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.     

Autophagyinhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation Schonewolf CA,Mehta M,Schiff D,Wu H,Haffty BG,Karantza V,Jabbour SK简

AIM: To investigate whether the inhibition of autophagy by chloroquine (CQ) sensitizes rectal tumors to radiation therapy (RT) or concurrent chemoradiation (chemoRT).METHODS: In vitro, HCT-116 and HT-29 colorectal cancer (CRC) cell lines were treated as following: (1) PBS; (2) CQ; (3) 5-fluorouracil (5-FU); (4) RT; (5) CQ and RT; (6) 5-FU and RT; (7) CQ and 5-FU; and (8) 5-FU and CQ and RT. Each group was then exposed to various doses of radiation (0-8 Gy) depending on the experiment. Cell viability and proliferative capacity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Clonogenic survival curves were constructed and compared across treatment groups. Autophagy status was determined by assessing the LC3-II to LC3-I ratio on western blot analysis, autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFP-labeled LC3 on fluorescence microscopy. Cell cycle arrest and cell death were evaluated by FACS and Annexin V analysis. All experiments were performed in triplicate and statistical analysis was performed by the student’s t test to compare means between treatment groups.RESULTS: RT (2-8 Gy) induced autophagy in HCT-116 and HT-29 CRC cell lines at 4 and 6 h post-radiation, respectively, as measured by increasing LC3-II to LC3-I ratio on western blot. Additionally, electron microscopy demonstrated autophagy induction in HT-29 cells 24 h following irradiation at a dose of 8 Gy. Drug treatment with 5-FU (25 μmol/L) induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction. CQ (10 μmol/L) alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation (8 Gy; P < 0.001 and 0.00001, respectively). Significant decrease in clonogenic survival was seen only in the HT-29 cell line, when CQ was combined with RT at doses of 2 and 8 Gy (P < 0.5 and P = 0.05, respectively). There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT.CONCLUSION: Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro, suggesting that addition of CQ to chemoRT improves CRC treatment response.     

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.     

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.     

Expression of the bifunctional suicide gene CDUPRT increases radiosensitization and bystander effect of 5-FC in prostate cancer cells

Purpose

To test the hypothesis that, with 5-fluorocytosine (5-FC) treatment, the co-expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) can lead to greater radiosensitization and bystander effect than CD-expression alone.

Methods and materials

R3327-AT cell lines stably expressing CD or CDUPRT were generated. The 5-FC and 5-FU cytotoxicity, and the radiosensitivity with/without 5-FC treatment, of these cells were evaluated under both aerobic and hypoxic conditions. The bystander effect was assessed by apoptosis staining and clonogenic survival. The pharmacokinetics of 5-FU and 5-FC metabolism was monitored in mice bearing CD- or CDUPRT-expressing tumors using ¹⁹F MR spectroscopy (MRS).

Results

CDUPRT-expressing cells were more sensitive to 5-FC and 5-FU than CD-expressing cells. CDUPRT-expression further enhanced the radiosensitizing effect of 5-FC, relative to that achieved by CD-expression alone. A 25-fold lower dose of 5-FC resulted in the same magnitude of radiosensitization in CDUPRT-expressing cells, relative to that in CD-expressing cells. The 5-FC cytotoxicity in co-cultures of parental cells mixed with 10-20% CDUPRT cells was similar to that in 100% CDUPRT cells. ¹⁹F MRS measurements showed that expression of CDUPRT leads to enhanced accumulation of fluorine nucleotide (FNuc), relative to that associated with CD-expression alone.

Conclusion

Our study suggests that CDUPRT/5-FC strategy may be more effective than CD/5-FC, especially when used in combination with radiation.     

Preliminary analysis of a randomized clinical trial of adjuvant postoperative RT vs. postoperative RT plus 5-FU and levamisole in patients with TNM stage II-III resectable rectal cancer

