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Objective: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1)., Methods: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC 50 ) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay., Results: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group ( P <0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC 50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells ( P <0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G 2 /M phase also decreased, while G0/G1 phase increased in cell cycle., Conclusion: The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Lichens are symbiotic organisms that have been traditionally used for treating different kinds of ailments. As there are only a few reports on the antiviral activity of lichens, we thought of evaluating the anti-Herpes simplex virus-1 (HSV-1) activity of methanolic extract of Roccella montagnei and their isolated compounds. Fractionation of crude methanolic extract of Roccella montagnei by column chromatography isolated two pure compounds. Antiviral activity was assessed using a CPE inhibition assay at non-cytotoxic concentrations on Vero cells. Molecular docking and dynamics studies were carried out against Herpes simplex type-1 thymidine kinase to understand the binding interactions of the isolated compounds with reference to acyclovir. Isolated compounds were characterized as methyl orsellinate and montagnetol by spectral methods. Methanolic extract of Roccella montagnei exhibited an EC 50 value of 56.51 µg/ml, while the compounds methyl orsellinate and montagnetol offered EC 50 values of 13.50 µg/ml and 37.52 µg/ml, respectively, against HSV-1 viral infection on Vero cell lines. The selectively index (SI) of montagnetol (10.93) was found to be higher when compared to that of methyl orsellinate (5.55), indicating its better anti-HSV-1 activity. The docking and dynamics studies showed montagnetol was stable throughout the 100 ns, having better interactions and docking scores with HSV-1 thymidine kinase than methyl orsellinate, as well as the standard. To understand the mechanism of montagnetol's anti-HSV-1 activity, more research is required, and this could lead to the discovery of new and effective antiviral agents.Communicated by Ramaswamy H. Sarma.

Background: Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge., Objectives: To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness., Patients and Methods: Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays., Results: Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to., Conclusions: The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8 + T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8 + T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8 + T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM-CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT-GM-CSF-danger signal system by means of artificial cancer specific promoters or a modified delivery system.

Background: Mesenchymal stem cells (MSCs) have been applied as a promising vehicle for tumour-targeted delivery of suicide genes in the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy against malignant gliomas. The efficiency of this strategy is largely dependent on the bystander effect, which relies on high suicide gene expression levels and efficient transportation of activated GCV towards glioma cells. However, up to now, the methods to enhance the bystander effect of this strategy in an efficient and safe way are still lacking and new approaches to improve this therapeutic strategy are required. Methods: In this study, MSCs were gene transfected using magnetosome-like ferrimagnetic iron oxide nanochains (MFIONs) to highly express HSV-tk. Both the suicide and bystander effects of HSV-tk expressed MSCs (MSCs-tk) were quantitatively evaluated. Connexin 43 (Cx43) expression by MSCs and glioma cells was measured under different treatments. Intercellular communication between MSCs and C6 glioma cells was examined using a dye transfer assay. Glioma tropism and the bio-distribution of MSCs-tk were observed. Anti-tumour activity was investigated in the orthotopic glioma of rats after intravenous administration of MSCs-tk followed by intraperitoneal injection of GCV. Results: Gene transfection using MFIONs achieved sufficient expression of HSV-tk and triggered Cx43 overexpression in MSCs. These Cx43 overexpressing MSCs promoted gap junction intercellular communication (GJIC) between MSCs and glioma cells, resulting in significantly inhibited growth of glioma through an improved bystander effect. Outstanding tumour targeting and significantly prolonged survival with decreased tumour size were observed after the treatment using MFION-transfected MSCs in glioma model rats. Conclusion: Our results show that iron oxide nanoparticles have the potential to improve the suicide gene expression levels of transfected MSCs, while promoting the GJIC formation between MSCs and tumour cells, which enhances the sensitivity of glioma cells to HSV-tk/GCV suicide gene therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: To establish the technique of intratumoral combination therapy of radiofrequency hyperthermia (RFH) with herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy for rat ovarian cancers. Material and methods: This study consisted of three parts: (1) in vitro experiments to establish the 'proof of principal' that combination of RFH and HSV-TK gene therapy has the synergistic effect on human ovarian cancer cells; (2) creation of bioluminescence imaging-detectable rat ovarian cancer model; and (3) in vivo experiments using this rat model to validate the technical feasibility of the combination therapy. Cells and nude rats were divided into four groups: (i) combination therapy (HSV-TK/GCV + RFH); (ii) RFH; (iii) HSV-TK/GCV; and (iv) phosphate-buffered saline (PBS). Data were analyzed using Dunnett t -test or Kruskal-Wallis test. Results: Cell proliferation assay demonstrated significantly greater reduction in viable cells with the combination therapy [0.52 (0.43, 0.61)] compared to other treatments [RFH 0.90 (0.84, 0.96), HSV-TK/GCV 0.71 (0.53, 0.88), PBS 1 (1, 1); p  < .05]. For 24 rat models with bioluminescence imaging-detectable orthotopic ovarian cancer ( n  = 6 per group), optical imaging demonstrated significantly decreased relative bioluminescence signal with the combination therapy [0.81 (0.52, 1.08)] compared to other treatments [RFH 3.60 (2.34, 4.86), HSV-TK/GCV 2.21 (1.71, 2.71), PBS 3.74 (3.19, 4.29); p  < .001]. Ultrasound imaging demonstrated the smallest relative tumor volume with the combination therapy [0.78 (0.45, 1.11) versus 3.50 (2.67, 4.33), 2.10 (0.83, 3.37), 3.70 (1.79, 5.61); p  < .05]. Conclusion: The feasibility of intratumoral RFH-enhanced HSV-TK/GCV gene therapy was established on a unique rat model with molecular imaging-detectable orthotopic ovarian cancer.

