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Hepatobiliary & Pancreatic Diseases International 2003年第1期2卷 147-151页

作者： Zhao-Shen Li Xue Pan Guo-Ming Xu Long Cui Guan-Rong Dai Yan-Fang Gong Zhen-Xing Tu the Department of Gastroenterology Department of General Surgery Changhai Hospital, Second Military Medical University, Shanghai 200433, China Department of Gastroenterology Changhai Hospital Second Military Medical University Shanghai 200433 China. zsli@***

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-l were recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

关键词： pancreatic cancer adenovirus verctor cytosine deaminase gene therapy

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

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World Journal of Gastroenterology 2002年第2期8卷 270-275页

作者： Li-Zong Shen Wen-Xi Wu Qiang Ding Yi-Bing Hua,Department of General Surgery,The First Affiliated Hospital of Nanjing Medical University,Nanjing,210029,Jiangsu Province,China De-Hua Xu Zhong-Cheng Zheng Xin-Yuan Liu,Shanghai Institute of Biochemistry and Cell Biology,The Chinese Academy of Sciences,Shanghai,200031,China Kun Yao,Department of Microbiology and Immunology,Nanjing Medical University,Nanjing,210029,Jiangsu Province,China Department of General Surgery The First Affiliated Hospital of Nanjing Medical University 300 Guangzhou Road Nanjing 210029 Jiangsu Province China.

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micr

关键词： Gene Therapy Genetic Vectors Adenoviridae Animals Antimetabolites Bystander Effect Carcinoembryonic Antigen Cell Line Colorectal Neoplasms cytosine deaminase Flucytosine Hela Cells Humans Nucleoside deaminases Promoter Regions (Genetics) Research Support, Non-U.S. Gov't Tumor Cells, Cultured

In situ transduction of cytosine deaminase gene followed by systemic use of 5-fluorocytosine inhibits tumor growth and metastasis in orthotopic prostate cancer mouse models

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Chinese Medical Journal 2002年第2期115卷 227-231,152-153页

作者：张正望殷莲华张永康赵凤娣复旦大学医学院(原上海医科大学)附属中山医院泌尿外科上海200032 复旦大学医学院病理生理教研室上海200032

OBJECTIVE: To investigate the antitumor and anti-metastatic effect of in situ transduction of adenovirus encoding cytosine deaminase (AdCD) followed by the systemic use of 5-fluorocytosine (5-FC) in the orthotopic (o.t.) prostate cancer mouse model. METHODS: The o.t. prostate cancer model of C57BL/6 mouse was developed by o.t. inoculation of RM-1 cells to the subcapsular area of the prostate gland. In situ transduction of the CD gene, followed by systemic use of 5-FC at a daily dosage of 300 mg/kg for 14 days, was performed two days later. RESULTS: Compared with mice treated with Adbeta-gal/5-FC, 5-FC and PBS, mice of the o.t. model receiving in situ treatment of AdCD/5-FC had significant prolongation of survival and suppression of local tumor growth. More importantly, pathological observations showed that metastatic activity occurred in all mice of the PBS, 5-FC and Adbeta-gal groups including metastasis to the iliac lymph node (10/10, 10/10, 10/10) and the lung (8/10, 7/10, 7/10). However, only two out of ten had iliac lymphatic metastasis in the AdCD/5-FC group with no systemic or preaotic lymphatic metastasis, suggesting a strong metastatic inhibitory effect. CONCLUSIONS: In situ transduction of AdCD followed by systemic use of 5-FC leads to the inhibitory effect on tumor growth and metastatic activity in the o.t. mouse model of prostate cancer. Clinically, it may be possible to treat metastatic or recurrent prostate cancer with a novel gene therapy using in situ injection techniques in future.

