Antitumoral vaccination with granulocyte-macrophage colony-stimulating factor or interleukin-12-expressing DHD/K12 colon adenocarcinoma cells

Immunomodulating gene therapy for the treatment of malignant diseases is under extensive investigation. In this study, we induced an antitumoral immune response with murine interleukin-12 (mIL-12) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cells in a model of peritoneal carcinomatosis. Intraperitoneal injection of DHD/K12 tumoral cells engineered to produce IL-12 or GM-CSF did not generate any tumors, whereas untransduced DHD/K12 cells gave rise to peritoneal carcinomatosis. IL-12-expressing DHD/K12 cells also protected against tumors derived from coinjected parental cells. To test whether cytokine-producing cells could elicit a memory antitumoral immune response, animals received a challenge with parental DHD/K12 cells 35 days after the injection of proliferating or irradiated DHD/K12 engineered cells. Under our experimental conditions, irradiated tumor cells did not generate any antitumoral immunity. In contrast, tumor development was delayed and survival increased in the animals vaccinated with cytokine-secreting proliferating cells. A specific cytotoxic T-lymphocyte response against DHD/K12 parental cells was observed after vaccination with GM-CSF-expressing cells. Our results demonstrated that intraperitoneal vaccination with IL-12- or GM-CSF-expressing adenocarcinoma cells induced a systemic immune antitumoral response that may be useful as an adjuvant therapy after surgical resection of colorectal cancer.     

Matrix metalloproteinase inhibition prevents colon cancer peritoneal carcinomatosis development and prolongs survival in rats.

Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation which play a role in cancer progression and metastatic spreading. We investigated the effects of the MMP inhibitor, batimastat, in vitro on the proliferation and invasiveness of the rat colon cancer cell line DHD/K12, and in vivo on the growth of an aggressive model of peritoneal carcinomatosis producing haemorrhagic ascites and metastases, obtained in the rat by i.p. injection of DHD/K12 cells. MMP production was studied in conditioned culture media, solid tumors and ascitic fluid. In vivo, after injection of tumor cells on day 0, rats received i.p. daily either batimastat (30 mg/kg) or equal volume of vehicle from day 2 until killing on day 43 (series I) or from day 13 until death (series II). The grade of peritoneal carcinomatosis, ascite volume, number and size of liver metastases were evaluated in both series, and survival in series II. MMPs-1, -2 and -9 were identified in culture media, tumors and ascites. In vitro, batimastat did not modify DHD/K12 cell proliferation and slightly reduced cell invasion. In vivo, in series I, batimastat treatment totally prevented peritoneal carcinomatosis and liver metastasis development. In series II, it significantly prolonged survival (P < 0.0002) and reduced peritoneal carcinomatosis (P < 0.001) and hepatic metastases number as compared with controls. However, batimastat-treated rats of the two series had peritoneal inflammation with marked ascites. Nevertheless, inhibition of MMP is a new therapeutic approach which may be promising in treatment of microtumors as in more advanced cancer stages.     

Heat-directed suicide gene therapy mediated by heat shock protein promoter for gastric cancer

The prognosis of patients with metastatic gastric cancer, particularly peritoneal carcinomatosis, remains poor despite intensive interventions. Gene therapy and hyperthermia can be promising strategies for such advanced disease. The study was conducted to explore the possible effective therapeutic approach of suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with hyperthermia for advanced gastric cancer. The heat shock protein (hsp) 70B gene promoter-oriented HSV-tk (HSP-tk)/ganciclovir (GCV) system directed by heat shock was developed. Hsp promoter activity under the control of heating was assessed by dual luciferase assay in gastric cancer cell lines and implanted tumors of nude mice. In vitro cytotoxic assay was performed using the HSP-tk/GCV delivered by the hemagglutinating virus of Japan (HVJ) liposome, with or without heating. Mice with subcutaneously xenografted tumors and peritoneal carcinomatosis were treated with hyperthermia and gene therapy using the HVJ-liposome-carrying HSP-tk. Assessment by luciferase assay demonstrated highly inducible and tumor-specific promoter activity in vitro and in vivo. Cytotoxic assays showed that cells transfected with HSP-tk became more sensitive to GCV with heating. A synergistic effect was also observed when treated with a non-heat-inducible cytomegalovirus (CMV) promoter-mediated HSV-tk/GCV and heating, indicating bystander killing. The HVJ-liposome-carrying HSP-tk/GCV combined with hyperthermia significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice with peritoneal carcinomatosis. We conclude that the combination of suicide gene therapy with hyperthermia can provide a promising treatment modality for advanced gastric cancer.     

