Y,et al.Inhibition of establishment of hepaticmetastasis in mice by combination gene therapy using both HSV-tk and GM-CSF genes inmurine colon cancer.Cancer Gene Ther,1997,4∶339

Received：2000-07-03