Background: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome., Patients and Methods: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression., Results: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker., Conclusion: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Retinoblastoma is an severe ophthalmic disease and the most common type intraocular malignant tumor, particularly in infants. Currently, few drugs and therapies are available. Gene therapy has been considered to be a potential treatment to cure cancer effectively and Herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV‑TK/GCV) is one type of suicide gene therapy that has been extensively studied. Numerous in vitro and in vivo studied have shown that this system can kill tumor cells, including liver and lung cancer cells. GCV is used as an antiviral drug, and the thymidine kinase, HSV‑TK can phosphorylate GCV to GCV‑TP, a competitive inhibitor of DNA synthesis, instead of guanine‑5'‑triphosphate in the process of DNA synthesis. This process prevents DNA chain elongation causing cell death via apoptosis. However, the toxic effects of HSV‑TK/GCV on retinoblastoma cells remain unknown, and the molecular mechanisms of its therapeutic effects have not been fully elucidated. Our results suggest that HSV‑TK/GCV can significantly cause the death of retinoblastoma cell lines, HXO‑RB44 and Y79. Further studies have reported that this cell death is induced by the inhibition of autophagy by activating the MAPK/ERK (mitogen‑activated protein kinase/ERK) signaling pathway. The mTOR inhibitor Torin1 can partially block the toxic effects of HSV‑TK/GCV on HXO‑RB44 cells. The above results demonstrate that the mechanism undertaken by HSV‑TK/GCV to exhibit therapeutic effects mechanism may inhibit autophagy by activating MAPK/ERK. The findings of the present study may provide novel insight for the exploration of HSV‑TK/GCV in the treatment of retinoblastoma.

Based on the hypothesis that the bulky selenium atom, with 4p orbitals, can sterically hinder the approach of a cellular kinase to 5'-OH for phosphorylation, 4'-selenonucleosides with one-carbon homologation were designed and synthesized via a novel seleno-Michael reaction, with the stereoselectivity controlled by steric effects. 5'-Homo-4'-selenonucleosides (n = 2) demonstrated potent antiherpes simplex virus (HSV-1) activity, indicating that the bulky selenium atom might play a key role in preventing phosphorylation by cellular kinases, resulting in no antiviral activity.

Hepatocellular carcinoma (HCC) has particularly high incidence rate in Asia and its resistance to the chemotherapeutic drugs and cell death make it intractable. Vaccinia virus (VV) is a potential vehicle and has been widely used in cancer therapy. SMAC/DIABLO is a critical factor in activating caspases and eliminating inhibition of IAPs when the programmed cell death is promoted. In this study, we constructed a tumor-targeted vaccinia virus carrying SMAC/DIABLO gene that was knocked in the region of viral thymidine kinase gene (VV-SMAC). Our results showed that VV-SMAC efficiently infected and destroyed HCC cells via triggering both caspase-dependent apoptosis and necroptosis with depletion of IAPs. Furthermore, ripoptosome, a prerequisite complex of necroptosis, was assembled and induced by VV-SMAC. In addition, the combination of VV-SMAC and vinblastine represented a synergistic effect on HCC cells. In summary, our data suggest that VV-SMAC is a potential candidate and combination of VV-SMAC and vinblastine may provide a new avenue in treatment of HCC.

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV(r)) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10 days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV(r) HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC(50)s) and inhibitory effects of these compounds on the replication of TAR among ACV(s) and ACV(r) HSV-1 clones. These results indicate that the EC(50)s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC(50) for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesion's samples or from virus isolates., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process.

Background: Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown., Findings: Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes., Conclusions: Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti-transplant, autoimmune, or anti-tumor immune responses in vivo and in vitro.

As gene therapy has matured from clinical trials to the first commercial products, understanding of the mechanisms of gene delivery has increased tremendously. This has also been reflected in viral vector development, creating a number of new approaches to tackle issues in transduction efficiency, biodistribution and viral safety. This review will highlight the most important issues and advancements in vector development, administration, surface modification, integration to host genome and safety. The gene therapy products currently available or near market approval, based on p53 expression (Gendicine and Advexin), conditionally replicative adenoviruses (Oncorine) and thymidine kinase + ganciclovir therapy (Cerepro), are introduced with emphasis on the molecular mechanisms of action.

Objective: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death., Methods: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated., Result: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect)., Conclusion: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

Objective: To evaluate the lethal effect of adenovirus-mediated HSV-TK-ganciclovir (GCV) gene therapy in combination with hydroxycamptothecin (HCPT) on hunman bladder carcinoma cell line T-24 cells., Methods: Human bladder carcinoma cell line T-24 was transfected with adenovirus expression vector containing TK gene and green fluorescent protein (GFP) gene, and the transfection efficiency was observed and TK expression detected by PCR. After successful cell transfection indicated by GFP expression, GCV and hydroxycamptothecin are respectively added into the cell culture with normal T-24 cells serving as the blank control group. The growth inhibition rate of hunman bladder carcinoma cells in response to HCPT treatment for 72 h and the cell survival rate of 24 h, 48 h and 72 h after transfection with different protocols were observed by MTT assay. The apoptosis of the cells treated with GCV (0.5 mg/ml)+HCPT (10 mg/L) for 4 h was observed by flow cytometry., Results: The cell inhibition rate increased gradually with increment of HCPT concentration, from 14% at HCPT concentration of 0.01 mg/L to 60% at 50 mg/L, but for a concentration above 100 mg/L, the inhibition rate did not exhibit further increase (P=0.216). GCV alone and GCV in combination with HCPT both resulted in significantly decreased survival rate of human bladder carcinoma cells (P=0.00), and the killing efficiency of the cells by GCV+HCPT protocol increased obviously with increment of HCPT concentration and prolongation of the action time. The cells treated with 0.5 mg/ml GCV alone for 72 h retained a cell survival rate of 34.6%, which was lowered to only 8.07% with combined treatment with GCV (0.5mg/ml) and HCPT (10 mg/L). Typical apoptotic peak before M1 phase of the cells appeared 4 h after treatment with GCV+10 mg/ml HCPT, which resulted in a apoptosis rate of 52.93%., Conclusion: HSV-TK/GCV in combination with HCPT can enhance the lethal effect of suicide gene therapy against human bladder carcinoma cells and effectively induce apoptosis of the cells.