OBJECTIVES: Two-hundred eighteen patients with TNM stage II-III resectable rectal cancer, enrolled into a randomized clinical trial, were assessed for efficacy and toxicity of adjuvant postoperative radiation therapy (RT) vs. those of combined RT and chemotherapy (CT), with 5-fluorouracil (5-FU) plus levamisole. End points were overall survival, disease-free survival, the rate of loco-regional recurrence, and treatment-related toxicity. METHODS: Patients in arm I underwent RT (50 Gy) in daily fractions of 2 Gy, 5 days/week for 5 weeks. Patients in arm II began with 5-FU (450 mg/m(2)/day intravenous bolus, days 1-5) plus levamisole (150 mg/day orally, days 1-3); postoperative RT was delivered during week 2 at the same dosage and schedule as in arm I. The other five cycles of CT (5-FU every 28 days and levamisole every 15 days for the length of 5-FU administration) continued after the end of RT if clinical and hemato-biochemical parameters were normal. RESULTS: RT was completed or modified in 170 (90%) of 189 evaluable patients undergoing RT (both treatment groups). Only 44 (59%) of 75 evaluable patients of arm II completed or had an adjustment of the CT schedule; the remaining 31 patients (41%) had to stop or never started the CT regimen. Patients undergoing combined RT and CT had more severe toxicity (enteritis, P = 0.03). There was one CT-related death (gastrointestinal bleeding) in this subset. No significant difference was observed in outcome of patients in the two study groups, nor for pattern of recurrence (heterogeneity chi(2) = 4.82; d.f. = 2; P = 0.08). CONCLUSIONS: These preliminary findings suggest a similar efficacy, coupled with less morbidity, of postoperative RT alone compared with a combined regimen of postoperative RT and CT in patients undergoing radical surgery for stage II-III rectal cancer.     

Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas

5-Fluorouracil (5-FU) is a potent antimetabolite used for chemotherapy of gastrointestinal (GI), breast, and head and neck malignancies. Although clinical trials have been conducted, the poor therapeutic index of 5-FU has precluded its clinical use for a number of other tumor types. It is unclear whether this lack of utility is due to problems with drug delivery or inherent insensitivity. Adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy has the potential to overcome pharmacokinetic issues associated with systemic 5-FU and is particularly well suited to use with tumors in which local control is paramount, such as recurrent, localized prostate cancer and malignant gliomas. In this study, the in vitro response by a panel of human tumor cell lines derived from both GI (colon, pancreas) and non-GI (prostate, glioma) tumors to 5-FU and to AdCMVCD (an Ad encoding Escherichia coli CD)/5-FC was examined. Whereas the sensitivity (IC(50)) of individual cell lines to these agents varied, no significant difference in median IC(50) for either 5-FU or AdCMVCD/5-FC was evident for the four tumor types tested (P > 0.1). The relevant contributions of Ad gene transfer efficiency and inherent 5-FU sensitivity in determining response to AdCMVCD/5-FC were then assessed. Multiple linear regression analysis revealed that whereas both factors significantly contribute to the response, inherent 5-FU sensitivity was substantially more important (beta= 0.78 versus 0.48; P < 0.001). Finally, the therapeutic efficacy of a single intratumoral injection of AdCMVCD followed by systemic 5-FC was assessed in three intracranial C.B17 severe combined immunodeficient mouse models of human glioma. AdCMVCD/5-FC efficacy was specific, virus dose-dependent, and closely paralleled in vitro 5-FU and CD/5-FC sensitivity in two of three models tested. These results reveal that glioma cells are as sensitive as GI tumor cells to the antineoplastic effects of 5-FU, identify inherent 5-FU sensitivity as an important factor in determining CD/5-FC efficacy, and confirm previous findings in rat models that demonstrate the potential clinical utility of AdCMVCD/5-FC gene therapy for gliomas.     

Gene transfer into human prostate adenocarcinoma cells with an adenoviral vector: Hyperthermia enhances a double suicide gene expression, cytotoxicity and radiotoxicity

We have previously developed a recombinant adenovirus containing a fusion gene of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK) controlled by a cytomegalovirus (CMV) enhancer-promoter. This replication-incompetent adenovirus effectively transduced the CD-TK gene into human prostate adenocarcinoma DU-145 or PC-3 cells. Interestingly, heat shock at 41 degrees C for 4 hours elevated the level of CD-TK by approximately 5- to 20-fold at a multiplicity of infection (MOI) of 1. Heat-enhanced expression of CD-TK promoted cytotoxicity by 23-, 9-, or 47-fold in the presence of 50 microg/mL ganciclovir (GCV), 500 microg/mL 5-fluorocytosine (5-FC), or 50 microg/mL GCV+500 microg/mL 5-FC, respectively, at an MOI of 1. Moreover, there was an increase in radiosensitivity when adenovirus-infected cells were heated at 41 degrees C for 4 hours followed by irradiation in the presence of the prodrugs. Virus+heat+1 microg/mL GCV treatment increased radiosensitivity by a dose-modifying factor (DMF) of 2.2, whereas virus+heat+10 microg/mL 5-FC exposure resulted in a DMF of 2.3. Radiosensitization was clearly enhanced as a result of combined prodrug exposure (DMF=4.4). Our results suggest that the efficiency in expression of suicide genes from an adenoviral vector used for cytotoxic anticancer therapy could be improved by combining heat treatment with radiation therapy.     

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.     

