Cytosine Deaminase Gene Research Articles

Recombinant adenoviral vector containing tumor-specific l-plastin promoter fused to cytosine deaminase gene as a transcription unit: generation and functional test

The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system (AdLP-) in which the expression of the transgene is directed by the tumor-specific L-plastin promoter (LP) (Chung et al., 1999). The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase (CD) gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter (AdLPCD). Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocytosine (5-FC) to 5-Fluorouracil (5-FU). HPLC analysis in conjunction with counting radioactivity for [6-3H]-5FC and [6-3H]-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies (Peng et al., 2001; Akbulut et al., 2003) suggest that the use of the AdLPCD/5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.

803. Functional Analysis of Various Gene Linkers Serving Proportional Coexpression of PET Marker and Therapeutic Genes

Top of pageAbstract Objective: To improve the overall level of gene expression of the second gene of interest in our universal HSV-1 amplicon vectors (Jacobs et al. 2003) using alternative gene linkers (eukaryotic IRES, 2A) and/or mutant HSV-1 thymidine kinase (tk39; Black et al. 1996). Background: The gene expression cassette cdIREStkgfp (Jacobs et al. 2003) was originally engineered to assess for the proportionality of co-expression of the therapeutic E. coli cytosine deaminase gene, cd, and the HSV-1-tkgfp fusion gene (fusion of the herpes simplex virus type 1 thymidine kinase gene serving as PET-marker gene and the gene coding for green fluorescent protein, gfp, as a reporter gene for cell culture; Jacobs et al. 1999). Functional evaluation had shown an attenuation of the IRES-mediated expression of the second fusion gene compared to the expression of the gene at the 5'-primed end of the IRES-element. Methods: First, the ECMV-derived IRES-element was replaced by an eucaryotic IRES-element (Ie; Chappell et al. 2000) as well as by the 2A-element of the food and mouth disease (FMD) virus (deFelipe et al. 1999). Thereafter, human Gli36dEGFR glioma cells (as well as BHK, Vero2-2, U87 cells) were infected with an MOI=1 of HSV-CITG, HSV-CIeTG or HSV-C2ATG, and relative GFP-expression was compared. 24 h after infection, a single cell analysis of relative GFP-expression was performed by means of the MPI-Tool imaging software (Jacobs et al. 2003). Second, the HSV-1-tk gene was replaced by the gene mutant HSV-1-tk39 showing a higher enzymatic efficiency than the wild type HSV-1-tk (Black et al. 1996). Gli36dEGFR cells were infected with an MOI=3 of HSV-CITG or HSV-CITk39G, and an MTT-assay was performed employing different concentrations of ganciclovir (0.01-10 g/ml). Results: In cell culture, the relative level of TKGFP-expression mediated by HSV-CIeTG-and HSV-C2ATG-infection was 65.2+21.9% and 86.6+12.4%, respectively, compared to HSV-CITG infected cells. These data indicate a further attenuation- instead of the expected improvement- of expression of the second gene in all tested cell lines. In contrast, the exchange of wild type HSV-1-tk to its mutant tk39 showed a significant increase in protein activity (LD50 HSV-CITG: 2.7 g/ml GCV; LD50 HSV-CITk39G: 0.023 g/ml GCV). Conclusion: No significant improvement of the expression of the second gene was achieved by replacing the ECMV-derived IRES-element with the alternative linkers, eukaryotic IRES and 2A-FMD, in all cell lines tested. However, an increasing activity of the second gene product with respect to its TK-activity could be achieved by replacing the wild type tk-gene with the tk39 mutant possessing a higher enzymatic efficiency.

599. Low-Dose Etoposide Enhances Tumor-Specific Adenovirus-Mediated Cytosine Deaminase Gene Therapy through Induction of Integrin αvβ3 and αvβ5 Overexpression and Upregulation of Human Telomerase Reverse Transcriptase Promoter

Purpose: The human telomerase reverse transcriptase (hTERT) promoter is used to restrict adenoviral expression of suicide or proapoptotic genes in telomerase-positive cancer cells. Etoposide can enhance intratumoral transgene expression in mice immunized with adenoviral vectors. The aim of this study is to evaluate whether greater therapeutic benefit can be achieved by combination treatment of low-dose etoposide and replication-incompetent adenoviral vectors encoding cytosine deaminase (CD) driven by the hTERT promoter for murine bladder carcinoma.

