Carcinoembryonic Antigen Promoter Research Articles

C-KIT-ERK1/2 signaling activated ELK1 and upregulated carcinoembryonic antigen expression to promote colorectal cancer progression.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c‐KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1‐overexpressed CRC cells. Furthermore, we explored the role of c‐KIT‐ERK1/2 signaling in activation of ELK1. Blocking c‐KIT signaling using Imatinib or ISCK03 reduced p‐ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c‐kit loss‐of‐functional Wadsm/m mice showed lowered c‐KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c‐KIT‐ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.

Specific driving of the suicide E gene by the CEA promoter enhances the effects of paclitaxel in lung cancer.

Classical chemotherapy for lung cancer needs new strategies to enhance its antitumor effect. The cytotoxicity, nonspecificity, and low bioavailability of paclitaxel (PTX) limits their use in this type of cancer. Suicide gene therapy using tumor-specific promoters may increase treatment effectiveness. We used carcinoembryonic antigen (CEA) as a tumor-specific promoter to drive the bacteriophage E gene (pCEA-E) towards lung cancer cells (A-549 human and LL2 mice cell lines) but not normal lung cells (L132 human embryonic lung cell line), in association with PTX as a combined treatment. The study was carried out using cell cultures, tumor spheroid models (MTS), subcutaneous induced tumors and lung cancer stem cells (CSCs). pCEA-E induced significant inhibition of A-549 and LL2 cell proliferation in comparison to L132 cells, which have lower CEA expression levels. Moreover, pCEA-E induced an important decrease in volume growth of A-549 and LL2 MTS producing intense apoptosis, in comparison to L132 MTS. In addition, pCEA-E enhanced the antitumor effects of PTX when combined, showing a synergistic effect. This effect was also observed in A-549 CSCs, which have been related to the recurrence of cancer. The in vivo study corroborated the effectiveness of the pCEA-E-PTX combined therapy, inducing a greater decrease in tumor volume compared to PTX and pCEA-E alone. Our results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically towards lung cancer cells, and may be used to enhance the effectiveness of PTX against this type of tumor.

Anti-colorectal cancer effect of interleukin-2 and interferon-β fusion gene driven by carcinoembryonic antigen promoter.

This study was designed to investigate the antitumor effects of combined interleukin-2/interferon-β-based gene therapy in colorectal cancer. Transfection of the fusion gene expression plasmid induced significant apoptosis of Lovo cells. Additionally, the fusion gene exhibited strong inhibitory activity against tumor growth and apoptosis when being injected into the nude mice implanted with human colon cancer cells. Furthermore, the tail-vein injection showed a more notable effect than direct injection into tumor. These results suggest that the combined interleukin-2/interferon-β-based gene therapy with the carcinoembryonic antigen promoter might be an effective antitumor strategy.

Enhanced antitumor effect of combining TRAIL and MnSOD mediated by CEA-controlled oncolytic adenovirus in lung cancer.

Lung cancer, especially adenocarcinoma, is one of the leading causes of death in the world. Carcinoembryonic antigen (CEA), a superb non-small-cell lung cancer marker candidate, showed a beneficial effect in cancer therapy with oncolytic adenovirus in recent studies. Cancer-targeting dual gene-virotherapy delivers two therapeutic genes, linked by a connexon, in the replication-deficient vector instead of one gene so that they can work in common. In this study, we constructed a tumor-specific oncolytic adenovirus, CD55-TRAIL-IETD-MnSOD. The virus has the fusion protein complementary DNAs for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and for manganese superoxide dismutase (MnSOD) complementary DNA linked through a 4-amino acid caspase-8 cleavage site (IETD), and uses a CEA promoter to control virus E1A express. This is the first work to use a CEA promoter-regulated oncolytic adenovirus carrying two therapeutic genes for cancer research. Its targeting and anticancer capacity was evaluated by in vitro and in vivo experiments. The results indicated that CD55-TRAIL-IETD-MnSOD caused more cell apoptosis than CD55-TRAIL or CD55-MnSOD alone, or their combination in vitro, with low cytotoxicity of normal cells. In the A549 tumor xenograft model in nude mice, data showed that CD55-TRAIL-IETD-MnSOD could effectively suppress tumor growth than single gene groups, with no histological damage in liver, spleen or kidney tissues. Thus, the CEA-regulated dual-gene oncolytic virus CD55-TRAIL-IETD-MnSOD may be a novel potential therapy for lung cancer.