胞嘧啶脱氨酶基因联合放射对食管癌细胞株的杀伤作用

目的：观察大肠杆菌胞嘧啶脱氨酶／5-氟胞嘧啶（CD／5-FC）自杀基因系统对食管癌细胞株（EC109细胞）的杀伤效应以及与放射联合应用时对食管癌肿瘤细胞协同杀伤效应。方法：采用PCR方法,从大肠杆菌基因组DNA中扩增出CD基因;构建重组真核载体pcDNA3．1-CD;用脂质体转染法转染食管癌ECl09细胞,观察CD／5-FC体系对EC109细胞的杀伤效应和杀伤时的旁观者效应以及对放射的增敏效应。结果：RTP—CR分析结果表明CD基因已转入ECl09细胞并开始转录。体外实验证实,5-FC对转CD基因后的食管癌细胞株有明显的细胞毒作用,CD／5-FC体系对肿瘤细胞对放射增敏效果明显,加5-FC及放射治疗组细胞存活曲线低于单纯加前药5-FC对照组或单纯放射对照组。结论：CD／5-FC体系对肿瘤细胞的自杀效应具有旁观者效应和放射增敏效果。     

双自杀基因CD和TK对K562细胞体内外杀伤作用的实验研究

目的:探讨双自杀基因CD和TK对K562细胞的体内外抑制作用及前体药物对肿瘤的杀伤作用。方法:将目的基因转染入K562细胞,MTT法观察细胞在体内外的增殖状况及5-FC/GCV对转染细胞的杀伤作用,电子显微镜观察其超微结构变化。将K562/CDgly TK和K562细胞接种于裸鼠皮下,观察各种肿瘤细胞在体内的成瘤情况及对前体药物治疗的敏感性。结果:单独使用GCV或5-FC对K562及K562/CDgly TK细胞产生明显的杀伤作用,联合应用该2种药物对肿瘤细胞的杀伤作用更强。将K562细胞和K562/CDgly TK细胞接种于小鼠皮下后小鼠成瘤率为100%,GCV或5-FC可明显抑制裸鼠体内的肿瘤形成,联合应用GCV和5-FC治疗K562/CDglyTK细胞在小鼠体内形成的肿瘤,较单独应用GCV和5-FC及对照组小鼠形成的肿瘤体积明显缩小,生存期也明显延长。结论:双自杀基因在体内外对K562细胞均有明显的抑制作用,可增加前体药物GCV和5-FC对瘤细胞的杀伤率。     

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用     

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.     

Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer

The bacterial enzyme cytosine deaminase (CD) catalyzes the conversion of 5-fluorocytosine (5-FC) to the lethal 5-fluorouracil (5-FU) and so provides a useful system for selective killing of gene-modified mammalian tumor cells. Cloning of the CD gene from Escherichia coli and expression in human tumor cell lines enabled these cells to convert ³H-labeled 5-FC into ³H-5-FU. Two CD-expressing human tumor cell lines (adenocarcinoma cell line KM12 and glioblastoma cell line T1115) became 200-fold more sensitive to 5-FC than the nonexpressing parental cell lines. At least 90% of the cells are killed within 7 days. CD-expressing cells are able to kill nonexpressing cells when grown in the same culture flask (bystander effect). The CD gene may be used as a suicide system for in situ chemotherapy or as a safety mechanism abrogating the expression of other genes. © 1996 Wiley-Liss, Inc.     

逆转录病毒介导的CD/5-FC对胰腺癌细胞的杀伤作用

目的 :探讨大肠埃希菌胞嘧啶脱氨基酶 (CD) /5 -氟胞嘧啶 ( 5 FC)系统对胰腺癌细胞的体外生长抑制作用。方法 :将含CD基因的重组逆转录病毒载体导入胰腺癌细胞形成转化细胞系 ,对转化细胞进行体外药物敏感实验 ,包括 :1)转化细胞在前体药物 5 FC作用下的细胞生长抑制率 ;2 )MTT法检测旁观者效应。结果 :体外实验 5 FC的有效浓度 >2mmol/L时 ,就表现出杀伤作用 ,当 5 FC的有效浓度 >6mmol/L时 ,几乎未见细胞生长 ,PA3 17/CD与TD2混育比例在 90 %时 ,杀伤作用最大 ,相同药物浓度下 ,混育 96h杀伤作用明显高于 2 4h。结论 :CD/5 FC系统对胰腺癌细胞系细胞具有实验性基因治疗作用     

Apoptosis Induction With 5-Fluorocytosine/Cytosine Deaminase gene Therapy for Human Malignant Glioma Cells Mediated by Adenovirus

Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36delta5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36delta5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.     

