Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTACTM) further expands the repertoire of genetic tools for conditional interrogation of cellular functions.

Noninvasive theranostic imaging of HSV-TK/GCV suicide gene therapy in liver cancer by folate-targeted quantum dot-based liposomes.

Theranostics is emerging as a popular strategy for cancer therapy; thanks to the development of nanotechnology. In this work, we have combined an HSV-TK/GCV suicide gene system and near-infrared quantum dots, as the former is quite effective in liver cancer treatment and the latter facilitates tumor imaging. A folate-modified theranostic liposome (FL/QD-TK) was developed, which is composed of an HSV-TK suicide gene covalently coupling with near-infrared fluorescent CdSeTe/ZnS core/shell quantum dots. The liver cancer-targeting and biosafety of FL/QD-TK were studied in vitro and in vivo. FL/QD-TK exhibited highly specific tumor imaging and strong inhibition of the folate receptor-overexpressed Bel-7402 mouse xenografts without systematic toxicity. This study may shed light on gene delivery and targeted cancer therapy.

The cell death and DNA damages caused by the Tet-On regulating HSV-tk/GCV suicide gene system in MCF-7 cells.

Ganciclovir (GCV) affects the molecular mechanism of cell death and DNA damage by the rAAV (recombinant adeno-associated virus)-mediated Tet-On/HSV-tk/GCV suicide gene system in human breast cancer cell line MCF-7. A rAAV/TRE/Tet-On/HSV-tk combining a Tet-On regulating system and a suicide gene HSV-tk was used to transfect human breast cancer cell line MCF-7, and therapeutic effects on this system were studied. Afterwards, we used RT-PCR, western blotting, and a modified comet-assay to explore the potential mechanism of the HSV-tk/GCV suicide gene system in breast cancer treatments. MTT assay has shown that the cell number of GCV+rAAV+Dox group was significantly decreased compared with that of other groups after treatment and flow cytometric analysis detected that there was a substantial increase of S phase cells in this group, which means the HSV-tk/GCV suicide gene system probably works on the cell cycle. RT-PCR detected the expression level of p21 increased and PCNA had an opposite trend. Western blotting detected the protein expression of p21 and p53 increased and PCNA, CDK1, cyclin B decreased in GCV+rAAV+Dox group. The modified comet-assay shown that the very small extra fragments generated by the GCV+rAAV+Dox group treatment are visible as a small cloud extending from the comet in the direction of electrophoresis. The therapeutic mechanism of the HSV-tk/GCV suicide gene system on human breast cancer cell line MCF-7 is probably by upregulating the expression of p21 through a p53-dependent DNA damage signalling pathway, leading the decrease of protein expression of PCNA, cyclin B, CDK1 in MCF-7 cells and promoting the cell cycle arrest at G1/S phase. In summary, the HSV-tk/GCV suicide gene system arouses the death of MCF-7 cells from blocking the cell cycle and DNA damage.

Targeted suicide gene therapy for glioma using human embryonic stem cell-derived neural stem cells genetically modified by baculoviral vectors.

Tumor-tropic neural stem cells (NSCs) can be used in the Trojan horse approach as cellular vehicles for targeted delivery of therapeutic agents to distant tumor sites. To realize this cancer therapy potential, it is important to have a renewable source to generate large quantities of uniform human NSCs. Here, we reported that NSCs derived from HES1 human embryonic stem cell line were capable of migrating into intracranial glioma xenografts after systemic injection or after intracranial injection at a site distant from the tumor. To test whether the HES1-derived NSCs can be used for cancer gene therapy, we used a baculoviral vector to introduce the herpes simplex virus thymidine kinase suicide gene into the cells and demonstrated that baculovirus-mediated transgene expression may last for at least 3 weeks in NSCs. After being injected into the cerebral hemisphere opposite the tumor site and in the presence of ganciclovir, NSCs expressing the suicide gene were able to inhibit the growth of human glioma xenografts and prolong survival of tumor-bearing mice. Our findings suggest that human embryonic stem cells could potentially serve as a clinically viable source for production of cellular vehicles suitable for targeted anticancer gene therapy.