Expression of the immediate early IE180 protein under the control of the hTERT and CEA tumor-specific promoters in recombinant pseudorabies viruses: Effects of IE180 protein on promoter activity and apoptosis induction

Since the pseudorabies virus (PRV) genome encodes for a single immediate-early protein, IE180, we reasoned that this strong transactivating protein could represent a key regulatory switch that could be genetically manipulated in order to alter its tropism towards cancer cells. We therefore initiated studies to test whether the human telomerase reverse transcriptase (hTERT) and carcinoembryonic antigen (CEA) tumor promoters could functionally replace the IE180 promoter. We show that both promoters can functionally substitute the IE180 promoter in plasmid constructs and recombinant viruses, and observed that IE180 differentially auto-regulated each promoter tested, with PRV IE180 negatively regulating the hTERT promoter but positively hyper-activating the CEA promoter. Interestingly, we also observed that the recombinant PRV-TER and PRV-CEA viruses preferentially replicated in diverse cancer cell lines compared to control non-cancer cells, and the PRV-CEA was capable of additionally inducing a profound apoptotic phenotype which we correlated to the overexpression of IE180.

Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter.

Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA) to direct E gene expression (pCEA-E) towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells.

Cytotoxicity of lymphocytes activated by superantigen toxic-shock-syndrome toxin-1 against colorectal cancer LoVo cells

Toxic-shock-syndrome toxin-1 (TSST-1), a superantigen, can stimulate T cell activation and be used for immunotherapy. In this study, we employed the carcinoembryonic antigen (CEA)-positive LoVo cells to test whether retrovirus-mediated TSST-1 expression could activate human T cells and promote cytotoxicity against tumor cells. We first generated plasmids of pLEGFP-N1-5HRE-CEAp-TSST-1-linker-CD80TM containing a fusion gene of the CEA promoter, 5 copies of the hypoxia-response elements (HRE) as an enhancer, the fragments for TSST-1, and the transmembrane domain of CD80 (CD80TM) and control pLEGFP-N1-5HRE-CEAp (without TSST-1) and generated retroviruses of 5HCTC and 5HC, respectively. After infection with 5HC and 5HCTC retroviruses to establish cell lines, the high levels of TSST-1 expression were observed on the membrane and cytoplasm of the 5HCTC-infected LoVo cells, particularly culture under a hypoxic condition, but not on CEA(-) HeLa cells. Furthermore, the TSST-1-expressing LoVo cell lysates, but not the control cell lysates, stimulated human T cell proliferation, and the co-culture of the TSST-1-expressing LoVo, but not control cells, with human peripheral blood mononuclear cells (PBMC) induced a high frequency of TNF-α- and IL-2-secreting T cells in vitro, particularly under hypoxic conditions. More importantly, co-culture of the TSST-1-expressing LoVo cells, particularly under hypoxic conditions, but not control cells, with different numbers of PBMC promoted potent cytotoxicity against LoVo cells in a dose-dependent manner in vitro. These data provide proof of the principle that selective induction of TSST1 expression in CEA(+) colorectal cancer (CRC) cells activates T cells that destroy tumor cells, particularly under a hypoxic condition. Therefore, our findings may aid in the design of new immunotherapy for the intervention of CRC at clinic.

Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD)

The application of gene therapy in cancer treatment is limited by non-specific targeting. In the present study, we constructed a recombinant plasmid, containing a carcinoembryonic antigen (CEA) promoter and double suicide genes thymidine kinase (TK) and cytosine deaminase (CD), henceforth referred to as pCEA-TK/CD. Our results showed that the CEA promoter can specifically drive target gene expression in CEA-positive lung cancer cells. In the presence of prodrugs 5-flucytosine and ganciclovir, pCEA-TK/CD transfection decreased inhibitory concentration 50 and increased apoptosis and cyclomorphosis. Our result suggests that gene therapy using pCEA-TK/CD may be a promising new approach for treating lung cancer.

