Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Replication-competent adenoviruses may provide a highly efficient means of delivering therapeutic genes to tumors. Previously, we evaluated in vitro a replication-competent adenovirus (Ad5-CD/TKrep) containing a cytosine deaminase (CD)/herpes simplex type 1 thymidine kinase (HSV-1 TK) fusion gene that allows lytic viral therapy to be combined with double suicide gene therapy. Both the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems enhanced the tumor cell-specific cytopathic effects of the Ad5-CD/TKrep virus in vitro and sensitized cells to radiation. To extend these in vitro findings in vivo, we evaluated the antitumor activity of the Ad5-CD/TKrep virus in combination with double prodrug therapy and radiation therapy. The Ad5-CD/TKrep virus independently demonstrated significant antitumor activity against C33A cervical carcinoma xenografts. Therapeutic outcome was dramatically improved with systemic administration of double, but not single, prodrug (5-FC + GCV) therapy. When used in a neoadjuvant setting, Ad5-CD/TKrep-mediated double suicide gene therapy dramatically potentiated the effectiveness of radiation therapy. The trimodal approach of Ad5-CD/TKrep viral, double suicide gene, and radiotherapies produced significant tumor regression and ultimately 100% tumor cure. The results demonstrate the high therapeutic potential of the trimodal approach and provide a solid foundation for future clinical trials.
...
PMID:Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization. 1064 40

Detection of a therapeutic response early in the course of cancer treatment, before tumor growth delay or regression, is not currently possible in experimental models or clinical medicine. New interim measures of therapeutic response would be particularly useful in the development of cancer chemosensitization gene therapy by facilitating optimization of gene transfer protocols and prodrug dosing schedules. Diffusion MRI is a sensitive technique producing quantitative and noninvasive images of the apparent mobility of water within a tissue. We investigated the utility of diffusion MRI for detecting early changes associated with a refined cytosine deaminase (CD)/5-fluorocytosine (5FC) chemosensitization gene therapy paradigm in orthotopic 9L gliomas stably expressing the recently cloned S. cerevisiae CD gene. Mean tumor diffusion increased 31% within 8 days of initiating 5-FC treatment, preceding tumor growth arrest and regression. Complete regression of the intracranial tumor was observed in four of five treated animals, and recurrent tumor in the remaining animal exhibited water diffusion behavior similar to primary, untreated tumors. These results demonstrate the efficacy of the yCD/5FC strategy for glioma and suggest that increased tumor water diffusion is an indicator of active therapeutic intervention.
...
PMID:Diffusion MRI detects early events in the response of a glioma model to the yeast cytosine deaminase gene therapy strategy. 1087 48

Replicating viruses for cancer gene therapy have beneficial antitumor effects, however, in the setting of an enzyme/prodrug system, the interactions between these viruses and the activated agents are complex. A replicating vaccinia virus expressing the cytosine deaminase gene (VVCD), which converts the prodrug 5-FC into 5-FU, was characterized in vitro and in vivo for its antitumor effects and pathogenicity. Replicating VVCD (+/-5-FC) at various MOIs was used to infect MC38 murine colon adenocarcinoma cells. At high MOIs (>0.1) virus alone was able to kill the majority (65-90%) of cells by day 5 with no additional benefit from prodrug. At low MOIs only the effect of prodrug is seen. Cell lysates demonstrated 300-fold reduced viral recovery from cells treated with both VVCD and 5-FC compared with controls treated with virus alone. Nude mice bearing subcutaneous MC38 tumors were injected with VVCD (or control) and treated with 5FC or control. Mice injected with VVCD (with or without 5FC treatment) had smaller tumors than the controls, suggesting that replicating vaccinia alone is cytotoxic to tumors in vivo. The addition of 5-FC improved the antitumor response when a low dose of virus was injected into tumors. Also, compared with mice that received virus alone, those that received VVCD and 5FC had significantly prolonged survival from virus-mediated death. In conclusion, the addition of an enzyme/prodrug system to a replicating virus can improve the antitumor response and decrease viral pathogenicity. Gene Therapy (2000) 7, 1217-1223.
...
PMID:Complex interactions between the replicating oncolytic effect and the enzyme/prodrug effect of vaccinia-mediated tumor regression. 1091 90

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.
...
PMID:In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. 1091 55

The efficacy of combination suicide gene therapy was evaluated using a Herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system and an Escherichia coli cytosine deaminase/5-fluorocytosine (CD/5-FC) system on the LNCaP human prostate cancer cell model. Two types of plasmid vectors with the HSV-TK gene were constructed. A constitutive chicken beta-actin promoter drove one and a prostate-specific antigen (PSA) promoter drove the other. Similarly, a pair of plasmids with the CD gene under a cytomegalovirus (CMV) promoter and the PSA promoter was also constructed. LNCaP cells were transfected in vitro with either or both of those plasmids using a cationic lipid reagent. Transfected cells were treated with GCV and/or 5-FC. The percentage of viable LNCaP cells 7 days after treatment with HSV-TK/GCV or CD/5-FC under a constitutive promoter was 40% and 41% of controls, respectively. The cell viability when two suicide genes were combined was 23%. The cell viabilities after four days with PSA promoter-HSV-TK vectors, CD vectors and a combination of both were 79%, 88% and 88%, respectively. Suicide gene therapy using either HSV-TK/GCV, CD/5-FC, or both, was effective in the LNCaP model. An additive effect was observed when the two suicide genes were used together. The PSA promoter did not seem to be effective enough to elicit cytotoxicity under the experimental conditions used here.
...
PMID:Suicide gene therapy on LNCaP human prostate cancer cells. 1144 69