All-trans retinoic acid enhances bystander effect of suicide-gene therapy against medulloblastomas.

In our previous study we evaluated the antitumor effect of herpes simplex virus-thymidine kinase gene (HSV-tk) on human medulloblastomas (MBs) in a therapeutic delivery system using the immortalized neural stem cell (NSC) line C17.2. However, our findings indicated that the bystander effect between C17.2tk and Daoy MB cells was weak compared to the bystander effect between NSCtk and C6 glioma cells. Gap junction intercellular communication (GJIC) is the main mechanism mediating the bystander effect in HSV-tk gene therapy. All-trans retinoic acid (ATRA) has been shown to up-regulate the expression of Connexin43 and GJIC. In this study we investigated the synergistic effect of ATRA and HSV-tk gene therapy in the treatment of MBs. We found that the expression of Connexin43 in Daoy cells was significantly increased when cells were exposed to 3µmol/l of ATRA (P<0.05). After co-culturing C17.2tk cells with Daoy cells at different ratios ranging from 1:1 to 1:16, ATRA significantly increased the bystander anti-tumor effect compared to ATRA-untreated cells (P<0.05). In intracranial co-implantation experiments, mice co-implanted with C17.2tk/Daoy cells and treated with a combination of ATRA and GCV had significantly smaller tumors compared to the animals treated with GCV alone (P<0.05). Together, our results show that ATRA enhanced the tumoricidal effect in HSVtk/GCV suicide gene therapy against Daoy MB cells by strengthening the bystander effect in vitro and in vivo.

Cancer cell targeting with mouse TERT-specific group I intron of Tetrahymena thermophila.

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

Radiosensitization and anti-tumour effects of cytosine deaminase and thymidine kinase fusion suicide gene in human adenoid cystic carcinoma cells.

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) can convert innocuous prodrugs into cytotoxic metabolites and are being investigated for use in gene therapy for cancer. Human adenoid cystic carcinoma (ACC-2) cells transduced with a CD/HSV-TK fusion gene (ACC-2/CD-TK cells) were found to be more sensitive to radiation than ACC-2 cells when exposed to 5-fluorocytosine (5-FC; 40 microg/ml) plus ganciclovir (0.1 microg/ml) for 48 h before irradiation. Analysis of radiation survival curves for cells exposed to 5-FC plus ganciclovir before irradiation showed that ACC-2 cells had a higher capacity for sublethal damage repair (D(q) value) and greater cellular radiosensitivity (D(0) value) than ACC-2/CD-TK cells. Colony formation rate after 2 Gy of irradiation was significantly greater for ACC-2 than for ACC-2/CD-TK cells when cells were treated with 5-FC plus ganciclovir before irradiation. This study, therefore, indicates that addition of radiation might substantially improve the therapeutic potential of CD-TK fusion gene therapy of human adenoid cystic carcinomas.

Novel caspase-suicide proteins for tamoxifen-inducible apoptosis.

Taking advantage of a mutant estrogen receptor ligand binding domain (ER(T2)), we developed novel Caspase fusion proteins for inducible apoptosis. We show that Caspase-ER(T2) fusion proteins become specifically activated by the synthetic ligand 4-OH- tamoxifen and rapidly induce apoptotic cell death in human, murine, and zebrafish cells. This novel tool for targeted cell ablation greatly facilitates the generation of disease models as well as developmental and regeneration studies in model organisms.

Suicide gene therapy with adenoviral delivery of HSV-tK gene for patients with local recurrence of prostate cancer after hormonal therapy.