Objective: 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., Methods: 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., Results: 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%., Conclusion: CD-TK fusion double suicidal gene system can produce significantly stronger toxic effect against RM-1 cells in vitro than either of suicidal genes.

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.

We have earlier demonstrated that inhibition of polyamine biosynthesis with difluoromethylornithine (DFMO) can be used to enhance the cytotoxicity of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in different tumor cell lines. Here, the utility of this treatment combination was tested in vivo in a nude mouse tumor model. First, the effect of DFMO was verified by treating mice bearing subcutaneous 9L rat glioma tumors with 2% DFMO in drinking water. The drug treatment induced almost complete suppression of ornithine decarboxylase activity, and as a result, a strong decrease in intratumoral putrescine and spermidine concentrations, which were normalized 4 days after drug removal. Consequently, the tumors displayed a significant reduction in the proliferation activity that was increased to 20% higher than the normal level at day 4 and returned to normal level 7 days after DFMO removal. Next, 9L tumors with 30% of TK-GFP fusion gene positive cells were induced and the animals were given DFMO and GCV in 2 treatment schemes, with the drug administration periods overlapping either 5 or 2 days. The analysis of tumor size at the end of the treatment revealed that DFMO can enhance HSV-TK/GCV cytotoxicity when the overlap between DFMO and GCV was 5 days, but the result was not significant. However, the 2-day overlap scheme yielded a significantly (p < 0.05, ANOVA) enhanced antitumor effect. In conclusion, the data here confirms that a novel combination of 2 clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV suicide gene therapy, can be used synergistically in vivo.

Background & Objective: The presence of Coxsackie and adenovirus receptor (CAR) on target cell surface is required for efficient adenovirus transfection; lack or down-regulated expression of CAR on cancer cells is the main cause of inefficiency of adenovirus-based gene therapy. This study was to evaluate enhancive effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the transfection efficiency of adenovirus in ovarian carcinoma cell line A2780, and explore its possible application to adenovirus-based gene therapy., Methods: mRNA and protein levels of CAR on A2780 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot before and after treatment of TSA. Transfection efficiency of adenovirus was valued by flow cytometry (FCM). In vitro antitumor effect of adenovirus/thymidine kinase (ADV/TK) was detected by MTT assay., Results: After treatment of TSA, mRNA and protein levels of CAR on A2780 cells were obviously increased. Transfection rates of adenovirus were (1.24+/-0.14)% in untreated group, (7.58+/-0.32)% in 5 nmol/L of TSA treated group, and (7.94+/-0.28)% in 100 nmol/L of TSA treated groups. In vitro antitumor effect of ADV/TK was 4-10 folds in TSA (5 or 100 nmol/L) treated groups compared with that in untreated group., Conclusion: TSA could enhance transfection efficiency of adenovirus in ovarian carcinoma cells, and may be useful in gene therapy for ovarian carcinoma.

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Although great efforts have been made to improve conventional therapy for diffuse malignant pleural mesothelioma, the median survival time of the patients after appearance of clinical symptoms remains poor. Due to confinement of the primary tumor to the pleural space, locoregional approaches are attractive strategies to improve the clinical outcome. In this context locoregional gene therapy using the recombinant adeno-associated virus 2 (rAAV-2) may be a new approach. Vectors were constructed containing a fusion gene, consisting of the Herpes simplex virus thymidine kinase (HSV-TK) and the green fluorescent protein (GFP) genes; the former serving as suicide gene by converting the prodrug ganciclovir (GCV) into a toxic agent, thereby killing infected cells. Among a number of different tumor cell lines, rAAV-2 achieved high GFP expression levels in three mesothelioma cell lines (H-Meso-1, MSTO-211H, NCI-H28). A variety of rAAV-2-constructs containing different promoters were tested. The vector with the elongation factor-1alpha (EF-1alpha) promoter showed the highest expression rates. Expression could be further increased by addition of the tyrosine kinase inhibitor genistein. Using the rAAV-2-based suicide system, a nearly complete eradication of transduced and GCV-treated mesothelioma cells was observed. rAAV-2-based suicide gene therapy may be a new approach for locoregional treatment of mesothelioma.

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.

The efficacy of HSV-TK/ganciclovir gene therapy largely relies on the bystander effect, i.e. the ability of transfected cells to kill the adjacent, untrasfected cells. This mechanism itself depends chiefly on the transfer via gap junctions of phosphorylated ganciclovir between cells, and is often deficient in glioblastomas. In this report, we demonstrate that n-butyrate markedly enhances the gap junction intercellular communication of GJIC-deficient glioma cells, and significantly increases the bystander effect in such cells. This effect of n-butyrate appears to be independent from its HDAC inhibitory effect, since trichostatin A does not reproduce it.

