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.     

Interaction of endothelial progenitor cells expressing cytosine deaminase in tumor tissues and 5-fluorocytosine administration suppresses growth of 5-fluorouracil-sensitive liver cancer in mice

The drug delivery system to tumors is a critical factor in upregulating the effect of anticancer drugs and reducing adverse events. Recent studies indicated selective migration of bone marrow-derived endothelial progenitor cells (EPC) into tumor tissues. Cytosine deaminase (CD) transforms nontoxic 5-fluorocytosine (5-FC) into the highly toxic 5-fluorouracil (5-FU). We investigated the antitumor effect of a new CD/5-FC system with CD cDNA transfected EPC for hepatocellular carcinoma (HCC) in mice. We used human hepatoma cell lines (HuH-7, HLF, HAK1-B, KYN-2, KIM-1) and a rat EPC cell line (TR-BME-2). Escherichia coli CD cDNA was transfected into TR-BME-2 (CD-TR-BME). The inhibitory effect of 5-FU on the proliferation of hepatoma cell lines and the inhibitory effect of 5-FU secreted by CD-TR-BME and 5-FC on the proliferation of co-cultured hepatoma cells were evaluated by a tetrazolium-based assay. In mouse subcutaneous xenograft models of KYN-2 and HuH-7, CD-TR-BME was transplanted intravenously followed by 5-FC injection intraperitoneally. HuH-7 cells were the most sensitive to 5-FU and KYN-2 cells were the most resistant. CD-TR-BME secreted 5-FU and inhibited HuH-7 proliferation in a 5-FC dose-dependent manner. CD-TR-BME were recruited into the tumor tissues and some were incorporated into tumor vessels. Tumor growth of HuH-7 was significantly suppressed during 5-FC administration. No bodyweight loss, ALT abnormality or bone marrow suppression was observed. These findings suggest that our new CD/5-FC system with CD cDNA transfected EPC could be an effective and safe treatment for suppression of 5-FU-sensitive HCC growth.     

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.     

Management of malignant pleural effusion by suicide gene therapy in advanced stage lung cancer: a case series and literature review

Gene therapy can be defined as the transfer of genetic material into a cell for therapeutic purposes. Cytosine deaminase (CD) transferred into tumor cells by an adenoviral vector (Ad.CD), can convert the antifungal drug fluorocytosine (5-FC) to the antimetabolite 5-fluorouracil (5-FU), which kills not only the transfected tumor cells but also their neighbors by the so-called 'bystander effect'. After testing a protocol for Ad.CD transfer and lung tumor burden control in a Lewis mouse model, we used this technique in the management of lung cancer patients with malignant pleural effusion (MPE): two cases are presented investigating the possible enhancement of anticancer effect in both non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) by local activation of the pro-drug 5-FC. Results were discussed in parallel to a literature review on the topic. 5-FC and Ad.CD were administered intratumorally to Lewis mouse lung carcinoma and the effect was monitored by tumor size and electromicroscopy. Two patients with advanced stage lung cancer (1SCLC, 1NSCLC), which developed MPE during first-line treatment were administered 10(12) plaque-forming unit (pfu) Ad.CD by intrapleural instillation, in two doses (day1 and day7). Instillation was performed when the pleural fluid was 200?ml. In addition, they received 5-FC 500?mg four times daily for 14 days. Lung tumor regression and successful transfer of adenoviral particles were observed in treated animals. Patients presented complete regression of pleural effusion as monitored by computerized tomography scan. Neutrapenia and anemia were the most severe adverse effect presented (grade III/grade IV 100%). The increased toxicity followed by the intrapleural gene therapy indicates the augmentation of anticancer effect of transformed pro-drug 5-FC to active 5-FU. The obtained data indicate that intrapleural gene therapy may be a useful tool, adjunct to chemotherapy, in the management of MPE related to lung cancer.     

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.     

Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy

Since neural progenitor cells can engraft stably into brain tumors and differentiate along the neuronal and glial line, we tested the hypothesis that transplanted cytosine deaminase (CD)-expressing ST14A cells (an immortalized neural progenitor cell line) can convert locally 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and produce a regression of glioma tumors. ST14A, retrovirally transduced with the E. coli CD gene, showed a strong bystander effect on glioma cells as assessed by in vitro assay. Intracerebral injection of C6 glioma cells generated a rapidly growing tumoral mass. DiI prelabeled ST14A, coinjected into the rat brain with C6 glioma cells, survived in the tumoral mass up to 10 days and their number was not affected by in vivo 5-FC treatment. In contrast, a significant decrease of the glioma tumoral mass (-50%) was observed in 5-FC-treated rats. 5-FC had no effect on the tumor in the absence of CD-expressing ST14A cells. Our results support the feasibility of systems based on intratumoral transplantation of prodrug-converting cells for brain tumor therapy.     

5—氟胞嘧啶对表达胞嘧啶脱氨酶的人肾母细胞瘤裸鼠异种移植瘤 …

OBJECTIVE: To study the effect of 5-fluorocytosine (5-FC) as prodrug in the treatment of Wilms' tumor xenografts transduced with cytosine deaminase (CD) gene. METHODS: An in vivo model of a poorly differentiated Wilms' tumor transplanted in nude mice was established. Expression adenoviral-vector of CD gene (Ad/CMV-CD) or lac gene (Ad/CMV-lac) was transduced to the tumor xenografts by intratumoral injections. Expression of the transduced genes were confirmed by RT-PCR. Mice with Wilms' tumor xenograft were treated with 5-FC (500 mg.kg-1.d-1 x 10 d). Tumor growth was monitored. RESULTS: The growth of tumor xenografts transduced with lac gene grew as quick as the untransduced ones. In contrast, the growth of the tumor xenografts transduced with CD gene was significantly inhibited as compared to untransduced and lac gene transduced xenografts. The average rate of inhibition was 65% according to the tumor weight at 8 wk. Cell necrosis was observed in the CD gene transduced tumors. CONCLUSION: Intratumoral cytosine deaminase gene transduction followed by systemic 5-fluorocytosine is effective in the treatment of Wilms' tumor.     

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.     

Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5-FC/CD + UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects.     

In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.     

Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy.Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10–30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone.Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization.Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers.     

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.     

Oncolytic herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation

Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional Herpes simplex virus 1 (HSV-1) expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. In animal models, 12 days of 5-FC administration was superior to 6 days, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing-schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10?ng?ml?1) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses.     

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.     

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.     

Efficacy of adenovirus-mediated CD/5-FC and HSV-1 thymidine kinase/ganciclovir suicide gene therapies concomitant with p53 gene therapy.

Recent evidence has suggested that tumor cells having a wild-type p53 status are more sensitive to chemotherapeutic agents and radiation than cells that lack functional p53. The heightened sensitivity of wild-type p53 cells is thought to be attributable to their propensity to undergo p53-mediated apoptosis after insult. Given that suicide gene therapy is essentially tumor-targeted chemotherapy, we examined the hypothesis that coexpression of wild-type p53 could enhance the efficacy of adenovirus-mediated suicide gene therapy. Human Hep3B and SK-OV-3 cells, which are null for p53, were infected with a pair of replication-deficient adenoviruses that expressed a cytosine deaminase/herpes simplex virus thymidine kinase (CD/HSV-1 TK) fusion gene without (fusion gene nonreplicative adenovirus, FGNR) or with (FGNRp53) the wild-type human p53 gene. The sensitivity of cells to the CD/5-fluorocytosine (CD/5-FC) and HSV-1 TK/ ganciclovir (GCV) enzyme/prodrug systems was determined in vitro and in vivo. Coexpression of p53 did not enhance the cytotoxicity of either the CD/5-FC or HSV-1 TK/GCV system in vitro. The failure to observe an effect of p53 could not be explained on the basis of insufficient or transient p53 expression, because FGNRp53-infected cells growth arrested in G1, induced Bax, and underwent apoptosis at an increased rate after prodrug treatment, particularly when the adenovirus E1A protein was present. Intratumoral injection of FGNRp53 concomitant with single or double pro-drug therapy resulted in a tumor growth delay that was equal to or less than that observed with the FGNR virus. Our results indicate that coexpression of p53 may not necessarily improve the efficacy of adenovirus-mediated CD/ 5-FC and HSV-1 TK/GCV suicide gene therapies in vivo.     

