A Potential Therapy Using Engineered Stem Cells Prevented Malignant Melanoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node–Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. MATERIALS AND METHODS: CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. RESULTS: In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. CONCLUSION: The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.

Human Sulfatase-1 Improves the Effectiveness of Cytosine Deaminase Suicide Gene Therapy with 5-Fluorocytosine Treatment on Hepatocellular Carcinoma Cell Line HepG2 In Vitro and In Vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human sulfatase-1 (Hsulf-1) is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans (HSPGs), altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation, cell motility, and apoptosis. We investigated the role of combined cancer gene therapy with Hsulf-1 and cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene on a hepatocellular carcinoma (HCC) cell line, HepG2, in vitro and in vivo.</p><p><b>METHODS</b>Reverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC. Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy. We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.</p><p><b>RESULTS</b>A significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system. A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed. Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene. In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone, and the combined treatment resulted in a significant increase in survival.</p><p><b>CONCLUSIONS</b>Hsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.</p>

Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

OBJECTIVES: Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system. METHODS: To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration. RESULTS: In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy. CONCLUSION: Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC.

Antitumor activity of the recombinant rClone30-CD/5-FC system / 药学学报

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.

Inhibitory effect of recombinant adenovirus containing CDglyTK double suicide gene driven by KDR promoter on human stomach adneocarcinoma SCG7901 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inhibitory effect of adenovirus-mediated fusion gene system driven by KDR promoter on the proliferation of human gastric adneocarcinoma SCG7901 cells and observe the bystander effect in vitro.</p><p><b>METHODS</b>SCG7901, ECV304 and HepG2 cells were infected with Ad-KDR-CDglyTK and Ad-CMV-CDglyTK at a multiplicity of infection (MOI) of 100, and the infection efficiency and the mRNA expressions of the transferred fusion gene were investigated. GCV and/or 5-FC at different concentrations were added into the culture medium of the infected cells to observe the targeted antitumor effect and bystander effect of CDglyTK suicide gene driven by KDR promoter.</p><p><b>RESULTS</b>With the MOI of the adenovirus of 100, the fluorescence emitted by green fluorescent protein (GFP) was observed in 95% of the infected SCG7901, ECV304 and HepG2 cells. All the cells infected by Ad-CMV-CDglyTK and SCG7901 and ECV304 cells infected by Ad-KDR-CDglyTK were highly sensitive to the prodrugs. In comparison, HepG2 cells infected with Ad-KDR-CDglyTK did not show much sensitivity to the two prodrugs. Following treatment with the prodrugs at the same concentration, the infected SCG7901 and ECV304 cells exhibited gradually lowered survival rates as the culture time was prolonged, whereas the transgenic HepG2 cells showed no such time-dependent changes. When the non-infected cells were cocultured with the transgenic cells, the bystander effect of CDglyTK gene was observed, which increased with the ratio of the transgenic cells. In these mixed cell culture systems, GCV and 5-FC showed obvious synergetic effect in suppressing the cell survival.</p><p><b>CONCLUSION</b>The CDglyTK fusion gene system driven by KDR promoter can inhibit the proliferation of SCG7901 and ECV304 cells with obvious bystander effect in vitro. The combination of the prodrugs produces obvious synergetic effect against the cell survival.</p>

Selective cytotoxic effect of lentivirus-mediated double suicide gene transfer on human gastric adneocarcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.</p><p><b>METHODS</b>SGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.</p><p><b>RESULTS</b>The infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.</p><p><b>CONCLUSION</b>Lentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.</p>