Adipose tissue-derived human mesenchymal stem cells mediated prodrug cancer gene therapy

Human adipose tissue-derived mesenchymal stem cells (AT-MSC) are considered to be a promising source of autologous stem cells in personalized cell-based therapies. Tumor tracking properties of MSC provide an attractive opportunity for targeted transgene delivery into the sites of tumor formation. In the present study, we addressed whether the suicide gene introduction into human AT-MSC could produce a tumor-specific prodrug converting cellular vehicle for targeted chemotherapy. We prepared yeast fusion cytosine deaminase::uracil phosphoribosyltransferase gene-expressing cells [cytosine deaminase (CD)-expressing AT-MSC (CD-AT-MSC)] by retrovirus transduction. We explored their therapeutic potential on a model of human colon cancer in the presence of prodrug 5-fluorocytosine (5-FC). Gene manipulation of human AT-MSC did not sensitize CD-AT-MSC to 5-FC, thus overcoming the inherent disadvantage of suicide effect on cellular vehicle. CD-AT-MSC in combination with 5-FC augmented the bystander effect and selective cytotoxicity on target tumor cells HT-29 in direct coculture in vitro. We confirmed directed migration ability of AT-MSC and CD-AT-MSC toward tumor cells HT-29 in vitro. Moreover, we achieved significant inhibition of s.c. tumor xenograft growth by s.c. or i.v. administered CD-AT-MSC in immunocompromised mice treated with 5-FC. We confirmed the ability of CD-AT-MSC to deliver the CD transgene to the site of tumor formation and mediate strong antitumor effect in vivo. Taken together, these data characterize MSC derived from adipose tissue as suitable delivery vehicles for prodrug converting gene and show their utility for a personalized cell-based targeted cancer gene therapy.     

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.     

Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts

The efficacy of cancer gene therapy using bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) enzyme/prodrug strategy is limited by the inefficiency of cytosine deaminase (CD)-catalyzed conversion of 5-FC into 5-fluorouracil (5-FU). We have shown previously that yeast CD (yCD) is more efficient at the conversion of 5-FC than bCD. In the current study, we hypothesized that the increased production of 5-FU by yCD would enhance the efficacy of the CD/5-FC treatment strategy by increasing the bystander effect as well as the efficacy of radiotherapy because of the radiosensitizing capacity of 5-FU. To test this hypothesis, we generated stable HT29 human colon cancer cell lines expressing either bCD (HT29/bCD) or yCD (HT29/yCD). The amount of 5-FU produced in HT29/yCD tumors after a single injection of 5-FC (1000 mg/kg, i.p.) was 15-fold higher than that produced in HT29/bCD tumors. In tumor-bearing nude mice, the average minimum relative tumor size (compared with pretreatment values) of HT29/bCD tumors treated with 5-FC and radiation (500 mg/kg i.p. and 3 Gy, 5 days a week for 2 weeks) was 0.55+/-0.1, compared with 0.01+/-0.01 in HT29/yCD tumors (P = 0.002). Moreover, an increased cytotoxic and radiosensitizing effect of 5-FC on bystander cells was observed in vitro and in vivo when yCD was expressed in HT29 cells instead of bCD. In mice bearing HT29 tumors containing 10% HT29/yCD cells, the combined treatment resulted in a minimum tumor size of 0.20+/-0.07 compared with 0.60+/-0.1 in 10% HT29/bCD cells (P < 0.001). These results demonstrate that the use of yCD in the CD/5-FC strategy has a high potential to improve the therapeutic outcome of combined gene therapy and radiotherapy in cancer patients.     