TAPET-CD, a genetically engineered Salmonella strain with chromosomal-incorporated cytosine deaminase (CD) gene, has been shown to selectively accumulate tumors, suppress tumor growth, and convert 5-fluorocytosine (5-FC, an antifungal agent) to the antitumor agent 5-fluorouracil (5-FU) in animals. The current studies investigated the safety of TAPET-CD, and TAPET-CD/5-FC combination, in animals. In C57BL/6 mice (n = 10 females/dose), the maximum nonlethal dose of TAPET-CD (intravenous [IV] bolus) was 1 x 10(6) colony-forming units (cfu)/mouse, or > 10,000 x that of wild-type Salmonella. In Sprague-Dawley rats (n = 4/sex/group), after treatment with 4 weekly cycles of TAPET-CD (an IV injection/cycle at 1 x 10(5), 3 x 10(5), 1 x 10(6), 3 x 10(6), or 1 x 10(7) cfu/rat on day 1) and 5-FC (per os twice daily [PO b.i.d.], 250 mg/kg on days 2-7/cycle), clinical signs and mortality were evaluated daily, body weight and clinical pathology weekly, and gross necropsy on day 29. No treatment-related toxicity, although occasional and mild clinical signs (e.g., dehydration), increased hepatic enzyme/function values and white blood cells, splenic enlargement, and bilateral red discoloration of the kidneys, were observed. In cynolmogus monkeys, Experiment 1 involved treatment with TAPET-CD (IV injection at 1 x 10(9) cfu/monkey). Clinical signs and mortality were evaluated daily, body weight weekly, and gross necropsy on days 2, 7, and 31 (1/sex/time point). Experiment 2 involved treatment with TAPET-CD (IV injection at 1 x 10(9) and 1 x 10(10) cfu/monkey in Groups 1 to 3 and Groups 4 to 6, respectively) on day 1 and 5-FC (PO b.i.d. at 250, 500, and 1000 mg/kg in Groups 1 to 3, and 500, 1500, and 0 mg/kg in Groups 4 to 6, respectively) on days 4 to 17 (n = 1/sex/group). Clinical signs and mortality were evaluated daily; body weight and clinical pathology on days 1, 2, 4, 14, and 18; body temperature on days 1, 4, and 18; ophthalmic examinations on days 3 and 17; and gross necropsy and histopathology on day 18. Experiment 1 indicated that TAPET-CD at 1 x 10(9) or 1 x 10(10) cfu/monkey was well tolerated, with only occasional mild clinical signs (i.e., emesis, vomiting, inappetance, loose/infrequent/absence of stool), increases in hepatic enzyme/function values, and splenic enlargement. Experiment 2 indicated that TAPET-CD/5-FC combination had a maximum tolerated dose (MTD) of 1 x 10(10) cfu/monkey for TAPET-CD and 500 mg/kg for 5-FC in monkeys. Supra-MTDs induced renal toxicity. In conclusion, TAPET-CD had a good safety profile (reflected by the extremely large amount of TAPET-CD needed to induce mortality or toxicity) in mice, rats, and monkeys. More adverse events were observed with TAPET-CD/5-FC combination when compared to TAPET-CD and these events were similar to the reported effects of 5-FU, suggesting the involvement of 5-FU.
...
PMID:Evaluation of the acute and subchronic toxic effects in mice, rats, and monkeys of the genetically engineered and Escherichia coli cytosine deaminase gene-incorporated Salmonella strain, TAPET-CD, being developed as an antitumor agent. 1156 16

The low transduction efficiency of viral and nonviral vectors is a major limitation in tumour gene therapy. The HSV-1 tegument protein VP22 has been shown to exhibit a novel intercellular transport property. VP22 wild-type as well as VP22 fusion proteins efficiently spread from the original expressing cell to numerous neighbouring cells, so that protein transport by VP22 chimaeric polypeptides into the surrounding cells offers a possible compensation for the inadequate gene transfer efficiencies. To improve the therapeutic efficacy of the E. coli cytosine deaminase (CD) suicide gene we made use of the VP22 transport property in CD transducing adenoviral (Ad) vectors. C- and N-terminal fusions of CD linked in-frame with VP22 were generated and cloned into recombinant adenoviral vectors. Following in vitro transduction immunofluorescence analysis of Ad-transduced producer cells coplated with naive cells confirmed that the characteristic foci pattern of central producer and adjoining neighbour cells displaying nuclear staining was retained. After transduction of rat hepatoma cells with adenoviral vectors and subsequent incubation with the prodrug 5-FC, we observed enhanced cell cytotoxicity when comparing the CD-VP22 fusion (Ad-CD-VP22) with Ad-vectors expressing the CD gene only (Ad-CD). Thereby employment of Ad-vectors encoding VP22 fusion proteins opens up new possibilities to potentiate the efficiency of suicide gene therapy for the treatment of solid tumours.
...
PMID:Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene. 1189 4

The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC.
...
PMID:Expression of Cytosine Deaminase Gene in Human Colon Carcinoma Cells by Recombinant Retroviral Vector. 1221 18

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.
...
PMID:Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy. 1242 9

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.
...
PMID:Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes. 1263 7


<< Previous 1 2 3 4 5 6 7 Next >>

---