A double suicide gene system driven by KDR promoter selectively kills human colon adneocarcinoma SW480 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the selective killing effect of adenovirus (Ad)-mediated double suicide gene system driven by the KDR promoter (KDR-CDglyTK) on human colon adneocarcinoma SW480 cells.</p><p><b>METHODS</b>KDR-expressing SW480 cells and LS174T cells that did not express KDR were infected by KDR-CDglyTK, and the infection efficiency and the expression of CDglyTK in the cells were detected by RT-PCR. The infected cells were treated with the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method. DNA content and the cell cycle changes in SW480 cells were detected by flow cytometry.</p><p><b>RESULTS</b>The expression of green fluorescent protein (GFP) was observed in 95% of the infected SW480 and LS174T cells with a multiplicity of infection (MOI) of 100. RT- PCR demonstrated that the product of CD/TK gene existed in SW480 cells infected by Ad- KDR- CD/TK, but not in infected LS174 cells. The infected SW480 cells exhibited high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected SW480 cells. At the MOI of 100, treatment of the infected cells with the prodrugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase and the prodrug-treated cells showed an apoptotic peak in flow cytometry.</p><p><b>CONCLUSION</b>CDglyTK fusion gene system driven by the KDR promoter selectively kills and induces the apoptosis of the KDR-CDglyTK SW480 cells.</p>

Effect of adenovirus-mediated CD/TK double suicide gene system on colorectal cancer growth and cytokines in the tumor microenvironment in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of adenovirus-mediated CD/TK double suicide gene system on tumor growth and cytokine levels in the tumor microenvironment in mice bearing transplanted colorectal cancer.</p><p><b>METHODS</b>CT26 cells were implanted subcutaneously into 30 Balb/c mice, which were subsequently randomized into the control (n=15) and experimental group (n=15). After the tumor formation, CD/TK double suicide gene system was administered for tumor treatment, and the changes in the tumor volume, tumor inhibition rate, and levels of cytokines in the tumor microenvironment were investigated.</p><p><b>RESULTS</b>CD/TK double suicide gene system resulted in a significant inhibition of the tumor growth and significantly increased levels of such cytokines as IL-2, IL-10, TNFalpha and IFNgamma in the tumor microenvironment.</p><p><b>CONCLUSION</b>CD/TK double suicide gene system produces significant tumor inhibition effect and causes obvious cytokine changes in the tumor microenvironment in mice bearing transplanted colorectal cancer.</p>

Effect of KDR recombinant adenovirus containing double suicide gene on the proliferation of human colon adneocarcinoma SW620 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of adenovirus (Ad)-mediated fusion gene system driven by the KDR promoter on the proliferation of human colon adenocarcinoma SW620 cells.</p><p><b>METHODS</b>The KDR-expressing SW620 cells and LS174T cells not expressing KDR were both infected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or ganciclovir at different concentrations. The effect of the transfection on the cell proliferation was evaluated.</p><p><b>RESULTS</b>The expression of green fluorescent protein (GFP) was observed in 95% of the infected SW620 and LS174T cells with a multiplicity of infection (MOI) of 100. Significant difference was not founded in the growth of SW620 and LS174T cells with or without the transfection. The infected SW620 cells exhibit high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). The CDglyTK fusion gene produced much stronger killing effect of on the target cells than either of the single suicide genes (P<0.01).</p><p><b>CONCLUSION</b>CDglyTK fusion gene system driven by the KDR promoter selectively kills the KDR-CDglyTK SW620 cells and inhibits the cell proliferation.</p>

Pharmacokinetics and the bystander effect in CD::UPRT/5-FC bi-gene therapy of glioma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.</p><p><b>METHODS</b>C6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA).</p><p><b>RESULTS</b>(19)F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased.</p><p><b>CONCLUSIONS</b>(19)F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes. The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.</p>

A double suicide gene system driven by KDR promoter selectively kills human hepatic carcinoma cells and human umbilical vein endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the selective killing effects of adenovirus (Ad)-mediated double suicide gene system driven by KDR promoter (KDR-CdglyTK) on the human hepatic carcinoma cells and human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>KDR-expressing BEL-7402 and HUVECs and HepG2 cells that did not express KDR were infected by KDR-CdglyTK, and the infection efficiency and the expression of CdglyTK in the cells was detected by RT-PCR. The infected cells were treated with the the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method.</p><p><b>RESULTS</b>At the multiplicity of infection (MOI) of 100, the recombinant AdKDR-CDglyTK showed similar infection efficiency in the 3 cell lines. RT-PCR demonstrated CDglyTK expression in the recombinant adenovirus and the 3 infected cell lines. BEL-7402 and HUVECs infected by the KDR-CdglyTK, but not the HepG2 cells, were highly sensitive to the prodrugs (P<0.001). Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected BEL-7402 and HUVECs.</p><p><b>CONCLUSION</b>The double suicide gene system driven by KDR promoter has specific killing effect on KDR-expressing hepatocellular carcinoma cells and HUVECs.</p>