Tissue Specific Cytotoxicity of Colon Cancer Cells Mediated by Nanoparticle-delivered Suicide Gene In vitro and In vivo

This study aimed to develop an efficient and safe strategy to introduce suicide genes into colon cancer cells. In this study, we fused an enhanced carcinoembryonic antigen promoter (CEA) to a suicide gene, cytosine deaminase (CD). This construct was delivered into colon cancer cells using calcium phosphate nanoparticles (CPNP). The cells were then treated with the prodrug 5-FC. The therapeutic effect was evaluated in vitro and in vivo. Our study showed that the CEA promoter-driven, CPNP-delivered suicide gene was only expressed in CEA-positive colon cancer cells, and resulted in significant cytotoxicity after administration of the prodrug 5-FC in vitro. Moreover, our in vivo study showed that CPNP-mediated CEA-CD delivery, together with 5-FC treatment, resulted in significant tumor growth delay in xenograft human colon carcinoma. Our study indicates that the combination of CPNP and CEA-CD gene expression represents a novel approach for CEA-positive tumor gene therapy.

E1A, E1B double-restricted replicative adenovirus at low dose greatly augments tumor-specific suicide gene therapy for gallbladder cancer

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.

Tumor-specific promoters and their application in cancer gene targeted therapy

Tumor-specific promoters can induce high-efficiency and specific expression of exogenous genes in tumor cells. At present, commonly used promoters include alpha-fetoprotein promoter, carcinoembryonic antigen promoter,prostate specific antigen promoter,human telomerase reverse transcriptase promoter and multidrug resistance gene promoter and etc. Dual promoters, enhancer, regulatory element and other physical or chem-ical factors could be used to reconstruct or modify promoters to increase the expression and location of exogenous genes in tumor cells. Tumor-specific promoters play an important role in cancer gene targeted therapy by cou-pling with suicide gene, anti- oncogene, antiseuse nucleic acid, apoptosis gene and RNA interference. Key words: Neoplasms; Transcription initiation site; Genes; Target therapy

Amplified interleukin-15 expression vectors for cancer immunogene therapy

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays a key role in the regulation of both innate and adaptive immune responses. It promotes the survival, proliferation, activation and maintenance of natural killer and CD8+ T cells. It also stimulates the function of neutrophils, macrophages and dendritic cells, and could therefore be a potential cytokine for cancer immune therapy. The therapeutic effects of cytokines are related to their expression levels. In this study, we investigated an amplified IL-15 expression plasmid vector, pHi2-spIL15-CMV-tat, and a carcinoembryonic antigen (CEA)-positive tumor-specific amplified IL-15 expression plasmid vector, pHi2-spIL15-CEA-tat. In pHi2-spIL15-CMV-tat, IL-15 expression was driven by HIV2 LTR, which was transactivated by CMV promoter-controlled expression of HIV tat. In addition, the native IL-15 signal peptide was replaced by the IL-2 signal peptide to enhance its secretion. A significant amount of IL-15 expression was achieved when transfected into tumor cells in vitro. In order to target IL-15 expression in CEA-positive cells, the CEA promoter positively-controlled IL-15 expression plasmid vector pHi2-spIL15-CEA-tat was constructed by replacing the CMV with the CEA promoter. Efficient and targeted IL-15 expression was achieved in CEA-positive tumor cells by pHi2-spIL15-CEA-tat.

Image-Guided Radioiodide Therapy of Medullary Thyroid Cancer After Carcinoembryonic Antigen Promoter-Targeted Sodium Iodide Symporter Gene Expression

In contrast to follicular cell-derived thyroid cancer, medullary thyroid cancer (MTC) remains difficult to treat because of its unresponsiveness to radioiodine therapy, or to conventional chemo- and radiotherapy. We therefore examined the feasibility of radioiodine therapy of MTC after human sodium iodide symporter (hNIS) gene transfer, using the tumor-specific carcinoembryonic antigen (CEA) promoter for transcriptional targeting. NIS gene transfer was performed in vivo in human MTC cell (TT) xenografts, using adenoviral vectors carrying the NIS gene linked to the cytomegalovirus promoter (Ad5-CMV-NIS) or a CEA promoter fragment (Ad5-CEA-NIS). Functional NIS expression was confirmed by immunostaining as well as in vivo (123)I gamma-camera imaging followed by application of a therapeutic (131)I dose. TT cell xenografts in nude mice injected intratumorally with Ad5-CEA-NIS accumulated 7.5 +/- 1.2% ID/g (percentage injected dose per gram tumor tissue; 5 x 10(8) PFU) and 12 +/- 2.95% ID/g (1 x 10(9) PFU) with an average biological half-life of 6.1 +/- 0.8 and 23.6 +/- 3.7 hr, respectively, as compared with accumulation of 8.4 +/- 0.9% ID/g with a biological half-life of 12 +/- 8 hr after application of Ad5-CMV-NIS (5 x 10(8) PFU). After Ad5-CEA-NIS-mediated NIS gene transfer in TT cell xenografts administration of a therapeutic dose of 111 MBq (3 mCi) of (131)I resulted in a significant reduction of tumor growth associated with significantly lower calcitonin serum levels in treated mice as well as improved survival. We conclude that a therapeutic effect of (131)I was demonstrated in vivo in MTC cell xenografts after adenovirus-mediated induction of tumor-specific iodide accumulation by CEA promoter-directed hNIS expression.