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。     

Mature results of a pilot study of pelvic radiotherapy with concurrent continuous infusion intra-arterial 5-FU for stage IIIB-IVA squamous cell carcinoma of the cervix.

PURPOSE: To evaluate the long-term results of continuous infusion intra-arterial 5-fluorouracil (CI IA 5-FU) given with concurrent pelvic radiotherapy (RT) for FIGO stage IIIB-IVA carcinoma of the cervix. METHODS AND MATERIALS: Between 1965 and 1974, 27 patients with extensive FIGO Stage IIIB (22 patients) or Stage IVA (5 patients) squamous cell carcinoma of the cervix were treated with CI IA 5-FU and RT. Twenty-one patients (78%) had bilateral pelvic wall involvement, 25 (93%) had massive tumors (> or =8 cm in diameter), 7 (27%) had involvement of the lower one-third of the vagina, and 15 (56%) presented with hydronephrosis. All patients underwent routine clinical staging, transperitoneal para-aortic lymph node dissection, and bilateral hypogastric artery catheter placement. 5-FU was continuously infused at a dose rate of 10 mg/kg/day on Days 1-15 of RT. The median dose of 5-FU was 376 mg/m2/day (range 270-692). All patients received concurrent pelvic RT to a median dose of 50 Gy at 2.0 Gy per fraction. Only 4 patients received intracavitary RT. The median follow-up of surviving patients was 190 months. RESULTS: The overall 5-year survival rate was 37%. For the 22 patients with FIGO Stage IIIB disease, the 5-year survival rate was 41%. The survival rate for 18 patients treated with only external beam radiation and chemotherapy for Stage IIIB disease was 33%. Four of 10 patients treated with only 50 Gy of external beam radiation and CI IA 5-FU were long-term survivors. Acute complications, including hematologic toxicity and skin reactions, were severe, with 1 death from neutropenic sepsis. Severe late complications were only observed in patients treated with > or =60 Gy of external beam radiation. CONCLUSIONS: While this series is small, the fact that 4 patients with massive Stage IIIB tumors survived after a total radiation dose of only 50 Gy suggests that RT with CI IA 5-FU deserves further study. Modifications in dose, technique, and route of administration should reduce toxicity, and the addition of intracavitary RT should improve the local effectiveness of combined treatment.     

Combined modality therapy of rectal cancer: decreased acute toxicity with the preoperative approach.

PURPOSE: We compared the combined radiation therapy (RT) plus chemotherapy segments of two separate parallel phase I trials to determine if combined pelvic RT, fluorouracil (5-FU), and high-dose leucovorin (LV) had less acute toxicity when delivered preoperatively versus postoperatively in patients with rectal cancer. PATIENTS AND METHODS: Patients with unresectable disease received preoperative RT plus LV and 5-FU followed by surgery and postoperative LV and 5-FU. Patients with resectable disease received identical doses, techniques, and schedules of RT and LV and 5-FU except all therapy was delivered postoperatively. On day 1, patients received LV and 5-FU times one cycle. RT began on day 8. A second cycle of LV and 5-FU was given concurrently with the fourth week of RT. RESULTS: Although more patients (75% v 32%; P = .02) received the higher dose level of 5-FU (250 mg/m2), significantly fewer experienced acute grade 3 to 4 toxicity with preoperative versus postoperative therapy (13% v 48%; P = .045). There was no grade 3 to 4 myelosuppression in either group. The two grade 3 toxicities in the preoperative group were gastrointestinal. The grade 3 toxicities in the postoperative group included seven gastrointestinal and two genitourinary; four patients had a grade 4 toxicity. CONCLUSION: Given the high incidence of grade 3 to 4 toxicity also reported in the postoperative combined modality adjuvant randomized trials, future adjuvant trials should explore the preoperative approach.     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗作用及免疫机理研究

本研究以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Ltn)基因体内联合转染,观察了其抗肿瘤效应并分析了免疫机理.小鼠皮下接种结肠腺癌CT26细胞后3天,肿瘤局部注射表达Ltn的重组腺病毒AdLtn和表达CD的重组腺病毒AdCD,然后连续10天给予5一氟胞嘧啶(5-FC)300mg/kg进行治疗,结果表明,联合治疗组荷瘤小鼠皮下肿瘤结节的生长受到明显抑制,小鼠存活期明显长于单用AdLtn治疗组或单用AdCD/5-FC治疗组.经联合治疗后小鼠脾细胞的NK活性和对(37结肠腺癌细胞的CTL杀伤活性明显增强.瘤体细胞FACS分析结果表明,经联合基因治疗后,肿瘤组织CD4~ 、CD8~ 细胞浸润增加,结肠腺癌细胞表达H-2Kd和B7-1分子明显增加.提示经CD自杀基因和Ltn基因联合治疗后,肿瘤细胞免疫原性增加.本研究结果表明联合应用自杀基因和Ltn基因治疗可以提高机体对肿瘤细胞免疫的应答,增加机体的抗肿瘤作用,是肿瘤基因治疗中一条新的途径.     