Adenovirus-mediated double suicide gene selectively kills breast cancer MCF-7 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of adenovirus-mediated double suicide gene (CD/TK) for selective killing of breast cancer cells.</p><p><b>METHODS</b>Vascular endothelial growth factor (VEGF)-expressing MCF-7 cells and normal human mammary epithelial cells that did not express VEGF were infected with the adenovirus containing VEGFP-CD/TK-GFP genes. CD/TK gene expression in the infected cells was detected by RT-PCR. After treatment of the infected cells with GCV and/or 5-FC, the cell growth status was evaluated using MTT assay, and the cell cycle changes were detected with flow cytometry. In nude mice bearing human breast cancer, the recombinant adenovirus vector was injected directly into the tumor followed by intraperitoneal injection of the prodrugs GCV and/or 5-FC, and the subsequent tumor growth was observed.</p><p><b>RESULTS</b>The recombinant adenovirus achieved similar infection rates in MCF-7 and human mammary epithelial cells, and the rates increased gradually with the multiplicity of infection (MOI) of the virus. RT-PCR demonstrated the presence of CD/TK gene product in infected MCF-7 cells, but not in the infected mammary epithelial cells. The infected MCF-7 cells, but not the mammary epithelial cells, were highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide gene in killing the target cells (P<0.01). At the MOI of 100, treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase. In nude mice bearing MCF-7 cell-derived subcutaneous tumor, treatment with the double suicide gene system significantly inhibited the tumor growth, showing much stronger effect than either of the single suicide gene (P<0.01).</p><p><b>CONCLUSION</b>The adenovirus-mediated CD/TK double suicide gene driven by VEGF promoter combined with GCV and 5-FC treatment can be an effective therapy against experimental breast cancer, and produces much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.</p>

Anti-tumor and bystander effects of cationic liposome-mediated CD/5-FC suicide gene system combined with interferon-gamma in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the antitumor and distant bystander effects of cationic liposome-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) suicide gene system combined with interferon-gamma (IFN-gamma) in vivo.</p><p><b>METHODS</b>Murine hepatoma 22 (H22) cells transfected by CD gene were inoculated subcutaneous in Kunming mice in the left axillary region, and the H22 cells without CD gene transfection were inoculated in the right axillary region. The mice were randomly divided into 4 groups and treated with normal saline , 5-FC, IFN-gamma, and 5-FC+ IFN-gamma on a daily basis. The tumor inhibition and distant bystander effects were observed in the mice.</p><p><b>RESULTS</b>Exposure of CD gene-transfected tumor to 5-Fc resulted in obvious tumor growth inhibition with an inhibition rate of 78.38%, which was significantly increased to 93.21% (P<0.01) with 5-Fc +IFN-gamma treatment. A notable distant bystander effect in the CD/5-FC suicide gene system was observed in vivo, with a tumor inhibition rate of was 54.42%; when combined with IFN-gamma, the inhibition rate increased significantly to 87.57% (P<0.05).</p><p><b>CONCLUSION</b>When combined with IFN-gamma, CD/5-FC suicide system has stronger anti-tumor and distant bystander effects. CD/5-FC suicide gene system combined with IFN-gamma may provide a potential therapy for malignant tumors.</p>

