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Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

ABSTRACT: The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas.

SUBMITTER: Khan Z

PROVIDER: S-EPMC2940637 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

Khan Zahidul Z Knecht Wolfgang W Willer Mette M Rozpedowska Elzbieta E Kristoffersen Peter P Clausen Anders Ranegaard AR Munch-Petersen Birgitte B Almqvist Per M PM Gojkovic Zoran Z Piskur Jure J Ekström Tomas J TJ

Neuro-oncology 20100213 6

The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymati ...[more]

PMID: 20154339

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Project description:The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant 'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Project description:BackgroundHuman mesenchymal stem cell (MSC)-based tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene delivery is regarded as an effective treatment for glioblastoma (GBM). However, adverse-free target site homing of the delivery vehicles to the tumor microsatellite nests is challenging, leading to erroneously sustained released of this suicide protein into the normal brain parenchyma; therefore, limiting off-target cytotoxicity and controlled expression of the suicide inductor is a prerequisite for the safe use of therapeutic stem cells.MethodsUtilizing the intrinsic expression profile of GBM and its elevated expression of TGF-β relative to normal brain tissue, we sought to engineer human adipose-derived MSCs (hAMSC-SBE4-TRAIL) which augment the expression of TRAIL under the trigger of TGF-β signaling. We validated our therapeutic technology in a series of functional in vitro and in vivo assays using primary patient-derived GBM models.ResultsOur current findings show that these biologic delivery vehicles have high tumor tropism efficacy and expression TRAIL gene under the trigger of TGF-β-secreting GBMs, as well as avoid unspecific TRAIL secretion into normal brain tissue. hAMSC-SBE4-TRAIL inhibited the proliferation and induced apoptosis in experimental GBMs both in vitro and in vivo. In addition, our improved platform of engineered MSCs significantly decreased the tumor volume and prolonged survival time in a murine model of GBM.ConclusionsOur results on the controlled release of suicide inductor TRAIL by exploiting an endogenous tumor signaling pathway demonstrate a significant improvement for the clinical utility of stem cell-mediated gene delivery to treat brain cancers. Harvesting immune-compatible MSCs from patients' fat by minimally invasive procedures further highlights the clinical potential of this approach in the vision of applicability in a personalized manner. The hAMSC-SBE4-TRAIL exhibit great curative efficacy and are a promising cell-based treatment option for GBM to be validated in clinical exploration.

Project description: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.

Dataset Information

Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

Publications

Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

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