1032. Massive Expression of a Suicide Gene Using a Replicative but Non-Disseminative Adenovirus for Treatment of Solid Tumors

Cancer gene therapy using suicide genes had shown great potential in animal models although the efficacy of this approach is still insufficient for complete tumor regression. To improve efficacies, recent attention has focused on the use of oncolytic vectors. To date this strategy has not resulted in vectors with anti-tumor activities sufficiently potent to sustain consistent complete tumor regression, although in a few instances complete regression has been observed. In this study, we pursued a modified approach that makes use of viral DNA replication but not of oncolysis. This approach is based on the protease-deleted adenovirus vector (AdV) (Oualikene et al. 2000). Such vectors are designed to convert transduced cells into mega-factories for suicide genes. To validate this strategy, we have introduced the suicide gene cytosine deaminase::uracil phosphoribosyl transferase (CD::UPRT) into the replicative but non-disseminative AdV. In AdV the CD::UPRT transgene and the E1A gene were introduced as a bicitronic cassette under the control of the CMV5 promoter. This adenovirus was generated by introducing the expression cassette in the E1 region of an AdV backbone deleted for E1, E3, and the protease gene. Due to the protease deletion, this AdV can no longer form viral particles, even following replication resulting from E1A expression. The resulting Ad(dPS)C::U-IRES-E1A was grown in protease-complementing 293 cells. When tested for anti-tumor activities in two glioblastomas cell lines and two ovarian cancer cell lines, this Ad(dPS)C::U-IRES-E1A outperformed its non-replicative counterpart, the AdC::U-IRES-GFP, by up to 45-fold. In bystander assays, the number of transduced cells sufficient to convey the anti-tumor activity to the entire cell population could be reduced by more than 25-fold when comparing Ad(dPS)C::U-IRES-E1A to AdC::U-IRES-GFP. In a three dimensional spheroid cutlure model, anti-tumor activity was obtained with 25 times less particles when the replicative non-disseminative AdV was compared to AdC::U-IRES-GFP. In conclusion, the replicative but non-disseminative AdV platform substantially improves anti-tumor activity of suicide gene therapy with CD::UPRT and probably occurs through the activation of viral gene expression and DNA replication although the intrinsic anti-tumor activity of the E1A protein could also be a factor in the improved potency of AdC::U-IRES-E1A. This work demonstrated that the protease-deleted AdV platform may provide vectors that are potentially safer than conditional oncolytic AdVs and yet generate sufficient anti-tumor activity to eradicate large tumors. Work with animal models is in progress and preliminary results will be discussed.

Electron microscopic observation of effects of 5-flurocytosine on CD gene transduced pancreatic carcinoma cells

目的 应用微观检查方法探讨5-氟胞嘧啶(5-flurocytosine,5-FC)对胞嘧啶脱氨酶(cytosine deaminase,CD)基因修饰的胰腺癌细胞体外、体内杀伤的作用机制.方法构建含大肠杆菌胞嘧啶脱氨酶基因的腺病毒载体,经包装、扩增后制备纯化高效的CD腺病毒液,体外培养胰腺癌细胞并建立胰腺癌裸鼠皮下移植瘤模型,电镜下观察转染CD基因及加入前药5-FC后胰腺癌细胞的微观变化情况.结果含CD基因腺病毒载体经酶切鉴定正确,病毒滴度为2×1011pfu/ml,电镜观察到CD/5-FC系统对胰腺癌的体外杀伤是一种凋亡现象而非坏死,而体内实验观察到坏死与凋亡并存,同时亦观察到淋巴细胞浸润.结论 CD/5-FC系统对胰腺癌的作用,有多种因素及旁观者效应参与其内.体外结果显示,凋亡小体的摄入和毒性代谢产物的直接作用可能起主要作用.体内实验结果显示细胞坏死与凋亡并存,免疫反应在抑制肿瘤生长中也起着重要作用。

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.

5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that express cytosine deaminase as a late gene.

Clinical studies with oncolytic adenoviruses have shown that existing viruses are safe but lack efficacy. To selectively increase the toxicity of oncolytic adenoviruses targeting colon tumours, we have inserted the yeast cytosine deaminase gene (yCD) after the fibre gene in the major late transcript. yCD was expressed using either an internal ribosome entry site (IRES) or by alternative splicing of a new exon analogous to the Ad41 long fibre exon. The IRES-CD virus gave higher yCD expression on Western blots. Both approaches result in yCD expression restricted to the period after viral DNA replication. Viral burst size was reduced by less than approximately 10-fold by 5-fluorocytosine (5-FC), showing that expression of yCD as a late gene is compatible with virus replication. Cytopathic effect assays in colon cancer cell lines showed that both yCD viruses have approximately 10-fold increased toxicity in the presence of the prodrug 5-FC, which is converted to 5-fluorouracil (5-FU) by yCD. Toxicity was higher following addition of 5-FC immediately after infection. The largest gain in toxicity was seen in HT29 colon cancer cells, which are the least permissive colon cancer cells for the parental virus, indicating that the new 5-FC/yCD viruses may have broader applications for colon cancer therapy than their predecessors.