Almost all the polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic activity. Environmental carcinogen [(3)H] benzo[a]pyrene (BP) is carcinogenic only after its metabolic transformation to a reactive intermediate, which can then bind to cellular macromolecules. Inhibition of dimethylbenz anthracene- (DMBA-) DNA binding generally accompanied inhibition of tumor initiation as most inhibitors of initiation interfere with the metabolic activation of the initiator. The importance of carcinogen-DNA interaction and the enzymes involved in the metabolism of carcinogenic polycyclic hydrocarbons has led to a search for inhibitors that would be useful in modifying the cancer-causing effects of the PAHs. We tested the effect of calcium glucarate (Cag), a naturally occurring nontoxic compound, on carcinogen metabolism and DNA interaction. Cag inhibited [(3)H] BP binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. Application of Cag to mouse skin caused a dose-dependent inhibition of [(3)H] BP binding to epidermal DNA. To establish the relevance of the in vivo results to the in vitro situation, we followed the in vitro effect of Cag on [(3)H] BP binding to calf thymus DNA and observed that Cag inhibited the [(3)H] BP binding to calf thymus DNA in the presence of microsomes prepared from animals treated with DMBA. We also studied related events like DNA synthesis and carcinogen metabolism. For assessing the DNA synthesis, thymidine kinase was used as marker. Cag caused a dose-dependent inhibition of DMBA-induced thymidine kinase activity. At the same time, Cag caused a marked inhibition of DMBA-induced aryl hydrocarbon hydroxylase (AHH) activity, an enzyme responsible for the metabolism of PAHs like BP, both in vivo and in vitro. Our study indicates that Cag exerted its antitumor effect possibly by inhibiting the carcinogen-DNA binding, which appears to be due to reduced DNA synthesis and AHH activity.

Background & Objective: Adenovirus vector-mediated herpes simplex virus thymidine kinase gene (ADV-TK) transfer is one of the major gene therapy strategies for tumor. This study was designed to determine the in vitro anti-tumor efficiency of ADV-TK, a recombinant construct previously developed in our laboratory., Methods: Herpes simplex virus thymidine kinase (TK) gene was transduced into 14 types of cultured tumor cells with different histological origins using adenovirus vector followed by ganciclovir (GCV) medication. The killing efficiency was measured using MTT assay., Results: At the dosage of 1x10(9) viral particles/per well in the presence of 100 microg/ml GCV, ADV-TK/GCV caused effective killing of 11 out of total 14 types of tumor cells with a rate higher than 74%, the other 3 tumor cells, laryngeal epithelial cancer cells (Hep-2), hepatic cancer cells (Bel7402), and human colon cancer cells (HCT-8) were less sensitive to the ADV-TK/GCV treatment with the killing rates of 55.3%+/-2.0%, 61.3%+/-2.0%, and 63.7%+/-2.5%, respectively. Except for Hep-2, the killing efficiency caused by ADV-TK/GCV treatment was similar to that caused by cisplatin at a dosage equal to the in vivo peak concentration (5 microg/ml) in tissue., Conclusion: ADV-TK is highly efficient for active killing of tumor cells in vitro and is promising for future clinical application.

Background: The fusogenic (F) membrane glycoprotein of the paramyxovirus SV5 allows virus to enter host cells and mediates fusion between neighboring cells, which leads to cell death. F glycoprotein is synthesized as an inactive precursor (F(0)) that is cleaved by cellular protease furine to form the active heterodimer F(1) + F(2). The active protein can induce syncytium formation in the absence of another integral glycoprotein (HN), a property that appears to be unique among paramyxoviruses., Methodology: We constructed a non-replicative adenovirus to express SV5 F protein in tumor cells, and its fusion capacity was analyzed by fluorescent and confocal microscopy. Cell viability and bystander effect were compared with the thymidine kinase/ganciclovir suicide gene therapy. The structure of F-expressing cells was studied using electron microscopy., Results: F glycoprotein expression induced syncytium formation to a maximum at 72 h, after which syncytia progressively lost viability and detached. The cell membrane was disrupted while nuclear structure was preserved. Over-expression of SV5 F protein in tumor cells led to high cytotoxicity comparable with that associated with the thymidine kinase/ganciclovir. A potent bystander killing effect was detected until the ratio of F-transduced to non-transduced cells was 1 : 100., Conclusions: These results indicate that the fusogenic glycoprotein of the paramyxovirus SV5 could be used to eliminate tumor cells and may encourage studies aimed at modifying its selectivity and combining its expression with other cytotoxic strategies to improve their efficacy., (Copyright 2003 John Wiley & Sons, Ltd.)

Purpose: Carcinoembryonic antigen expression is increased in more than 80% of patients with colorectal cancer. Values are especially higher in patients with advanced stage disease. Virus directed prodrug/enzyme therapy (VDEPT) using genetically engineered viral vectors has been considered as one of the more notable cancer gene therapies for the transduction of various enzymes into cancer cells. We made adenovirus vectors under the control of a CEA promoter that included the HSV-tk gene and investigated its usefulness to specifically target human CEA-producing colorectal cancer cells., Methods: An adenovirus vector with the lacZ or HSV-tk gene under the control of a CAG or CEA promoter was designed for the VDEPT experiment. Human colorectal cancer cell lines were used for in vitro experiments to assure the transduction efficacy of the inserted genes by these vectors. To conduct the in vivo experiment, liver metastases of the cell line were created in CB17 SCID mouse. We then performed intrasplenic injections of adenovirus vectors and intraperitoneal injections of the prodrug, ganciclovir., Results: RCM-1, the CEA-producing human rectal cancer cell line, was more strongly stained by X-gal staining. Furthermore, COLO320 was faintly stained secondary to a shortage of CEA production. The in vivo VDEPT experiment with RCM-1 and the adenovirus vector driven by the CEA promoter revealed attenuation of liver metastases in the treatment group., Conclusions: Adenovirus vectors under the control of the CEA promoter can transduce inserted genes effectively into targeted human colorectal cancer cells according to the amount of expressed CEA protein. We anticipate the future use of VDEPT of the HSV-tk/GCV system using this vector in the treatment of advanced colorectal cancers.

