Construction Of Co-expression Vectors Containing HSV-tk And HIL-12 Genes And Study On Killing Effects Of HSV-tk/IL-12 On Human Colorectal Cancer

Background: Human colorectal carcinoma (HCC) is one of the most lethal cancersin humans. The majority of patients died of local recurrence despite aggressive medicalintervention including surgery, radiotherapy, chemotherapy. In recent years, gene therapyof tumors has become the most noticeable research field. It's the fifth model followedsurgical therapy, radiotherapy, chemotherapy and immunotherapy. The suicide genetherapy is one of the most promising gene therapies for the tumor. Interleukin-12 (IL-12)is a cytokine with many immunobiological activities which is disulfide-linkedheterodimer molecule produced predominantly by professional antigen presenting cells. Itnot only activates CTL and NK cells, but also promotes the induction of sundrybiological effects with significant relevance to anticancer immunity, such as enhancementof T (H) 1 helper response, an in vivo antiangiogenic effect, and induction of adhesionmolecules that assist in lymphocyte homing to sites of tumor growth. Also Interleukin-12(IL-12) can not only directly induce and enhance the activity of lymphokine activatedkiller (LAK) cells, but also stimulates IFN-gamma production in both T cells and NKcells. Therefore we constructed co-expression vectors containing HSV-tk and mIL-12genes. We evaluated the killing effects of HSV-tk/IL-12 on human colorectal cancer.Methods:(1) :After the full length cDNAs of both P40 and P35 subunit genes werecloned respectively into pcDNA3.1(±) to get P(+)/P40 and P(-)/P35.By the method ofdirectional cloning and introduction of IRES, the sequenced genes were subcloned intocorresponding restriction sites of pLXSN in turn to generate HSV-tk , hIL-12co-expression vectors pLXSN-tk-IRES-IL-12 named pL(TI)SN. (2) HSV-tk gene wassubcloned into retroviral vector pLXSN by recombinant DNA technology. pL(TI)SN andpL(tk)SN were packaged with PA317 and selected in G418 to obtain the positive clones,which was able to produce stable retrovirus. LoVo was infected by the recombinantretrovirus. The positive clones were obtained after G418 selection and were termedLoVo/TI and LoVo/TK .The integration and expression of tk gene in LoVo/TI andLoVo/TK cells were identified by PCR , RT-PCR , SDS-PAGE , western-blot ,immunocytochemical. The GCV killing effects on gene-modified cells were assessed byobserving growth state of LoVo/TI and LoVo/TK, MTT and FCM. (3) LoVo/TI cells weresuspended in phosphated-buffed saline at a concentration of 1×108 cells/ml and injectedsubcutaneously (100u1 inoculum volume) into the right back of syngeneic femaleBALB/C nude mice (4-5 weeks old,living in SPF conditions, purchased from the animalexperiment center of the first military medical university). Ten days after the inoculation,mice were randomly divided into three groups. There were five mice in each group. GCVwas administered intraperitoneally into the mice of the first group;Mice receivedtreatment twice daily for five days. Every five days tumor volumes were calculated usingthe simplified formula (L×W2×0.5). On 30th day all mice were killed, tumor volumeswere measured. Inhibitory rate of tumor growth was obtained according to the formula(1-the tumor volume of treatment group/the tumor volume of control group)×100%.Tissues of tumor and organs (such as heart, liver, kidney and lung) were removed andfixed in 10% buffed formalin and stained with hematoxylin and eosin forhistopathological analysis. tumor volumes of the group treated with HSV-tk/GCV,which was smallest. No pathologic changes were observed in organs. (4) To investigatethe distant bystander effect of herpes simplex virus thymidine kinase ganciclovir(HSV-TI/GCV)system on human colorectal carcinoma cell in vivo. Methods:TK+carcinoma were induced on left flank while TK-carcinoma were induced in rightflank ,then the GCV treatment was given to the urime and the effect on the growth of thetumor was subsequent observed. Results:In treatment group ,the TK+ tumor on the flantgrow slowly significant to the tumor on the cantrol group ,and the survival of the micewas prolonged. Histotlgically ,there were intratumoral necrosis in the (TK+) carcinoma.And the TK-tumor on the right flant grow slowly to the tumor on the cantrol group ,andthe survival of the mice was prolonged. Histotlgically ,intratumoral necrosis wereobviously in the (TK-) carcinoma. (5) In our experiment we want to culture thenon-LoVo/TI cells vaccine by irradiating at isotope Co60 50Gy, study the vaccine'scharacter non-Oncogenic of LoVo/TI contrast withLoVo/TI cells and it's antitumor effects.So we could provide the base theory in gene therapy of human colon carcinoma.Results: (1) By sequencing,restriction digestion and PCR we confirmed that thesequence, length, position and orientation ofinserted genes were all correct. Then targetvectors, pL(TI)SN and pL(TT)SN were constructed successfully. (2) The retroviral vectorpL(tk)SN was generatedand stable titer producing cell C26 was established(1.6×106/ml).PCR and RT-PCR demonstrated that tk gene was integrated into the LoVo/TI genome andexpressed at the mRNA level. There was no significant difference in cell proliferationamong LoVo/TI, LoVo/TK and LoVo cells. LoVo/TI cells were highly sensitive to GCV ,especially in low concentration of GCV. (3)LoVo/TI, LoVo/TK and LoVo cells (1×106cells /0.lml) were inoculated separately into subcutaneous tissue of different nudemice. Begin to inject GCV(50mg/kg, 2 times/d) to abdominal cavity of nudethe volumeof tumor from the 8th day. At the 20th day, finish the experiment, kill nude mice and weightumor. The results showed that the average of volume and weight of tumors in LoVo/TIgroup were significant smaller than that in LoVo/0and LoVo groups (p<0.05). (4)HSV-TI/GCV oncolysates can induce the kill effect to the carcinoma cell ,and the distantbystander effect to the distant carcinoma cell in other organ were obviously. (5) Thecellproliferation LoVo/TI cells that were gave a dose isotope Co60 50Gy, when comparedwith LoVo/TI cells, had been inhabited. A dose isotope Co60 50Gy not only assurancenon-oncogenic LoVo/TI vaccine cells, but also made it can transcript IL-12 gene. Thespleen cells could secret IFN-γ when we cultured LoVo/TI cells and irradiated LoVo/TIcells, the spleen cells ability of secreting IFN-γ had no different. Initially, we confirmedthat the vaccine of irradiated LoVo/TI cells in subcutaneour can inhabit volume of LoVotumor, the anti-tumor effects have obvious relationship with the number of vaccine ofirradiated LoVo/TI cells. But the mechanism needs to be studied further.Conclusions: We successfully constructed co-expression vectors containing HSV-tk,IL-12 genes. In vitro study showed that human colorectal carcinoma cells were sensitiveto HSV-tk/IL-12 system .These results suggest that HSV-tk/IL-12 system is an effectivegene therapy and will lay some foundation for clinical treatment of human colorectalcarcinoma. HSV-tk/GCV system can induce apoptosis of LoVo cells of human colorectalcarcinoma, and the percentage of apoptosis has a time effect. HSV-tk+/LoVo cells areblacked in S phase. Synergistic use of HS-Vtk/GCV system and 5-FU could improve theefficacy of present cancer suicide gene therapy approach. Initially, we confirmed that thevaccine of irradiated LoVo/TI cells in subcutaneour can inhabit volume of LoVo tumor,the anti-tumor effects have obvious relationship with the number of vaccine of irradiatedLoVo/TI cells.