The nitroreductase/CB1954 combination in Epstein-Barr virus-positive B-cell lines: induction of bystander killing in vitro and in vivo

Epstein-Barr virus (EBV)-based gene delivery vectors that preferentially express toxic genes in EBV-infected cells could be used to target EBV-positive tumors for destruction. We have shown previously that the cytosine deaminase (CD) enzyme, which converts the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil efficiently kills EBV-positive cells in the presence of 5-FC, with a substantial bystander killing effect in vitro and in vivo. To identify the optimal enzyme/prodrug combination for treating EBV-positive lymphomas, we have compared the effectiveness of the CD/5-FC combination with the nitroreductase (NTR)/CB1954 combination for killing EBV-positive B-cell lines. NTR metabolizes CB1954 into an alkylating agent that cross-links DNA. When the CD gene or the NTR gene were transfected into two different EBV-positive B-cell lines in vitro, approximately 90% of cells were killed in a prodrug-dependent manner, although the transfection efficiency was <5%. However, severe combined immunodeficient mouse tumors containing either 30% or 100% of NTR-expressing Burkitt lymphoma (Jijoye) cells were growth inhibited, but not cured, by treatment with intraperitoneal CB1954 (20 mg/kg/day) for 10 days. These results suggest that the NTR/CB1954 combination induces efficient bystander killing of EBV-positive B-cell lines in vitro but may not be as effective as the CD/5-FC combination for treating B-cell lymphomas in vivo.     

OSM对黑色素瘤的抑制作用与基因-放射治疗效果

目的：通过细菌内同源重组,制备含绿荧光（GFP）基因的CD腺病毒,初步观察其体外杀瘤作用。方法：先构建含CD基因的腺病毒梭载体pAdTrack-CMV-CD,与骨架载体pAdEasy-1在细菌内重组为pAd-CD,经293细胞包装为增殖缺陷性腺病毒,体外感染人结肠癌细胞株LoVo,并给予前药5－FC,观察其体外杀瘤效果。结果：pAdTrack-CMV-CD和同源重组的pAd-CD经酶切鉴定正确,pAd-CD转染293细胞,包装扩散腺病毒后,感染LoVo细胞,予5－FC治疗,4d后在荧光温微镜下观察到转染LoVo和未转染细胞胞均被杀死,并且 这种旁观者效应不依赖于细胞间接解。结论：细菌内质粒同源重组法可以高效制备含绿荧光（GFP)基因的CD腺病毒,体外腺病毒介导CD／5－FC可以杀伤肿瘤细胞,其旁观者效应不依赖于细胞间接触。     

Carcinoembryonic antigen-specific suicide gene therapy of cytosine deaminase/5-fluorocytosine enhanced by the cre/loxP system in the orthotopic gastric carcinoma model

Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 10(9) plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.     

Cytosine deaminase/5-fluorocytosine exposure induces bystander and radiosensitization effects in hypoxic glioblastoma cells in vitro

PURPOSE: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. METHODS AND MATERIALS: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. RESULTS: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. CONCLUSION: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.     

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS_2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS_2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.     

Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy

One important feature of human solid tumors is the presence of a hypoxic microenvironment. Under hypoxia, genes that contain a hypoxia-response element (HRE) can be activated by the binding of hypoxia-inducible factor-1. To reach the goal of selectively killing tumor cells in a hypoxic microenvironment using a gene therapy approach, we developed a cytosine deaminase (CD) gene construct (pH9YCD2) that contains an HRE gene enhancer. CD is an enzyme that catalyzes the conversion of noncytotoxic 5-fluorocytosine (5-FC) to the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU). Yeast CD was cloned into an SV40 promoter-based mammalian expression vector, and an HRE enhancer was inserted in front of the promoter. Human glioblastoma U-87 MG cells were transfected with pH9YCD2. Western blots revealed that CD was strongly expressed under hypoxic conditions (0.3-1% O2), whereas only minor CD expression was seen under normoxic conditions. To confirm that the expressed CD enzyme retains catalytic activity, we performed a 5-FC/5-FU-conversion assay in which 5-FC was incubated with the lysates of pH9YCD2-transfected cells. The percentage of conversion from 5-FC to 5-FU was 63% under hypoxia versus 13% under normoxia. In vitro, cell viability and colony-forming efficiency assays demonstrated that the gene construct was able to significantly kill glioblastoma cells in a hypoxia-dependent manner. In addition, 5-FC treatment of hypoxic pH9YCD2-transfected cells produced a marked bystander effect, which could be a distinct advantage for gene therapy. If this construct exhibits antitumor efficacy in vivo, it may have promise as an antitumor agent in humans.