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.

Cancer gene therapy based on the use of suicide genes, such as the thymidine kinase gene, is not producing satisfactory results. Several approaches have been delineated to enhance the therapeutic responses, including augmentation of the bystander effect, the combination of the herpes simplex virus thymidine kinase-ganciclovir (HSVTK-GCV) system into replication competent adenoviruses and others. Moreover, because usually less than 20% of human malignant cells are in S-phase, the HSVTK-GCV system is not as efficient as expected. To increase the cytotoxic effects of the HSVTK-GCV system, we hypothesized that concomitant expression of E1a protein, which drives cells to proliferation and S-phase, could increase the effects of the HSVTK-GCV system. Several retroviruses were constructed carrying bicistronic sequences of TK and E1a 12S genes under the control of the CMV promoter. The constructions were tested in murine (NIH-3T3, MSC11A5) and human cells (IMR90, HeLa, MDA-MB435). A clear increase of the HSVTK-GCV system killing effect in nonconfluent cells was observed in the cells studied, especially in NIH-3T3, MSC11A5, IMR90, and MDA-MB435 expressing cells. In confluence, the NIH3T3 and IMR90 E1a-TK-expressing cells were also very sensitive and most malignant E1a-TK-expressing cells showed an irreversible G2-M cell cycle arrest. Moreover, the concomitant expression of adenovirus E1a and the HSVTK-GCV system increased the sensitivity to anticancer agents such as cisplatin. These results show that adenovirus E1a protein expression clearly enhances the cytotoxic effects of the HSVTK-GCV system and the response to treatment with cisplatin.

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded products. Here we have developed a transduction and selection strategy for generating large numbers of polyclonal T cells transduced with a retroviral vector encoding the human low-affinity nerve growth factor receptor (LNGFR) for selection and a Fas-based suicide construct (LV'VFas). Ligation of CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to the generation of gene-modified T cells that retained clonal diversity, functional properties, and a homing receptor profile similar to untransduced peripheral blood lymphocytes. Microbeads conjugated directly to antibody specific to LNGFR significantly improved the immunomagnetic selection of LV'VFas-modified T cells and assisted in scaling of the selection procedure to therapeutic cell numbers. Thus, these studies identified a strategy that requires only a brief ex vivo culture and does not use drug selection to obtain large numbers of functional gene-modified polyclonal T cells that can be used for adoptive immunotherapy.

To test the concept that a replication-competent retrovirus carrying a suicide gene could have potential utility in the control of the natural virus infection in mammalian species, we constructed derivatives of a feline leukemia virus (FeLV) that is commonly associated with leukemia-lymphomas in this species. The FeLV, Rickard strain, subgroup A (FRA) genome contained at the 3' end of the envgene, an insert of an internal ribosomal entry site (IRES) linked to cDNA sequence of either herpes simplex virus thymidine kinase (HSV-TK) or a truncated HSV-TK (HSV-ATK) or yeast cytosine deaminase (CD). These constructs were transfected into feline fibroblast cells (H927). The viruses produced were determined to be replication-competent. The stable propagation of the full-length transgene was, however, dependent on the size of the insert, IRES-CD being the smallest in size (1031 bp) exhibiting maximal stability for at least up to six months. The protein products of the transgenes could be detected, despite the appearance of deleted proviruses at late passages. The transduced cells were susceptible to cytotoxic killing when the appropriate prodrug, ganciclovir (GCV), acyclovir (ACV) or 5-fluorocytosine (5-FC) was added to the culture medium. H927 cells, infected with another subgroup of FeLV, namely, FeLV-B or FeLV-C, could be superinfected by the FRA-suicide gene viruses and thus, subjected to killing. Interestingly, at an early stage of infection by the parental FRA, H927 cells could also be reinfected by the same subgroup FRA constructs to induce the suicide effect. Among the three constructs, the vector with the CD gene was determined to be superior to others in terms of stability, therapeutic index and bystander effect in the cell culture test system. While the in vivo correlates of the therapeutic effect in the feline model remain to be determined, our results do encourage investigation of the same concept in the control of HTLV and, perhaps even, HIV infection in humans.