In vitro evaluation of cancer-specific NF-κB-CEA enhancer–promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines

Nuclear factor-kappa B (NF-κB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer–promoter system, κB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-κB DNA-binding sites (κB4). In combination with a κB4 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 κB4-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, κB4, κB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in κB4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The κB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and κB4-CEA205-driven TP cDNA transiently transfected cells were 8- to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5′-deoxy-5-fluorouradine (5′-DFUR), in contrast to only 1.5- to 2-fold sensitised by the κB4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and κB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-κB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.

Transcription activity of ectogenic human carcinoembryonic antigen promoter in lung adenocarcinoma cells A549

The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08+/-0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27+/-3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer

We investigated the feasibility of using radioiodine therapy in colon carcinoma cells (HCT 116) following tumor-specific expression of the human sodium iodide symporter (hNIS) using the carcinoembryonic antigen (CEA) promoter. HCT 116 cells were stably transfected with an expression vector, in which hNIS cDNA has been coupled to a CEA promoter fragment. This promoter is responsible for tissue-specific expression of CEA in gastrointestinal tract epithelium, and has been shown to target therapeutic genes to colorectal cancer cells. Functional NIS expression was confirmed by iodide uptake assay, Western blot analysis, immunostaining and in vitro clonogenic assay. The stably transfected HCT 116 cells concentrated (125)I about 10-fold in vitro without evidence of iodide organification. In contrast, transfection of control cancer cells without CEA expression did not result in iodide accumulation. Western blot analysis using a hNIS-specific antibody revealed a band of approximately 90 kDa. In addition, immunostaining of stably transfected HCT 116 cells revealed hNIS-specific membrane-associated immunoreactivity. In an in vitro clonogenic assay approximately 95% of stably transfected HCT 116 cells were killed by exposure to (131)I, while only about 5% of NIS-negative control cells were killed. Further, using an adenovirus carrying the NIS gene linked to the CEA promoter, high levels of tumor-specific radioiodide accumulation were induced in HCT 116 cells. In conclusion, a therapeutic effect of (131)I has been demonstrated in colon carcinoma cells following induction of tumor-specific iodide uptake activity by CEA promoter-directed NIS expression in vitro. This study demonstrates the potential of NIS as a therapeutic gene allowing radioiodine therapy of colon cancer following tumor-specific NIS gene transfer.

CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter.

Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes. However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E. coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC). Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC. The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells. Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells. Safety is the major issue in gene therapy. Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed.

CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter.

Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes. However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E. coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC). Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC. The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells. Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells. Safety is the major issue in gene therapy. Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed.

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

Improvement of carcinoembryonic antigen-specific prodrug gene therapy for experimental colon cancer

Background. Improvement of tumor-specific gene expression is very important for achieving successful effects in prodrug gene therapy for advanced cancer with metastatic lesions. We used the Cre/loxP system for enhancing carcinoembryonic antigen (CEA)-specific prodrug gene therapy of cytosine (CD)/5-fluorocytosine (5-FC) for the treatment of a colon cancer model accompanied with liver metastases. Methods. Orthotopic colon cancer models were developed. Seven days later, adenovirus vector (3×109 pfu)was injected into the abdominal cavity, and 5-FC was administered for the next 10 days. Results. In these models, the double administration of 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-mediated switching system, completely inhibited liver metastases, and significantly reduced primary tumor volume compared to the administration of Mock or AxCEACD (P <.001). The survival periods of the mice treated with AxCEANCre and AxCALNLCD were longer than mice treated with Mock or AxCEACD, and longer than the mice treated with AxCACD (P <.05). Conclusions. The enhanced CEA-specific prodrug gene therapy using the Cre/loxP system was useful for the treatment of advanced colon cancer with liver metastases, implicating clinical application. (Surgery 2003;133:309-17.)