Mechanism of thermochemotherapy with 5-fluorocytosine on human colon cancer cell line SW480 transfected cytosine deaminase gene

Mechanism of thermochemotherapy with 5-fluorocytosine on human colon cancer cell line SW480 transfected cytosine deaminase gene

Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter.

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-flucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors.

Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.

Insect population control using female specific pro-drug activation

A system for population control of insects is proposed. It is based on transgenic insects expressing an enzyme which converts an inactive pro-drug into an active, toxic form. A model system is presented which relies on transposon-mediated integration of a bacterial cytosine deaminase (CD) gene into the genome of Drosophila melanogaster. We demonstrate female-specific sterility and transgene-dependent lethality when flies carrying the CD gene under a Drosophila female-specific promoter/enhancer are treated with 5-Fluorocytosine, a low-toxicity nucleoside analogue which is converted to toxic 5-Fluorouracil by the enzyme. The approach can be used with existing pro-insecticides and appropriate converting enzymes in combination with established mass rearing technology, for targeted, environmentally acceptable control of insects of economic and public health importance.

Combination with CD/5-FC gene therapy enhances killing of human bladder-cancer cells by radiation.

Resistance to radiation and chemotherapy is a significant obstacle to the treatment of advanced bladder cancer. Gene therapy combined with radiation represents a new approach to cancer treatment. In the present study, we investigated whether adenovirally directed, cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy could induce cell toxicity and radiosensitization through the intracellular production of 5-fluorouracil (5-FU) in bladder-cancer cells. Three human bladder-cancer cell lines, KK47 (wild-type p53+), T24 (p53 mutated) and 5637 (p53 mutated), were investigated. A recombinant adenovirus vector containing the CD gene (Ad-RSV-CD) was used. Cells were infected with Ad-RSV-CD and treated with 5-FC. Forty-eight hours after infection, the cells were irradiated and cytotoxicity assays performed to determine the extent of increase in in vitro cytotoxicity. A KK47 subcutaneous tumor-xenografts model was used in an animal study to examine the tumor growth inhibitory effect of this combination therapy. Ad-RSV-CD was directly injected into the tumor and daily 5-FC was intraperitoneally injected. Forty-eight hours after injection of Ad-RSV-CD, the tumor was irradiated. The tumor volume was measured every day. In all three cell lines, the combination treatment enhanced the cell killing of human bladder-cancer cells in vitro. It also enhanced the tumor-growth inhibition in the KK47 tumor model. In the present study, we demonstrated that CD/5-FC gene therapy combined with radiation therapy enhances cell killing of human bladder-cancer cells in in vitro and in vivo animal models.

Purging of leukemia-contaminated bone marrow grafts using suicide adenoviral vectors: an in vivo murine experimental model.

Autologous bone marrow transplantation is an alternative therapeutic option for acute myeloid leukemia patients lacking a compatible donor. However, bone marrow from these patients may contain residual leukemic cells that should be ideally eliminated prior to the infusion of the graft. With the aim of developing more efficient protocols of graft purging, adenoviral-mediated gene transfer protocols have been conducted. We studied whether suicide adenoviral vectors expressing the cytosine deaminase gene (AdCD) could be used for selectively killing leukemic WEHI-3B cells. The AdCD transduction followed by the 5-fluorocytosine exposure abrogated the growth of WEHI-3B cells in vitro, with a minimal effect on normal hematopietic progenitors. To test the efficacy of the purging protocol in vivo, bone marrow cells were mixed with syngenic WEHI-3B cells and this chimeric cell population was transduced with AdCD vectors. Infected cells were injected into myeloablated Balb-c mice, which then received a 5-fluorocytosine treatment for 4 days. All mice transplanted with unpurged bone marrow developed leukemia and died. However, 90% of recipients receiving the purging treatment were healthy up to 9 months post-transplantation and had a perfectly re-established hematopoietic system, without any signal of leukemic cell presence. In conclusion, suicide adenoviral vectors are proposed as a tool for the purging of Adenoviral-susceptible myeloid leukemia cells contaminating autologous bone marrow grafts.

Human nerve growth factor receptor and cytosine deaminase fusion genes.

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.