Recombinant adenoviruses, carrying herpes simplex virus thymidine kinase (HSVtk) genes, were developed to evaluate the possibility of tissue-specific gene therapy for thyroid carcinomas. The HSVtk gene was driven by a minimal thyroglobulin (TG) promoter (AdTGtk) and a tandemly repeated minimal TG promoter (Ad2 x TGtk) to obtain thyroid-specific cell killing ability. The transduction of HSVtk genes by infection with Ad2 x TGtk followed by ganciclovir (GCV) treatment showed more powerful cytotoxicity for TG-producing FRTL5 cells, a rat normal thyroid cell line, and FTC-133 cells, a human follicular thyroid carcinoma cell line, than when infected with AdTGtk in vitro. The cell killing ability of Ad2 x TGtk was 10- to 30-fold higher than that of AdTGtk and similar to that of AdCMVtk, which carries HSVtk under the control of CMV promoter. Whereas after treatment with adenovirus/GCV to non-TG-producing cell lines (undifferentiated thyroid carcinoma cell lines and carcinoma cell lines from other tissues), Ad2 x TGtk and AdTGtk needed more than 100-fold concentrated GCV to reach IC(50) compared to AdCMVtk. We confirmed the enhanced efficacy of Ad2 x TGtk for tissue-specific cytotoxicity in vivo. After adenovirus/GCV treatment for FTC-133 tumor-bearing nude mice, Ad2 x TGtk enhanced tumor growth inhibition and survival rates compared to AdTGtk. Tumor growth inhibition and survival rates by Ad2 x TGtk were similar to that by AdCMVtk. Moreover, any toxic effect for rat normal tissues was not revealed after intravenous injections with Ad2 x TGtk and intraperitoneal administrations with GCV in vivo, whereas severe liver damages were observed after treatment with AdCMVtk/GCV. These data indicate a beneficial effect of Ad2 x TGtk for tissue-specific gene therapy for TG-producing thyroid carcinomas without toxicity for normal tissues.

Suicide gene therapy using thymidine kinase/ganciclovir (Tk/GCV) yields highly variable results, in vitro and in vivo. To determine the reasons for such variations, we examined cellular mechanisms mediating its cytotoxicity in view of their interaction with adenoviral vectors (Ad) used for gene delivery. Here we report that the presence of adenovirus early region 4 (AdE4)-encoded viral proteins significantly decreases toxicity of Tk/GCV. The E4 region-encoded proteins exerted this effect when found on the adenoviral delivery vector and when provided in trans in Tk retrovirally transduced cells. The apoptotic response was assessed in GCV-treated cells. The decrease in toxicity caused by AdE4 proteins was not correlated with apoptotic response, as measured by internucleosomal DNA degradation and TUNEL assays. Our results indicate that apoptosis is not the only mechanism of Tk/GCV-induced cell death and that other mechanisms equally important in determining the success of such a gene therapy strategy should be considered when optimizing treatment conditions.

Nasopharyngeal carcinoma (NPC) is a human cancer of epithelial cell origin. Infection by Epstein-Barr virus has been shown to be closely associated with this tumor. Recent studies have indicated that another common epitheliotropic virus, human papillomavirus (HPV), is also found in a significant number of NPC cases. In this study, we evaluated the feasibility of using the HPV regulatory long control region (LCR) to drive the expression of the thymidine kinase (tk) gene to achieve chemosensitivity for gene therapeutic treatment of NPC. Testing HPV-11-LCR-tk constructs in NPC cell lines in the presence of ganciclovir (GCV) led to 50-60% cell death of transfected cells. The therapeutic efficacy was further tested in an in vivo model using nude mice transplanted with tumors derived from transfected NPC cells. Injection of 50 mg/kg body weight GCV twice daily for 14 days resulted in visually complete regression of the transplanted NPC tumor loads within 20 days after GCV treatment. Taken together, results from this pilot study indicate the feasibility of the development of a gene therapeutic protocol based on the chemosensitive gene constructs described in this paper., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)

After allogeneic hematopoietic stem cell transplantation (HSCT), mature transplanted T cells play a major role in restoration of the immune system. However, they can also induce a life-threatening complication: graft-versus-host disease (GVHD). Suicide gene therapy of GVHD aims to selectively eliminate alloreactive T cells mediating GVHD while sparing nonalloreactive T cells that should contribute to immune reconstitution. It was demonstrated previously that treatment with ganciclovir (GCV) can control GVHD in mice by killing donor T cells engineered to express the thymidine kinase (TK) suicide gene. TK allows phosphorylation of nontoxic GCV into triphosphate GCV, which is selectively toxic for dividing cells. Thus, in the TK-GCV system, the specificity of cell killing depends on the cycling status of TK T cells rather than allogeneic recognition. This is a potential drawback because in recipients of lymphopenic allogeneic HSCT, alloreactive and homeostatic signals drive the proliferation of donor T cells. It is shown here that the onset of alloreactive T-cell division occurs earlier than that of nonalloreactive T cells, thus establishing a time frame for GCV administration. A 7-day GCV treatment initiated at the time of HSCT allowed efficient prevention of GVHD, while sparing a pool of nondividing donor TK T cells. These cells later expanded and contributed to the replenishment of the recipient immune system with a diversified T-cell receptor repertoire. These results provide a rationale for designing the therapeutic scheme when using TK-GCV suicide gene therapy in allogeneic HSCT.

The presence of Epstein-Barr virus (EBV) in the tumor cells of some EBV-associated malignancies may facilitate selective killing of these tumor cells. We show that treatment of an EBV(+) Burkitt's lymphoma cell line with 5-azacytidine led to a dose-dependent induction of EBV lytic antigen expression, including expression of the viral thymidine kinase (TK) and phosphotransferase (PT). Azacytidine treatment for 24 h modestly sensitized the cell line to all nucleosides tested. To better characterize EBV TK with regard to various nucleoside analogues, we expressed EBV TK in stable cell clones. Two EBV TK-expressing clones were moderately sensitive to high doses of acyclovir and penciclovir (PCV) (62.5 to 500 microM) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 microM) compared to a control clone and were shown to phosphorylate GCV. Similar experiments in a transient overexpression system showed more killing of cells transfected with the EBV TK expression vector than of cells transfected with the control mutant vector (50 microM GCV for 4 days). A putative PT was also studied in the transient transfection system and appeared similar to the TK in phosphorylating GCV and conferring sensitivity to GCV, but not in BVdU- or PCV-mediated cell killing. Induction of EBV kinases in combination with agents such as GCV merits further evaluation as an alternative strategy to gene therapy for selective killing of EBV-infected cells.