关键词： Adenoviridae Animals Cell Division cytosine deaminase Disease Models, Animal Flucytosine Lymphatic Metastasis Male Mice Mice, Inbred C57BL Neoplasm Transplantation Nucleoside deaminases Prostatic Neoplasms Survival Rate Transfection Tumor Cells, Cultured

Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma

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Acta Pharmacologica Sinica 2005年第5期26卷 629-634页

作者： Cheng YI~2,Ying HUANG~3,Zhi-ying GUO~3,Shu-ren WANG~(3,4) ~2Cancer Center,West China Hospital,Sichuan University Chengdu,610041,China ~3Department of Pathophysiology,West China School of Preclinical and Forensic Medicine,Sichuan University,Chengdu 610041,China CancerCenter WestChinaHospitalSichuanUniversityChengdu610041China

Aim:To construct a Bifidobacterium infantis/CD targeting gene therapy systemand observe the antitumor effect of cytosine deaminase/5-fluorocytosine(CD/5-FC)suicide gene therapy system mediated by Bifidobacterium infantis on mela-noma in vitro and in ***:A recombinant CD/pGEX-1LamdaT plasmidwas transfected into Bifidobacterium infantis by ***-terium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid was in-cubated with 5-FC *** the supernatant fluid was collected andadded to melanoma B 16-F10 cells to observe the killing effect for B 16-F10 *** were inoculated with melanoma B 16-F10 cells to establish animal *** mice were then injected with 5-FC and Bifidobacterium infantis transfectedby recombinant CD/pGEX-1LamdaT ***:Two segments of approxi-mate 4.9 kb and 1.3 kb were extracted from the 6.2 kb recombinant plasmid,whichwere equal to the size of the pGEX-1LamdaT plasmid and CD gene,*** results showed that the full length and sequence of nucleotide acid ofthe inserted gene in extracted recombinant plasmid was completely identical to theCD *** vitro,B16-F10 cells treated by supernatant fluid were remarkablydamaged morphologically,and the cell growth was significantly ***-ments on the mice melanoma model showed that after treatment with a combina-tion of transfected Bifidobacterium infantis and 5-FC,the tumor volume wassignificantly inhibited compared with ***:The foreign gene,CD gene,was correctly inserted into pGEX-1LambdaT plasmid and transferredinto Bifidobacterium ***/5-FC suicide gene therapy system mediated byBifidobacterium infantis demonstrated a good antitumor effect on melanoma invitro and in vivo.

关键词： Bifidobacterium infantis cytosine deaminase gene therapy

Science Letters:Brain metastases of melanoma-mechanisms of attack on their defence system by engineered stem cells in the microenvironment

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2007年第9期8卷 609-611页

作者： DIMITROV Borislav D. ATANASSOVA Penka A. RACHKOVA Mariana I. BORDANI Alzano Lombardo Bergamo 24022 Italy Department of Neurology Medical University Plovdiv 4000 Bulgaria ASSON-93 Research Co. Chicago IL 60056 USA

This report gives a better emphasis on the role of targeted effectors (e.g. a combination of 5-FC with CD-NSPCs as compared to the application of NSPCs alone) and how such delivery of pro-drug activating enzymes and other tumor-killing substances may overcome melanocytic defence system, interact with and promote the host defence and immune response modulations not only in melanoma but, potentially, in other highly-metastatic cancers.

关键词： Melanoma Brain metastases Neural stem/progenitor cells (NSPCs) cytosine deaminase

Augmentation of antitumor effect of adenovirus-mediated CD suicide gene therapy by cotransfer of interleukin 2 gene in melanoma-bearing mice

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Chinese Medical Journal 1999年第2期 67-70页

作者：鞠佃文曹雪涛陶群王宝梅陈国友于益芝

Objective To investigate the antitumor effect of combined adenovirus encoding E. coli cytosine deaminase (AdCD) and adenovirus encoding murine interleukin 2 (AdIL 2) on murine melanoma. Methods C57BL/6 mice were inoculated *** B16F10 melanoma cells and 3 days later received injections of AdCD and/or AdIL 2 at the site of tumor inoculation followed by administration of 5 flurocytosine (5FC) 300 mg/kg per day for 10 days. Results Mice receiving AdCD/5FC/AdIL2 therapy developed tumors more slowly and survived much longer when compared with mice treated with AdCD/5FC, AdIL2, AdlacZ/5FC, or PBS. Immunological analysis illustrated that combined treatment could enhance NK activity and CTL activity. Flow cytometry demonstrated that AdCD/5FC/AdIL2 therapy increased the expression of MHC I and CD80 molecules on freshly isolated tumor cells. The CD4 + and CD8 + T cell infiltration in the tumor increased significantly after the combined therapy. Conclusions Our data showed that combined transfer of CD suicide gene and IL 2 gene could inhibit the tumor growth more significantly. The increased specific and non specific antitumor immunity might be responsible for the enhanced therapeutic effect.