氟胞嘧啶/胞嘧啶脱氨酶自杀基因疗法结合热休克蛋白-多肽复合物治疗小鼠胃癌

目的；研究氟胞嘧啶／胞嘧啶脱氨酶（5－FC／CD）基因疗法与热休克蛋白－多肽复合物（FSP70－PC）免疫疗法联合抗肿瘤效果。方法；将携带CD基因的重组腺病毒注射到小鼠MFC瘤体内，腹腔注射5－FC，同时皮下接种HSP70－PC。结果；经联合治疗后，70％荷瘤小鼠肿瘤体答缩小，消退，小鼠存活期延长，细胞毒T淋巴细胞（CTL）杀伤活性增高，CD4^ 及CD^8 T细胞浸润明显。结论：5－FC／CD基因疗法结合HSP－PC免疫疗法抗小鼠MFC瘤作用显著，具有临床应用前景。     

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.     

Phase I and II trial of five-day infused 5-fluorouracil and radiation in advanced cancer of the head and neck

Eighteen patients with advanced epithelial cancers of the head and neck region were studied for their tolerance and response to combined cycles of 120-hour infused 5-fluorouracil (5-FU) and external-beam radiation therapy. 5-FU infusions were given under conditions where radiosensitization would be expected at the higher infusion doses. Coincident radiation treatments were given as four sequential daily fractions of 250 rad each administered during days 1 through 4 of each five-day infusion cycle. The patients were rested for at least nine days after each cycle or longer until toxicity was resolved. The regimen was then repeated in each patient for a total of five treatment cycles. Thereafter therapy was consolidated, usually by boost radiation without drug. In sequential patient subsets the infusion load was progressively escalated in a phase I format. The complete response rate for stage IV patients was 75% with survival benefit compared to prior results. 5-FU dose-dependent combined modality loco-regional toxicity was demonstrated without significant enhancement of systemic toxicity of any form; 5-FU dose-dependent enhanced responsiveness and survival benefit is also suggested. Further scheduling and response studies of 5-FU under radiosensitizing conditions appear warranted.     

Bystander effect from cytosine deaminase and uracil phosphoribosyl transferase genes in vitro: A partial contribution of gap junctions

Among gene therapy strategies elaborated to kill cancer cells, one uses the CodA gene, coding for cytosine deaminase (CD) that converts 5-fluorocytosine (5-FC) into toxic 5-fluorouracil (5-FU). To enhance 5-FC metabolic activation, we prepared a vector carrying CodA and upp (uracil phosphoribosyl transferase) genes which rendered HeLa cells sensitive to 5-FC and enhanced a bystander effect not mediated by gap junctions. However, 1% CD⁺–UPP⁺ cells were able to kill 40% of the cell population if the cells were communicating. This suggests that, at very low percentages of CD⁺–UPP⁺ cells, CodA and upp induce a bystander effect through gap junction-dependent mechanisms.     

Enzyme/prodrug gene therapy for human colon cancer cells using adenovirus-mediated transfer of the Escherichia coli cytosine deaminase gene driven by a CAG promoter associated with 5-fluorocytosine administration

Escherichia coli cytosine deaminase (CD), which is a prokaryotic enzyme, converts nontoxic prodrug 5-fluorocytosine (5-FC) into the toxic chemotherapeutic agent 5-fluorouracil (5-FU). To investigate an enzyme/prodrug gene therapy for colorectal cancer, using adenoviral gene transfer of the E. coli CD gene associated with administration of 5-FC, we constructed replication-defective adenovirus vectors expressing the E. coli CD gene or lacZ gene driven by a CAG promoter (composed of a cytomegalovirus immediate early enhancer and a chicken beta-actin promotor). The present study demonstrated that an adenoviral gene transfer system using a CAG promoter induced sufficient gene expression of CD to confer the cytotoxicity of 5-FC to HT29 human colon cancer cells by converting it into 5-FU even at an moi of one. Furthermore, experimental gene therapy using intratumoral injection of the CD-expressing adenovirus with systemical administration of 5'-FC successfully suppressed the growth of established HT29 subcutaneous tumors in nude mice. These results suggest that enzyme/prodrug gene therapy using the adenoviral gene transfer of the E. coli CD gene with concomitant administration of 5-FC may be an effective strategy in the local control of colorectal cancer.