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk) / ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F / K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F / K20), and compared the cytopathic effect of Adv-TK-F / K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F / K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F / K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F / K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F / K20 may be a promising therapeutic modality for the treatment of gliomas.

Liver-directed gene therapy is a promising alternative for the treatment of various liver diseases. Pseudotyped (VSV-G) retroviruses can be produced in high titres which is essential to overcome the problem of low gene transfer efficiency detected previously with first generation Moloney murine (MMLV) retroviruses and plasmid vectors. We compared the lacZ gene transfer efficiency of MMLV retroviruses and VSV-G retroviruses in Watanabe heritable hyperlipidaemic rabbit liver using an intraportal administration route. Hepatocyte proliferation was stimulated by a partial (10%) liver resection and a thymidine kinase-ganciclovir treatment. We also studied the safety of the gene transfer by clinical chemistry, tissue pathology and PCR analysis of lung, kidney, spleen and gonads. Gene transfer efficiency with the VSV-G retrovirus was significantly higher than with the traditional MMLV-based retrovirus (9.5+/-5.26 vs 0.21+/-0.10 positive hepatocytes mm(-2), P<0.05). After a 12-month follow-up period no lacZ expression was detected in liver samples. No transgene was detected in plasma or in lung, kidney, spleen and gonads by PCR analysis 7 days after gene transfer. Transient increases were found in plasma c-reactive protein, aspartyl aminotransferase and alanine aminotransferase levels shortly after the operation with both types of retroviruses. VSV-G retrovirus was well tolerated and may become an efficient new tool in liver gene therapy. The absence of transgene in systemic circulation or in extrahepatic tissues including gonads is an important safety feature required for in vivo gene therapy., (Copyright 1999 Academic Press.)

An established principle of antineoplastic chemotherapy is that multidrug regimens are generally superior to single-agent therapy. This prompted us to elucidate whether the topoisomerase inhibitor topotecan (TPT) could enhance the efficacy of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system for the treatment of cancer. We assessed the interaction between these two treatments in murine MC38 and human HT-29 colon carcinoma cell lines that were genetically modified to constitutively express HSV-tk, sensitizing them to GCV. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (Adv. Enzyme Regul. 1984; 22:27-55). Subcutaneous tumor models, using the same cell lines in C57BL/6 and athymic nude mice, respectively, demonstrated that the combination of GCV and TPT resulted in statistically significant enhanced survival relative to single-agent treatment. In addition, nude mice bearing HT-29 tumor xenografts were treated with an Ad5 E1b Mr 55,000 attenuated replication-competent adenovirus expressing HSV-tk (Ad.TK(RC)) either alone or in combination with GCV and/or TPT. These experiments demonstrated that Ad.TK(RC) followed by GCV and TPT was more efficacious than any other treatment tested. Our results suggest that for antineoplastic therapy, molecular chemotherapy based on the HSV-tk/GCV system combined with traditional chemotherapy is a logical and practical future direction to pursue. Suicide gene therapy is the approach whereby genetically altering a cell makes it susceptible to an otherwise relatively nontoxic prodrug. By this approach it is possible to achieve relatively high concentrations of the toxic metabolites in the transduced cells while maintaining low systemic levels of the active drug. The most often used metabolic suicide gene transfer system is the HSV-tk/GCV paradigm, which is currently being used in cancer therapy or as a safety modality. The low response rate observed in the early clinical HSV-tk cancer trials may be due to failure in achieving adequate transduction efficiency and/or prodrug concentration within the tumor. The combination of such suicide gene prodrug systems with adjunctive drugs resulting in synergistic cytotoxicity might improve the clinical utility of this approach.

Objective: To compare the killing effects of herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system versus cytosine deaminase (CD)/5-fluorocytosine (5-FC) system on human pancreatic carcinoma PC-2 cells., Methods: Recombinant retroviral vectors expressing HSV-TK and CD genes were constructed and transduced into pancreatic carcinoma cell line. Prodrug sensitivity and IC50 values (concentration of drug at which cell growth is inhibited by 50%) of the transduced cells were measured by MTT method. The bystander effects in the two systems were also compared., Results: The IC50 value of HSV-TK-transduced cells to GCV was (1.06 +/- 0.12) micromol/L and 558-fold lower than parental PC-2 cells, while the IC50 value of CD-transduced cells to 5-FC.00 was (33.00 + 0.95) micromol/L and 258-fold lower than parental PC-2 cells. Mixed cells containing 10% of transduced cells showed 39% and 50.3% growth inhibition in TK/GCV and CD/5-FC systems respectively., Conclusion: Both HSV-TK/GCV and CD/5-FC systems showed effective antitumor activity in vitro to pancreatic carcinoma PC-2 cells. The therapeutic index of HSV-TK/GCV system is higher, but its bystander effect is lower than that of CD/5-FC system.

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV +/- apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60-70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells.