Enhanced suicide gene therapy by chimeric tumor-specific promoter based on HSF1 transcriptional regulation

Two tandem cassettes, one containing the telomerase reverse transcriptase gene (hTERT) promoter upstream of a constitutively activated form of heat shock transcription factor 1 (cHSF1) and followed by the other containing the heat shock protein 70B (hsp70B) promoter (HSE) upstream of the cytosine deaminase (CD) gene, could greatly enhance the efficiency of CD gene therapy while retaining tumor specificity in vitro and in vivo. This hTERT-cHSF1/HSE promoter could restrict gene expression in tumor cells and was about 1.5–3-fold more potent than the cytomegalovirus (CMV) promoter. hTERT-cHSF1/HSE-CD transfection led to tumor cells more sensitive to 5-fluorocytosine compared with hTERT-CD and its toxicity was comparable to that of CMV-CD. Besides enhancement of promoter activity, cHSF1 overexpression itself could enhance the bystander effect of CD gene therapy that could be reversed by anti-Fas antibody. This system also led to activation of stress-related genes such as hsp70 in tumor cells, which in the presence of cell killing by the cytotoxic gene is a highly immunostimulatory event. Furthermore, a more potent anti-tumor effect of hTERT-cHSF1/HSE-CD was observed in nude mice inoculated with Bcap37 cells. No obvious activity of the hTERT-cHSF1/HSE promoter was observed in normal tissues after intravenous administration. These results indicate that the hTERT-cHSF1/HSE promoter is highly tumor-specific and strong with potential application in targeted gene therapy, and therefore may be useful for construction of vectors for systemic therapy.

Killing effect of CD/5-FC system on human colon cancer cell lines SW 480 and LoVo

AIM:To investigate the killing effect of carcinoembryonic antigen (CEA) and tissue-specific cytosine deaminase (CD)/5fluorocytosine (5-FC) system on human colorectal carcinoma cell lines LoVo and SW480 in vitro. METHODS:Recombinant retroviral vector G1CEACDNa was constructed,in which the CD gene was controlled under the CEA promoter, and retroviral vector pCD2 were introduced through liposome technique respectively to the human colorectal carcinoma cell lines LoVo and SW480. Expression of CEA was high and low in both the cell lines respectively. The cells were selectively cultured in G418. The proliferative colonies were treated with 5-FC. RESULTS:After the transfection, LoVo-CEACD cells and LoVo-CD cells were more sensitive to 5-FC than their parental cells (P <0.01, t =5.688, n =9; P <0.01, t =3.136, n =9), and SW480-CEACD cells and SW480-CD cells were more sensitive than their parental cells as well (P <0.01,t =3.437,n =9;P <0.01, t =3.516, n =9). Furthermore, the LoVo-CEACD cells were more sensitive to 5-FC than the LoVo-CD cells (P <0.05, t =2.183,n =9) while the SW480-CEACD cells were less sensitive than SW480-CD cells.The LoVo-CEACD cells displayed a higher anti-tumor effect than SW480-CEACD cells in vitro. The bystander effect in all cells transfected with CD gene were observed in this study. CONCLUSION:The CEA tissue-specific CD/5-FC system displays an obvious targeting anti-tumor effect on human colorectal carcinoma cell lines LoVo and SW480, but the killing effect on the LoVo-CEACD cells is higher than that on the SW480-CEACD cells in vitro. Li CJ, Ma QJ, Lai DN, Lu JG, Wang XJ, Wang Q, Pan BR, Wu YZ, Li JM. Killing effect of CD/5-FC system on human colon cancer cell lines SW 480 and LoVo. Shijie Huaren Xiaohua Zazhi 2003;11(5):535-539

Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC.

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

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.

Adenovirus-mediated gene transfer in the treatment of pancreatic cancer.

Suicide gene therapy is a new experimental form of cancer chemotherapy that is currently being evaluated in human trials. To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by an adenovirus vector on human pancreatic carcinoma in vitro and in vivo. The CD gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD, and pAdEasy-1, which underwent recombination in bacteria BJ5183. The newly recombinant Ad-CD containing green fluorescent protein was propagated in 293 cells and purified by cesium chloride gradient centrifugation. The human pancreatic carcinoma cell line PaTu8988/SW1990 was infected with this virus, and then 5-fluorocytosine (5-FC) was added; XTT assay was used to estimate relative numbers of viable cells. An in vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) PaTu8988/SW1990 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor, and 5-FC was administered. The positive clones were selected by endonuclease digestion of the combinations, and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu/mL. It was found that significant cytotoxic activities were possessed by 5-FC for CD gene-transduced PaTu8988/SW1990 cells, but there was little effect on the nontransduced pancreatic carcinoma cells. The antitumor effect was observed in PaTu8988/SW1990 xenografts from nude mice with in situ CD gene transduction. These results indicate that the CD gene mediated by adenovirus has a high level of infectivity and is efficient for gene therapy for pancreatic carcinoma.