Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity (original) (raw)
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Sensitivity of 5-fluorouracil-resistant cancer cells to adenovirus suicide gene therapy
Cancer Gene Therapy, 2006
A promising approach for cancer gene therapy is the combination of adenovirus vectors (AdV) with the suicide gene cytosine deaminase and uracil phosphoribosyl transferase (CDHUPRT). While such vectors have been tested in tumor cell lines and xenograft models, it is not clear how these therapeutic vectors would perform in primary human tumors. We, thus, examined the effect of the combination of a recombinant adenovirus expressing the CDHUPRT (AdCU) with 5-fluorocytosine (5-FC) on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. In such models, we have found a direct correlation between the patients' response to 5-FU and the response shown by the cancer cells in vitro, confirming the clinical relevance of this methodology. Our findings demonstrated that this combination was able to kill primary tumor cells, including those that had developed resistance to 5-FU. Furthermore, while proliferating cells were more susceptible to 5-FU, the combination was effective in both rapid and slow proliferating samples. Our study demonstrated that this gene therapy approach could provide an effective therapeutic option for cancers and is not affected by acquired 5-FU resistance. Also of importance is the effectiveness of this gene therapy approach on slower proliferating cells that is typical of the majority of cancers in vivo. This suggests a greater likelihood that it will be effective in a clinical setting.
2002
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)/5fluorocytosine (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 ( ؍ 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.
Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU
Cancer Immunology, Immunotherapy, 2007
We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological eYcacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an eVective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L eYciently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu signiWcantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor speciWc immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3 + CD8 + T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy. Keywords Hepatoma • Colon cancer • 5-Fu • Flt3L • Immunity Abbreviations Flt3L Fms-like tyrosine kinase 3 ligand NK Natural killer HCC Hepatocellular carcinoma 5-Fu 5-Fluorouracil MEM Minimal essential medium Ad-mFL Adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide FBS Fetal bovine serum CMV Cytomegalovirus EFU Expression-forming unit CFU Colony-forming unit BFU Burst-forming unit MOI Multiplicity of infection IHC Immunohistochemistry Sheng Hou, Geng Kou, and Xiaoqiang Fan are contribute equally to this paper.
Molecular cancer therapeutics, 2001
We reported previously that 5-fluorouracil (FUra) efficacy could be enhanced by increasing tumoral thymidine phosphorylase (TP) activity. Potentiated TP yield was achieved by either transfecting cells with human TP gene (A. Evrard et al., Br. J. Cancer, 80: 1726-1733, 1999) or associating FUra with 2'-deoxyinosine (d-Ino), a modulator providing the tumors with TP cofactor deoxyribose 1-phosphate (J. Ciccolini et al., Clin. Cancer Res., 6: 1529-1535, 2000). The purpose of the present work was to study the effects of a combined modulation (TP gene transfer + use of d-Ino) on the sensitivity to FUra of the LS174T human colorectal cell line. Results showed a near 4000 times increase of cell sensitivity in vitro after double (genetic + biochemical) modulation. This potentiation of tumor response was accompanied by a total change in the FUra anabolic pathway with a 5000% increase of cytosolic fluorodeoxyuridine monophosphate, a stronger and longer inhibition of thymidylate synthase, a...
British Journal of Cancer, 2007
Nuclear factor-kappa B (NF-kB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kB DNA-binding sites (kB4). In combination with a kB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three-to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kB4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kB4, kB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kB4-CEA205and CMV-driven TP cDNAtransfected cancer cell lines (H630 and RKO). The kB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV-and kB4-CEA205-driven TP cDNA transiently transfected cells were 8-to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5 0-deoxy-5-fluorouradine (5 0-DFUR), in contrast to only 1.5-to 2-fold sensitised by the kB4and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV-and kB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.
International Journal of Cancer, 1999
Transferring a gene into cancer cells in order to sensitize them to drugs is an important approach in human cancer gene-therapy research. Thymidine phosphorylase (TP) is the first enzyme in the metabolic activation pathway of 5-fluorouracil (5-FU) to fluorodeoxyribonucleotides, thus, it could be used to increase the sensitivity of cancer cells to this antipyrimidine agent. In this study, an expression vector containing the human TP cDNA was transfected into C26 murine colon-carcinoma cells. Stable transfectants were selected; all showed increased TP activity, ranging from 2-to 10-fold when compared with wild-type cells. The in vitro sensitivity of transfectants to 5-FU and 5Ј-deoxy-5-fluorouridine (5Ј-DFUR) was enhanced, in agreement with the observed increase in TP activity. Then, tumors were generated by s.c. injection of TP-transfected or wild-type C26 cells in syngeneic BALB/c mice. 5-FU (25 mg/kg, i.p.) induced a growth delay of TP-transfected C26 tumors as compared with C26 wild-type tumors. These data suggest that TP could be transfected in tumor cells to increase the sensitivity to 5-FU for subsequent cancer gene therapy. Int.
International Journal of Radiation Oncology*Biology*Physics, 2007
Background We aimed to evaluate the efficacy of gene-directed enzymeprodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'.
BMC Cancer, 2014
Background: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1). Methods: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase. Results: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression. Conclusions: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.