Combination therapy of continuous venous infusion (CVI) of 5-FU and low dose consecutive cisplatin (CDDP), and the new oral anti-cancer drug S-1 for advanced gastro-intestinal cancer

Highly effective treatment is required for patients with advanced GI cancer. Returning to the starting point for reconsideration of cancer chemotherapy, with the aim of attaining a therapy (self rescuing concept: SRC) with more potential efficacy and less toxicity than current therapy, we report two kinds of chemotherapy in the present paper. They were set up preclinically using the theory of 5-FU biochemical modulation, and demonstrated their usefulness in clinical practice. S-1 is a newly developed oral anti-cancer drug which is a combination of Tegafur (FT), a prodrug of 5-FU and two modulators (CDHP, an inhibitor of 5-FU degradation and Oxo, a selective inhibitor GI toxicity by 5-FU) at a molar ratio of 1:0.4:1. In combination with CDHP, 5-FU gradually released from FT remained longer in plasma, and consequently had high anti-tumor activity, while the combined Oxo significantly suppressed GI toxicity due to 5-FU. The response rate to S-1 of stomach cancer in a phase II study was 46.5% (60/129). Toxicity at more than G3 was less than 10%. In the combination therapy employing 5-FU by CVI (5-FU: 250-350 mg/body for 24 h, 4-6 wks) and low dose consecutive CDDP, CDDP acts mainly as a modulator of 5-FU (to increase 5-FU sensitivity for tumor by inhibition of intracellular Met incorporation). For this purpose, it was found that daily consecutive administration is required, even at low dose of CDDP (3-5 mg/body/day for 5 days). A high response rate (40-60%) was obtained for advanced GI cancer. Toxicity at more than G3 was less than 10%. On the other hand, the possibility has been suggested that so far as 5-FU is concerned, CVI every other day (500-750 mg/body/day for 3 days) is more favorable than long term CVI, with regard to decreasing GI and myelotoxicities based upon the difference in generation time between normal cell (GI mucous membrane and stem cell) and tumor cell cycles. The possibility is suggested that the above-mentioned chemotherapy can become a standard therapy for GI cancer.     

Mechanisms of thymidine kinase/ganciclovir and cytosine deaminase/ 5-fluorocytosine suicide gene therapy-induced cell death in glioma cells

Suicide gene transfer using thymidine kinase (TK) and ganciclovir (GCV) treatment or the cytosine deaminase (CD)/5-fluorocytosine (5-FC) system represents the most widely used approach for gene therapy of cancer. However, molecular pathways and resistance mechanisms remain controversial for GCV-mediated cytotoxicity, and are virtually unknown for the CD/5-FC system. Here, we elucidated some of the cellular pathways in glioma cell lines that were transduced to express the TK or CD gene. In wild-type p53-expressing U87 cells, exposure to GCV and 5-FC resulted in a weak p53 response, although apoptosis was efficiently induced. Cell death triggered by GCV and 5-FC was independent of death receptors, but accompanied by mitochondrial alterations. Whereas expression of Bax remained unaffected, in particular, GCV and also 5-FC caused a decline in the level of Bcl-2. Similar findings were obtained in 9L and T98G glioma cells that express mutant p53, and also underwent mitochondrial apoptosis in both the TK/GCV and CD/5-FC system. Upon treatment of 9L cells with 5-FC, Bcl-xL expression slowly declined, whereas exposure to GCV resulted in the rapid proapoptotic phosphorylation of Bcl-xL. These data suggest that TK/GCV- and CD/5-FC-induced apoptosis does neither require p53 nor death receptors, but converges at a mitochondrial pathway triggered by different mechanisms of modulation of Bcl-2 proteins.     

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