Establishment of Surfactant-associated Protein A Suicide Gene System and Analysis of Its Activity

Summary： Alveolar epithelial type II （AT II） cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells （fibroblasts）. Progenitor ceils are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A （SPA） suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase （rAAV-SPA-TK） system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells （H441） transfected with TK gene had higher sensitivity to ganciclovir （GCV） than SPA low expression cells （A549）. In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell （AT II） niche in vitro and in vivo.

Experimental Studies on PNP Suicide Gene Therapy of Hepatoma

Summary: To investigate the killing effect of PNP/MeP-dR suicide gene system on hepatoma cells, pcDNA3.0/PNP, an eukaryotic expression vector harboring E. coli PNP gene, was transfected into human hepatoma HepG2 cells by liposome-mediated method. A HepG2 cell line with stable PNP gene expression, HepG2/PNP, was established with presence of G418 selection. The cell growth curves were determined with trypan blue staining. The sensitivity of HepG2/PNP to MeP-dR and bystander effects were assayed by MTT and FCM methods. The enzymatic activity of the product of PNP gene was determined by HPLC method. The cytotoxic effects of MeP-dR on HepG2/PNP cells were obvious (IC 50=4.5 μmol/L) and all HepG2/PNP cells were killed 4 days after the treatment with 100 μmol/L MeP-dR. In mixed cultures containing increasing percentages of HepG2/PNP cells, total population killing was demonstrated when HepG2/PNP cells accounted for as few as 5 % of all HepG2 cells 8 days after the treatment with 100μmol MeP-dR. High-pressure liquid chromatography (HPLC) demonstrated that the PNP enzyme could convert MeP-dR into 6-MP. PNP/MeP-dR suicide gene system had an advantage over traditional suicide gene systems for hepatoma gene therapy. Our e results suggest that high-level bystander effects of this system result in significant anti-tumor responses to hepatoma gene therapy, especially in vivo.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model

Objective： Intratumoral administration of adenoviral vector encoding herpes simplex virus （HSV） thymidine kinase （TK） gene （Ad-TK） followed by systemic ganciclovir （GCV） is an effective approach in treating experimental hepatocellular carcinoma （HCC）. However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy, miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. Methods： By inserting miR-122 target sequences （miR-122T） in the 3＇ untranslated region （UTR） ofTK gene, we constructed adenovirus （Ad） vectors expressing miR-122-regulated TK （Ad-TK-122T） and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Results： Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. Conclusions： miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

RETROVIRAL-MEDIATED SUICIDE GENE THERAPY OF EXPERIMENTAL GLIOMA

Objective: To establish a retroviral mediated suicide gene therapy system for experimental glioma and test its efficacy. Methods: C6 rat glioma cells were infected with recombinant retrovirus containing HSV tk gene. The C6/tk cell line which stably expressed tk was selected and cloned. The sensitivities of C6/tk cells to several nucleoside analogues, such as GCV, BVdU, ACV were compared by the growth inhibition studies. Antitumor effects were also observed after GCV treatment in nude mice bearing tumors derived from C6/tk cells. Results: The growth inhibition studies showed that GCV was the most efficient prodrug in this system. C6/tk cells were highly sensitive to GCV, with an IC 50 <0.2 μmol/L, being 500 fold less than that in tk negative C6 cells. In vivo studies showed significant tumor inhibition in the treatment group. Conclusion: Glioma cells can be eradicated by using retroviral mediated suicide gene system in vitro as well as in vivo .

ENHANCED ANTITUMOR EFFECTS OF SUICIDE GENE THERAPY BY SIMULTANEOUS TRANSFER OF GMCSF GENE IN LEUKEMIA-BEARING MICE

In the present report, antitumor effect of combined transfer of suicide gene and cytokine gene was studied. Adenovirus engineered to express E. Coli. Cytosine deaminase (AdCD) and/or adenovirus engineered to express murine granulocytemacrophage colonystimulating factor (AdGMCSF) were used for the treatment of leukemiabearing mice. The mice were inoculated s.c. with FBL3 erythroleukemia cells and 3 days later received intratumoral injection of AdCD in the presence or absence of AdGMCSF followed by intraperitoneal 5fluorocytosine (5FC) treatment. The results demonstrated that mice received combined therapy of AdCD/5FC and AdGMCSF developed tumors most slowly and survived much longer when compared with mice treated with AdCD/5FC alone, AdGMCSF alone, AdlacZ/5FC or PBS. Combined transfer of CD gene and GMCSF gene achieved higher specific CTL activity than control therapies. Pathological examination illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration in mice after combined therapy. The results demonstrated that combined transfer of suicide gene and cytokine gene could synergistically inhibit the growth of leukemia in mice and induce antitumor immunity of the host. The combination therapy might be a potential approach for cancer gene therapy.

Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells （A549） were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene （Ad-CD/UPRT） was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC （10-1000 μg/mL） administration. Furthermore, Ad-CD/UPRTinfected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

A STUDY ON THE TREATMENT OF GASTRIC CANCER BY CD GENE COMBINED WITH 5-FC IN VITRO AND IN VIVO

Objective: To study the killing effect of suicide gene CD on mouse gastric cancer. Methods: CD gene was transduced with the retroviral vector. The killing effect and bystander effect of CD gene on mouse gastric cancer cell line MFC were observed. The mouse gastric cancer model was used for in vivo study. The CD gene containing virus was injected into the tumors. The volumes of the tumors in every group were measured in time. Results: Significant killing effect and bystander effect were observed by CD gene in vitro, 70–80% cell death resulting from 20% of CD gene transduction. In vivo, CD/5-Fc caused tumor to diminution. Conclusion: CD/5-Fc system has significant killing effect on mouse gastric cancer

Suicide gene therapy of human breast cancer in SCID mice model by the regulation of Tet-On

Background RevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficie ncy (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On.Methods Herpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR).Results MCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 μg/ml at 1 μg/ml of Dox after 72 hours of GCV administration. At 1 μg/ml of GCV concentration, the cell numbers decreased from 7×10 4 cells/ml to 2×10 4 cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10 %-25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control.Conclusion The human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.

Therapeutic effect of AdCMVCD/5-FC system and metabolism of 5-FC in the treatment of human tongue squamous cell carcinoma

To investigate the therapeutic effect and metabolism of 5 fluorocytosine (5 FC ) in human tongue squamous carcinoma cells after treatment with adenovirus medi ated cytosine deaminase (AdCMVCD)/5 FC system Methods Human tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5 FC system The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay Tumor inhibition effect and histopathological changes were observed in vivo High performance liquid chromatography (HPLC) was performed to determine the m etabolism of 5 FC in vitro and in vivo Results AdCMVCD/5 FC system had strong killing effect and bystander effect on Tca8113 c ells Both condition media and cell extracts showed two peaks identified as 5 FC and 5 fluorouracil (5 FU) by HPLC and a time dependent generation of 5 FU and concomitant time dependent decreases of 5 FC Compared to the control gr oups, mice treated with AdCMVCD/5 FC system demonstrated significant tumor regr ession ( P <0 001); the tumor doubling time prolonged and inhibition rate was 92 62% There were substantial tumor necrotic areas and infiltrative lymphocy tes around necrotic areas in the AdCMVCD/5 FC treated group under light microsc ope There was a significantly low concentration of 5 FC and high concentratio n of 5 FU in tumor tissue, but only 5 FC was found in blood Conclusion AdCMVCD/5 FC suicide gene system had significant in vitro and in vivo anti tum or effect on human tongue squamous cell carcinoma due to convert 5 FC into 5 F U

Influence on the immune function of the human peripheral blood mononuclear cells transfected by retrovirus-mediated HSV-tk gene

Graft-versus-host disease （GVHD） is a severe complication and a major source of morbidity and mortality after allogeneic hematopoietic stem cell transplantation （HSCT）. Although T cell depletion of the allogeneic HSCT can efficiently prevent GVHD, it is associated with increased graft rejection and relapse of the malignant disease. To preserve the beneficial effects of donor T cells and avoid their GVHD effects, some approaches have been explored. One of them is to transfer a special suicide gene into the donor T lymphocytes so that they become more sensitive to a specific drug that is ordinarily not toxic. The most commonly used suicide gene is the thymidine kinase-encoding gene of herpes simplex virus （HSV-tk）.

Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells

Background Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E.coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells. Methods Adenoviral vectors, including Ad-EF1α-CD-cytomegolovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1α-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA+E.coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis. Results Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1α-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1α-CD single gene groups. The rate of cell survival was only (9±3)% in the Ad-EF1α-CD-CMV-TK group, but (37±3)% in the Ad-CMV-TK and (46±4)% in the Ad-EF1α-CD groups (P<0.05). Flow cytometry analysis indicated that the killing mechanisms of the groups were different. Necrosis and apoptosis were involved in the mechanism of the double gene group. Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1α-CD-CMV-TK [(11.0±2.1)%] and Ad-CMV-TK [(12.0±2.2)%] groups were higher than those in Ad-CMV-LacZ [(1.2±0.11)%] and Ad-EF1α-CD [(5.0±1.8)%] groups (P<0.05, respctively). DNA smear could be observed in both Ad-CMV-TK and Ad-EF1α-CD-CMV-TK groups after administration of prodrugs. Conclusions The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene. The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis.

Non-Viral Deoxyribonucleoside Kinases——Diversity and Practical Use

Deoxyribonucleoside kinases （dNKs） phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds, dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of great medical interest. However, during the last 20 years, research on dNKs has gone into non- mammalian organisms. In this review, we focus on non-viral dNKs, in particular their diversity and their practical applications. The diversity of this enzyme family in different organisms has proven to be valuable in studying the evolution of enzymes. Some of these newly discovered enzymes have been useful in numerous practical applications in medicine and biotechnology, and have contributed to our understanding of the structural basis of nucleoside and nucleoside analogue activation.