Pulmonary metastases are the main cause of death of patients with several types of cancer, including osteosarcoma, renal cell carcinoma, malignant melanoma, and breast cancer. Previously, we demonstrated that intralesional injection of the recombinant adenovirus (Ad) vector containing the herpes simplex virus thymidine kinase (TK) gene driven by an osteocalcin (OC) promoter (Ad-OC-TK) effectively suppressed the growth of osteosarcoma cells in vitro and tumors in vivo in a tumor-specific manner when supplemented with the prodrug acyclovir (ACV). In this communication, we studied the potential efficacy of the treatment of osteosarcoma pulmonary metastases with a systemic delivery route of Ad-OC-TK supplemented with ACV. We established osteosarcoma lung metastases in nude mice by the intravenous injection of rat osteosarcoma cells, ROS 17/2.8. These cells colonized and formed tumor nodules within 1 week in the lungs of nude mice. Whereas systemic delivery of a recombinant Ad vector containing the Escherichia coli beta-galactosidase (beta-gal) gene driven by a Rous sarcoma virus universal promoter (Ad-RSV-beta-gal) resulted in the nonspecific expression of beta-gal activity in the lung parenchyma, Ad-OC-beta-gal administration resulted in specific beta-gal expression in tumor cells deposited in the lung. When nude mice bearing ROS 17/2.8 lung tumors were treated with systemic Ad-OC-TK through tail vein administration, subsequent intraperitoneal ACV treatment significantly decreased the number of tumor nodules (P < .0001) and the net lung wet weight (P = .0005) while significantly increasing (.005 < P < .01) the survival of animals, when compared with untreated and Ad-OC-TK- or ACV-treated control groups. These results suggest that Ad-OC-TK/ACV may be used as a systemic therapy for the treatment of osteosarcoma lung metastasis.

Objective: To establish the replicated-deficient recombinant adenovirus-mediated thymidine kinase/acyclovir (Adtk/ACV) system and to evaluate its suicide effect on rat C6 brain gliomas in vitro and in vivo., Methods: The plasmid pAdtk and pJM17 were co-infected into 293 cells (adenovector packaging cells) and the results were identified by polymerase chain reaction (PCR) assay. After the glioma C6 cells were transduced by Adtk at different multiplicity of infection (MOI) and exposed to different concentrations of ACV or gancyclovir (GCV), the cell survival curves were studied, and the cell surface was observed with scanning electronic microscopy (SEM). C6 gliomas in vivo at different inoculation days were injected with Adtk intratumorally and ACV intraperitoneally daily, and the survival duration and histologic changes of the rats were observed., Results: The infectious Adtk virions had a suicide effect which was enhanced with the increase in MOIs of Adtk and ACV doses along with bystander effect. Under scanning electronic microscope, special pathologic changes were observed. ACV had a similar effect as GCV but a higher dose was used. The survival duration in day 3, day 6 and day 8 groups exceeded 90 days, and the rats in day 10 group survived 28.5 +/- 4.6 days, but the survival duration in untreated C6 group and AdLacZ/ACV (adenovirus-mediated LacZ/ACV) treated group were 16.8 +/- 3.1 and 14.0 +/- 2.2 days respectively., Conclusion: Adtk/ACV system can effectively kill the rat brain gliomas in vitro and in vivo.

Background: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects., Methods: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied., Results and Conclusions: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.

Peritoneal dissemination is one of the most common complications of the malignancies of the digestive system, such as gastric or pancreatic cancers. Yet, no effective therapy has been established so far to alleviate this devastating and often fatal end-stage condition. Here we describe a novel approach of intraperitoneal (i.p.) lipofection of a suicidal gene to the pancreatic cancer cells in a mouse peritoneal dissemination model. A human pancreatic cancer cell line, PSN-1, was inoculated into the peritoneal cavity of nude mice. Eight days later, a herpes simplex virus thymidine kinase (HSV-TK) gene expression plasmid under a potent hybrid promoter CAG was injected as a DNA-lipopolyamine complex. Ganciclovir (GCV) was then administered for 8 days, and the mice were examined for tumor development at the 24th day after the tumor inoculation. Although all 24 control mice showed macroscopic peritoneal dissemination and solid tumors on the pancreas, 8 of the 14 mice treated with HSV-TK and GCV were free of tumors, and only a few small tumors were observed in the remaining 6 mice. Treatment-related toxicity was not observed. The semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis suggested that the HSV-TK transgene was expressed in about 10% of tumor cells but not in the normal pancreas or in the small intestine. When the lacZ gene was transduced in place of the HSV-TK gene, the blue-stained cells were identified only in tumor nodules and not in normal organs. This preclinical study suggests the therapeutic feasibility of the i.p. lipofection-based suicidal gene/prodrug strategy for peritoneal dissemination of pancreatic cancer.

Transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene into cancer cells followed by treatment with ganciclovir (GCV) is an attractive strategy of cancer gene transfer. HSV-TK-transduced cells are efficiently killed by the direct cytotoxic effect of GCV-triphosphate, which is generated by HSV-TK from GCV. In addition, the bystander effect kills adjacent nontransduced tumor cells, although this mechanism is not fully understood. We addressed whether the systemic immune response is involved in tumor regression in vivo. Renca cells, from a renal carcinoma cell line transduced with a retroviral vector bearing the HSV-TK gene driven by the cytomegalovirus early promoter, were inoculated into BALB/c mice. After complete regression of inoculated tumors with GCV treatment, the animals were challenged with nontransduced tumor cells. Rejection or significant growth inhibition of challenged tumor cells was observed. In these animals, tumor-specific cytotoxic T cells were efficiently induced. CD8+ cells appeared to be a main component in this cytotoxic T cell fraction. Expression of class I major histocompatibility complex antigens increased in HSV-TK+ cells treated with GCV. These results suggest that suicide gene therapy may be useful not only for short-term tumor regression mediated by direct cell killing and bystander effect, but also, due to the vaccination effect, may be an aid in long-term tumor regression and prevention of recurrence.