Toxic effects of CD-TK double suicidal gene system against prostate carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the toxic effects of the CD-TK fusion gene systems against prostate carcinoma cell line RM-1 for assessing the value of suicidal gene therapy for prostate carcinoma.</p><p><b>METHODS</b>CD-TK fusion gene and green fluorescent protein (GFP) gene were transfected into RM-1 cells through adenovirus vectors. RT-PCR was used to demonstrate successful transfection and transcription of the suicidal genes. The toxic effects of 5-FC and GCV used alone or in combination on the transfected cells were observed by MTT assay, with the non-transfected RM-1 cells serving as control.</p><p><b>RESULTS</b>Cytotoxic activity of CD/5-FC and TK/GCV systems against RM-1 cells was observed, and combined treatment with the two drugs resulted in significantly lowered survival of CD-TK-expressing cells (P<0.05). After exposure to 5-FC and GCV for 72 h, the survival rate of the transfected cells decreased to 71.56% and 47.27%, respectively, and their combined use resulted in a survival rate as low as 18.46%.</p><p><b>CONCLUSION</b>CD-TK fusion double suicidal gene system can produce significantly stronger toxic effect against RM-1 cells in vitro than either of suicidal genes.</p>

Targeted killing of colorectal tumor cells by lentiviral constructs containing CD/TK suicide genes and KDR promoter / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.</p><p><b>METHODS</b>293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.</p><p><b>RESULTS</b>The recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).</p><p><b>CONCLUSION</b>The double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.</p>

Killing effect of double suicide genes mediated by retroviral vector on k562 cells / 中国实验血液学杂志

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.

In vitro experimental study of killing Hep-2 cells of laryngeal cancer with suicide fusion gene CDglyTK / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the killing effect of suicide gene CDglyTK combined with GCV or 5-FC on the human laryngeal carcinoma Hep-2 cell line in vitro.@*METHOD@#Constructed plasmid pcDNA3.1 (-) CMV. CDglyTK was verified by enzyme digestion of Xho I /Hind III and automatic sequence analysis, then it was introduced into Hep-2 cells by electroporation to yield cells expressing CDglyTK stably after selecting with G418(400 ng/L) for 14 da. The expression of CDglyTK mRNA in transfected Hep-2 cells was tested by RT-PCR. Compared with Hep-2 cells transferred with pcDNA3.1(-), in vitro chemosensitivity of CDglyTK-expressing Hep-2 cells to 5-FC, GCV or 5-FC + GCV was detected by MTT assay.@*RESULT@#The recombinant plasmid contained full-length coding region sequence of CD and TK gene. A anticipated 707 bp fragment was amplified from total RNA of CDglyTK-expressing Hep-2 cells by RT-PCR and a fusion protein of 59 000 was detected in cell extract from transfected Hep-2 cells. In vitro study growth of CDglyTK-positive Hep-2 cells were inhibited by 5-FC, GCV or 5-FC + GCV respectively, and the antitumour effect of 5-FC + GCV is superior to 5-FC or GCV.@*CONCLUSION@#CDglyTK may be a candidate for treating human laryngeal cancer.

[Construction of a novel recombinant vector carrying herpes simplex virus thimidine kinase and yeast cytosine deaminase suicide genes to manipulate leishmania genetically]

Leishmaniasis is a group of diseases with various clinical pictures, which is caused by Leishmania spp. This parasite causes disease in human and about one hundred animal species. The disease is widely distributed in Iran and also across the world. One of the best approaches in the development of vaccine against Leishmania is genetically modification of the parasite. Therefore, the aim of this study was to design a novel genetic construct in order to insert Herpes Simplex Virus thimidine kinase [HSV-tk] and Yeast cytosine deaminase [Yeast-cd] suicide genes into the Leishmania genome. In this work, at the first step, HSV-tk and Yeast-cd fragments along with a gene of Leishmani, alpha-tubuline were cloned into pBluescript vector and the arrangement of tk-aplha tub-cd was created. Subsequently, these fragments were cut off from the plasmid and sub cloned into the plasmid pF4X1.4.4sat. The final construct was confirmed by digestion with relevant restriction enzymes. The fragments tk-alpha tub-cd was cloned successfully in the plasmid pBluescript and its authenticity was confirmed. Subsequently, this genetic collection was inserted into the plasmid pF4X1.4sat and finally, the orientation of it was checked. The construct designed in this study was able to insert two cellular suicide genes HSV-tk and Yeast-cd into the Leishmanai genome. Thus, this is an approach in achievement of vaccine against Leishmanai in the coming up researches