关键词： gene therapy cytosine deaminase 5 fluorocytosine interleukin 2 adenovirus antitumor immunity

Pharmacokinetics and the bystander effect in CD：：UPRT/5-FC bi-gene therapy of glioma

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Chinese Medical Journal 2009年第11期122卷 1267-1272页

作者： SHI De-zhi HU Wei-xing LI Li-xin CHEN Gong WEI Dong GU Pei-yuan Department of Neurosurgery First Affiliated Hospital of Nanjing Medical University Nanjing Jiangsu 210029 China

Background cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides （F-Nuctd）. For the C6 mixture, only the 5-FC peak was detected. In vivo serial ^19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about

关键词： glioma cytosine deaminase uracil phosphoribosyltransferase pharmacokinetics magnetic resonance spectroscopy bystander effect

Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

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World Journal of Gastroenterology 2005年第44期11卷 6910-6919页

作者： Florian Graepler Marie-Luise Lemken Wolfgang A Wybranietz Ulrike Schmidt Irina Smirnow Christine D GroB Martin Spiegel Andrea Schenk Schenk Hansj(o|¨)rg Graf Ulrike A Lauer Reinhard Vonthein Michael Gregor Sorin Armeanu Michael Bitzer Ulrich M.Lauer Department of Internal Medicine Ⅰ Section of Experimental Radiology Department of Medical Biometry

AIM： To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model. METHODS： To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine （5-FC）, we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase （YCD） and the yeast uracil phosphoribosyltransferase （YUPRT） gene. RESULTS： In vitro stably transduced Morris rat hepatoma cells （MH） expressing the bifunctional SuperCD suicide gene （MH SuperCD） showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH cells stably expressing YCD solely （MH YCD） or the cytosine deaminase gene of bacterial origin （MH BCD）, respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions （P〈0.01） under both high dose as well as low dose systemic 5-FC application, whereas MH tumors without transgene expression （MH naive） showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness （SuperCD〉〉 YCD〉〉BCD〉〉〉negative control） was defined as a result of a direct in vivo comparison of all three suicide genes. CONCLUSION： Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

关键词： YCD/YUPRT fusion cytosine deaminase GDEPT Suicide gene therapy Hepatoma therapy

Adenoviral mediated suicide gene transfer in the treatment of pancreatic cancer

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Chinese Medical Journal 2002年第8期115卷 1205-1208,153页

作者：潘雪李兆申许国铭崔龙张素贞龚燕芳屠振兴上海第二军医大学附属长海医院消化科上海200433 上海第二军医大学附属长海医院普外科上海200433

OBJECTIVE: To determine the efficacy of adenovirus　mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: cytosine deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.

关键词： Gene Therapy Adenoviridae Animals cytosine deaminase Gene Transfer Techniques Genetic Vectors Humans Mice Mice, Inbred BALB C Nucleoside deaminases Pancreatic Neoplasms Research Support, Non-U.S. Gov't

Effects of radiation-induced suicide gene therapy under radiation of radionuclide 125I on human hepatocarcinoma cells in vitro

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Chinese Medical Journal 2014年第7期127卷 1385-1387页

作者： Li Ling Zhang Chunli Yan Ping Yin Lei Kang Lei Zhao Qian Wang Rongfu Key Laboratory of Nuclear Medicine Ministry of Health Jiangsu Key Laboratory of Molecular Nuclear Medicine Jiangsu Institute of Nuclear Medicine Wuxi Jiangsu 214063 China Department of Nuclear Medicine Peking University First HospitalBeijing 100034 China

There are few efficient therapeutic approaches to *** et al1 developed iodine （131I） metuximab injection （Licartin）,a novel 131I-labeled HAb18G/CD147-specific monoclonal antibody Fab＇2 ***18G/CD147 is a hepatocellular carcinoma-associated *** their results have no obvious improvements in survival rate of patients with ***：radiation effects; promoter regions, genetic lentivirus; cytosine deaminase; fluorocytosine; HepG2 cells

关键词： radiation effects promoter regions, genetic lentivirus cytosine deaminase fluorocytosine HepG2 cells