We conducted a Phase I study of in situ herpes simplex virus thymidine kinase (HSV-tk) plus ganciclovir (GCV) gene therapy, which was approved by the Japanese government as the first prostate cancer gene therapy trial. Major inclusion criteria were local recurrence of prostate cancer after hormonal therapy and no metastasis. Adv.HSV-tk was injected directly into the prostate in escalating doses from 10(9) to 10(10) infection units, followed by intravenous administration of GCV for 14 days. Eight patients received nine courses of this gene therapy. The detection of vector DNA in blood/urine was only transient and no remarkable adverse events were observed in any patient. With regard to clinical response, significant prolongation of the median serum prostate-specific antigen (PSA) doubling time from 2.9 to 6.2 months (P = 0.041) was detected. In five patients (six injections), a clear decrease of PSA values was observed. One patient showed repeated clinical response after repeated injections. Serum cytokine analysis showed no notable changes after treatment. Fluorescence-activated cell sorting analysis also showed no influence on phenotypic distribution in peripheral blood samples, except for an increasing trend of CD8(+)/HLA-DR(+) after therapy. This study confirmed the safety profile and possibility of clinical response at the surrogate marker level in a clinical trial of HSV-tk gene therapy for hormone-refractory prostate cancer.

Mechanisms of thymidine kinase/ganciclovir and cytosine deaminase/ 5-fluorocytosine suicide gene therapy-induced cell death in glioma cells.

Suicide gene transfer using thymidine kinase (TK) and ganciclovir (GCV) treatment or the cytosine deaminase (CD)/5-fluorocytosine (5-FC) system represents the most widely used approach for gene therapy of cancer. However, molecular pathways and resistance mechanisms remain controversial for GCV-mediated cytotoxicity, and are virtually unknown for the CD/5-FC system. Here, we elucidated some of the cellular pathways in glioma cell lines that were transduced to express the TK or CD gene. In wild-type p53-expressing U87 cells, exposure to GCV and 5-FC resulted in a weak p53 response, although apoptosis was efficiently induced. Cell death triggered by GCV and 5-FC was independent of death receptors, but accompanied by mitochondrial alterations. Whereas expression of Bax remained unaffected, in particular, GCV and also 5-FC caused a decline in the level of Bcl-2. Similar findings were obtained in 9L and T98G glioma cells that express mutant p53, and also underwent mitochondrial apoptosis in both the TK/GCV and CD/5-FC system. Upon treatment of 9L cells with 5-FC, Bcl-xL expression slowly declined, whereas exposure to GCV resulted in the rapid proapoptotic phosphorylation of Bcl-xL. These data suggest that TK/GCV- and CD/5-FC-induced apoptosis does neither require p53 nor death receptors, but converges at a mitochondrial pathway triggered by different mechanisms of modulation of Bcl-2 proteins.

The gene suicide system Ntr/CB1954 causes ablation of differentiated 3T3L1 adipocytes by apoptosis.

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss.

Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene.

Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70-95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.

"Suicide" gene therapy of breast cancer cells is only cytostatic in vitro but anti-tumoral in vivo on breast MCF7-ras tumor.

Gene therapy with Herpes Simplex Virus thymidine kinase gene (HSV-tk) is effective in various tumor models in vitro and in vivo. We compared the efficacy of the HSV-tk gene therapy in vitro and in vivo in MCF-7 and MCF7-ras cells which form tumor in athymic mice. After viral infection, cells were treated with GCV (Ganciclovir) and live cells were counted. The in vitro treatment significantly inhibited cell growth but did not induce early and late apoptosis, measured, respectively, by annexin or by propidium iodide staining and a significant cell death. The HSV-tk/GCV treatment of MCF7-ras tumor in athymic mice showed a significant inhibition of tumor development until 60 days post-treatment. Some mice showed a complete tumor eradication without tumor regrowth after the end of treatment. In conclusion, we demonstrated that the HSV-tk/GCV system is not very efficient in vitro, but very efficient in vivo in our animal breast cancer model.

The gene suicide system NTR/CB1954 causes ablation of differentiated 3T3L1 adipocytes by apoptosis

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss.