C1300 Neuroblastoma Cells Research Articles

Comparison of Cytotoxic and Ototoxic Effects of Lipoplatin and Cisplatin in Neuroblastoma In Vivo Tumor Model.

Background:This study aimed to compare the cytotoxic, cytostatic, and ototoxic effects of lipoplatin compared to cisplatin application in the subcutaneous xenograft nude mouse neuroblastoma tumor model.Methods:In this study, C1300 neuroblastoma cells were administered subcutaneously to 21 male nude mice. When the tumor reached 150 mm3 diameter, mice were randomized into 3 groups. Saline, cisplatin, and lipoplatin were given intraperitoneally. The auditory function tests were performed before administration and 72 hours after administration. Mice were sacrificed and the tumor and cochlea were removed after 72 hours. Histopathologic evaluation of necrosis and apoptosis was determined by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Cyclooxygenase 2, superoxide dismutase 2, and inducible nitric oxide synthase levels were determined by immunohistochemistry in tissue samples.Results:Apoptosis and necrosis rates were higher in lipoplatin group than in cisplatin group (P = .035 and P = .010, respectively) in tumor tissue. In the spiral ganglion, apoptosis and necrosis were lower in the lipoplatin group than in cisplatin group (P = .002 and P = .002, respectively). Cyclooxygenase 2 pattern in the cochlea was positive in both control and lipoplatin group and negative in cisplatin group (P = .001). Superoxide dismutase 2 and inducible nitric oxide synthase 2 protein expressions showed no difference between groups. The auditory functions were similar to baseline values and had a better threshold value in lipoplatin group than cisplatin group.Conclusion:For the treatment of neuroblastoma, the use of lipoplatin seems to be beneficial in reducing side effects of cisplatin. We recommend that the mechanism of these properties of lipoplatin should be evaluated in further studies.

Isometric Retraction and Invisible Nerve Cell Processes

Many physiological studies in recent years on stress and hibernation have noted an unusual morphological phenomenon consisting of the rapid disappearance and restoration of the apical dendrites of pyramidal neurons in the hippocampus, prefrontal cortex, and other parts of the brain. The present article seeks to explain this on the basis of morphological analysis of the natural elastic properties of the neuroplasm and the kinetics of partially preserved processed of living isolated neurons. Movement of neuroplasm in processes has been demonstrated with a bidirectional flow. A new physiological phenomenon is described – the isometric retraction of nerve cell processes, in which flows of neuroplasm are in opposite directions, leading to marked thinning of the middle parts and thickening of both ends. It is suggested that extreme thinning of processes can shrink them to submicroscopic dimensions, such that they become invisible under the light microscope. Repeated reversible “disappearance” and “reappearance” of processes has been demonstrated in neuron cultures and C-1300 neuroblastoma cells. Decreases in process diameter to the limits of visibility have been demonstrated using natural stretching of processes as an example. This same effect is seen in areas between reversible varicosities along processes. These areas become extremely thin and then invisible. By becoming thinner, the processes are able to become much longer. This review of the existing literature and our own data leads to the suggestion that the phenomenon of disappearing apical dendrites can be explained in terms of their isometric retraction, leading to the development of “invisible processes.”

Identification of the Cluster Control Region for the Protocadherin-β Genes Located beyond the Protocadherin-γ Cluster

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

Testosterone attenuates morpho‐functional alterations by 2‐methoxyestradiol exposure and induces differentiation in C6 cells

2-Methoxyestradiol (2ME) is a cytotoxic drug that interacts with tubulin and alters microtubule dynamics. It has been reported that testosterone (T) has a neuroprotective effect against oxidative stress and induces differentiation in mouse C1300 neuroblastoma cells. Here, we investigated the ability of T to attenuate the cytotoxic effects of 2ME and to induce cell differentiation in an immortalized rat glial cell line, known as C6. C6 cells were exposed for 5 days to 5 µM 2ME, 50 nM T, or both. We evaluated the morphological changes, growth rate, vitality, catalase activity, and glial fibrillary acidic protein (GFAP) immunoreactivity in control and treated C6 cells. Western blot analyses were used to quantify expression of tyrosinated tubulin (Tyr-Tub), acetylated tubulin (Acet-Tub), total α-tubulin (TOT-Tub), and GFAP. After 2ME exposure, the cells displayed a globular, shrunken shape, and retraction or absence of cytoplasmic processes; moreover, 2ME treatment significantly decreased cell growth, cell viability, catalase activity, and expression of both Tyr-Tub and Acet-Tub. However, when T was added, the cells exhibited a glial-like shape, elongated cell processes, and enhanced cell growth, cell vitality, catalase activity, and GFAP immunoreactivity. Densitometric values of Tyr-Tub, Acet-Tub, and GFAP increased significantly when T was present, while Tot-Tub values were unaltered. These results indicate that, in C6 cells, T: (i) attenuated the morpho-functional changes caused by 2ME exposure; (ii) induced glial differentiation; and (iii) exerted a direct action on the microtubule system.

Prickle promotes neurite outgrowth via the Dishevelled dependent pathway in C1300 cells

Murine Prickle1 and Prickle2 belong to the planar cell polarity genes. Prickle2 but not Prickle1 gene expression was induced in C1300 neuroblastoma cells during neurite-like process formation induced by retinoic acid (RA). Over-expression of Prickle1 or Prickle2 in C1300 cells induced striking neurite-like process formation in the absence of RA. Since Prickle binds to Dishevelled (Dvl) to induce its degradation in Drosophila, we examined the participation of Dvl protein in the neurite-like process formation of C1300 cells. Upon induction of the neurite-like process formation, the amount of Dvl1 protein decreased. Prickle1 and Prickle2 could associate with Dvl1 and over-expression of Prickle1 or Prickle2 resulted in the reduction of Dvl1 protein in C1300 cells. Furthermore, over-expression of Dvl1 in C1300 cells prevented the neurite-like process formation induced by Prickle1 or Prickle2 over-expression. Thus, Prickle1 and Prickle2 promote neurite-like process formation of C1300 cells via the Dvl1 dependent mechanism.

D-glucose induces microtubular changes in C1300 neuroblastoma cell line through the incorporation of 3-nitro-L-tyrosine into tubulin.

The microtubular network of neurons is involved in several functions such as formation and tropism of cellular processes, cell division and intracellular transport. A lot of evidences testify that the microtubular network of neurons can be impaired by oxidative stress. A condition of oxidative stress is often possible when D-glucose overloads its metabolic pathway, resulting in an increase in reactive oxygen species and subsequent neurological disorders. The aim of this work was to check in undifferentiated mouse neuroblastoma cells (C1300) the possible oxidative effects of D-glucose on microtubules. Using a concentration of 110mM D-glucose, cell morphology, growth rate, viability and catalase activity were seriously altered. Noteworthy, an increase in 3-nitro-L-tyrosine and a downregulation of tubulins was found in D-glucose-exposed cells, whereas another cytoskeletal proteins, namely actin, did not show any changes. In conclusion, microtubular network can be impaired by D-glucose through specific nitrosative effects, suggesting a possible mechanism at the basis of hyperglycemia-induced neuronal damage.

Neuroprotective effect of ultra-low doses of antibodies against S100 protein in neuroblastoma culture during oxygen and glucose deprivation

Antibodies against S100 protein applied in high and ultra-high dilutions possess neuroprotective activity and maintain survival of neuroblastoma C-1300 cells under conditions of oxygen and glucose deprivation. The examined antibody preparations stimulated differentiation in neuroblastoma culture thereby demonstrating pronounced neurotrophic activity.

New Neuritogenic Steroid Glycosides from the Vietnamese Starfish Linckia Laevigata

Two new steroid glycosides, linckosides L1 (1) and L2 (2), were isolated, along with the previously known echinasteroside C (3) from the ethanolic extract of the Vietnamese blue starfish, Linckia laevigata. The structures of the new compounds were elucidated by spectroscopic methods (mainly 2D NMR) and chemical transformations. Lifetime observations and analyses of silver impregnated preparations on the culture of neuroblastoma C-1300 cells showed that glycosides 1, 2, and 3 are capable of inducing neuronal differentiation similar to that of neurotrophins and of enhancing substantially the neuritogenic activity of NGF.

Neurotrophic effects of polyhydroxylated steroids and steroid glycosides in cultured neuroblastoma cells

The effects of steroid compounds from Pacific Ocean starfishes were studied using cultured neuroblastoma C-1300 cells. Vital observations and examination of silver-impregnated preparations showed that the test substances in a concentration of 2-10 microM stimulate differentiation and improves survival of neuroblastoma cells under adverse conditions (similarly to neurotrophins). These substances in high concentrations (20-40 microM) had no effect or exhibited cytotoxic activity. The screening test allowed us to select several compounds for further studies of neurotrophic and neuroprotective properties.

Superior Protective and Therapeutic Effects of IL-12 and IL-18 Gene-Transduced Dendritic Neuroblastoma Fusion Cells on Liver Metastasis of Murine Neuroblastoma

Fusion vaccine of dendritic cells (DCs) and tumor cells has the advantage of inducing an immune response against multiple tumor Ags, including unknown tumor Ags. Using the liver metastasis model of C1300 neuroblastoma cells, we assessed the protective and therapeutic effects of fusion cells transduced with the IL-12 gene and/or the IL-18 gene. Improving the fusion method by combining polyethylene glycol and electroporation increased loading efficiency. In the A/J mice vaccinated with fusion cells modified with the LacZ gene (fusion/LacZ), IFN-gamma production and CTL activity increased significantly compared with that of DCs/LacZ, C1300/LacZ, or a mixture of the two (mixture/LacZ). With the transduction of IL-12 and IL-18 genes into the fusion cells (fusion/IL-12/IL-18), the level of IFN-gamma increased more than five times that of other fusion groups. In addition, NK cell activity and CTL activity increased significantly compared with that of mixture/LacZ, fusion/LacZ, DC/LacZ, or C1300/LacZ. In the protective and therapeutic studies of fusion cell vaccine, mice vaccinated with fusion/LacZ, fusion/IL-12, fusion/IL-18, or fusion/IL-12/IL-18 showed a significant decrease in liver metastasis and a significant increase in survival compared with mice given a mixture/LacZ, DCs/LacZ, or C1300/LacZ. In particular, the mice receiving fusion/IL-12/IL-18 vaccine showed a complete protective effect and the highest therapeutic effects. The present study investigates the improved loading efficiency of fusion cells and suggests that the introduction of IL-12 and IL-18 genes can induce extremely strong protective and therapeutic effects on liver metastasis of neuroblastoma.

Plasma Membrane Voltage-dependent Anion Channel Mediates Antiestrogen-activated Maxi Cl– Currents in C1300 Neuroblastoma Cells

The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl- channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl- channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl- current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin beta subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl- currents, suggesting that VDAC forms the plasma membrane Maxi Cl- channel or a part thereof.

Glioma-specific cytotoxic T cells can be effectively induced by subcutaneous vaccination of irradiated wild-type tumor cells without artificial cytokine production

Effective induction of systemic antitumor immunity is a crucial step for success of immune gene therapy for intracerebral gliomas. We examined in this study the ability to induce glioma-specific cytotoxic T lymphocytes (CTL) by subcutaneous (s.c.) immunization of irradiated whole-tumor cell vaccine with or without artificial cytokine production, and also examined in vivo efficacy of the induced CTL against a rat brain tumor model with 9L gliosarcoma cells. Murine neuroblastoma C1300 cells transduced with the interleukin-2 (IL-2), IL-4 or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (C1300/IL-2, C1300/IL-4 or C1300/GM-CSF) were used as cytokine-producers. Glioma-specific CTL activity was equivalently induced in the rats vaccinated s.c. with irradiated 9L, irradiated IL-2-producing 9L cells or the mixed population of irradiated 9L and C1300/IL-2 cells, while the activity was relatively lower in the rats vaccinated with irradiated 9L cells mixed with either C1300/IL-4 or C1300/GM-CSF cells. In the rats immunized s.c. with irradiated 9L cells, intracerebral (i.c.) 9L tumors implanted together with either C1300/IL-2 or C1300/IL-4 were completely rejected. Pre-established brain tumor also could be eliminated by the s.c. immunization of irradiated 9L cells and i.c. transplantation of IL-2-producers. These results suggest that glioma-specific CTLs could be effectively induced by s.c. immunization of irradiated wild-type tumor cells without artificial cytokine production.

Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth.

Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo.

Osteopontin induces angiogenesis of murine neuroblastoma cells in mice.

Angiogenesis is an essential process for tumor growth and is regulated by tumor-derived angiogenic cytokines. Osteopontin (OPN) is one of the cytokines produced by various tumor cells and is suggested to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with vascular endothelial cell growth factor (VEGF). To provide evidence of OPN involvement in a causal role in tumor angiogenesis, we generated a stable transfectant from murine neuroblastoma C1300 cells to constitutively secrete high levels of murine OPN. The OPN mRNA expression and protein secretion were confirmed by RT-PCR and ELISA, respectively. The biological activity of secreted OPN was determined with migration assay by using human umbilical vein endothelial cells (HUVEC). Transfection with OPN gene did not increase VEGF production and did not affect gene expression of other angiogenic factors confirmed by complementary DNA macroarray system. To demonstrate the effect of OPN on tumor-induced angiogenesis in vivo, millipore chambers containing OPN-transfected or control cells were implanted to the dorsal air sac of mice. The OPN-transfected cells significantly induced neovascularization in comparison to the control cells in mice. Conclusively, these results provide direct evidence of OPN involvement in the role of tumor angiogenesis.

Okadaic acid-sensitive activation of Maxi Cl(-) channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells.

1. The regulation of Maxi Cl(-) channels by 17beta-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl(-) channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. 2. Whole-cell Maxi Cl(-) currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. 3. Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl(-) channels. The inhibitory effect of 17beta-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. 4. Current activation was unaffected by the removal of intracellular Ca(2+) and Mg(2+), but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl(-) channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. 5. Although the role of these Maxi Cl(-) channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.

Inhibitory Effect of TNP-470 on Hepatic Metastasis of Mouse Neuroblastoma

TNP-470 is a strong inhibitor of angiogenesis. The present study was designed to determine whether the angiogenesis inhibitor TNP-470 inhibits metastasis of mouse neuroblastoma cells to the liver and thus increases survival. A murine neuroblastoma cell line, C1300, and A/J mice were used in this study. First, to demonstrate the inhibitory effects of TNP-470 on angiogenesis, we quantified the area of angiogenesis on images made with SP-500 image analyzer (Olympus) 7 days after implanting a millipore chamber and compared the areas for the TNP-470-treated mice and control mice. Next, to determine the inhibitory effect of TNP-470 on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting C1300 cells (1 x 10(6)) in the spleen of the mice and compared histologic findings, sizes, and weights of the livers of treated mice and control mice 14 days after the beginning of a 7-day infusion of TNP-470 (60 mg/kg). We also compared survival rates using the Kaplan-Meier method. When the angiogenesis inhibitor TNP-470 was infused into mice that received tumor cells, the area of angiogenesis in the TNP-470-treated mice was smaller than that in the control mice (52.5 +/- 6.3 SD vs 94.1 +/- 27.6 mm(2), P < 0.001). After the same treatment in other mice, no histologic evidence of metastasis was found, whereas control mice had countless tumor cell masses. Similarly, the weight of the liver was less in TNP-470-treated mice (0.8 +/- 0.1 g vs 4.5 +/- 0.3 g, P < 0.001). Survival was longer in the TNP-470-treated mice than in controls (80% of treated mice were alive more than 60 days after treatment, whereas all control mice died by Day 20). TNP-470 inhibits metastasis of mouse C1300 neuroblastoma cells to the liver, and thus increases survival. TNP-470 inhibits metastasis by inhibiting angiogenesis.

Retroviral transfer of the ?-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for beta-galactosidase (beta-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from beta-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0-200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, beta-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation.

Retroviral transfer of the β-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for β-galactosidase (β-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from β-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0–200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, β-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation. J. Neurosci. Res. 59:265–275, 2000 © 2000 Wiley-Liss, Inc.

Inhibition of voltage-gated cationic channels in rat embryonic hypothalamic neurones and C1300 neuroblastoma cells by triphenylethylene antioestrogens

The effect of the non-steroidal antioestrogens tamoxifen and toremifene on voltage-gated cationic currents was examined in primary cultures of rat hypothalamic neurones and the C1300 mouse neuroblastoma cell line using the whole-cell patch clamp technique. When applied to the external bathing solution both tamoxifen and toremifene were able to inhibit TTX-sensitive sodium currents with IC 50 values of 1–2 μM and delayed rectifier type potassium currents (IC 50, 2–3 μM). However, only toremifene showed a significant inhibition of the I A current (IC 50 3 μM). Inhibition of voltage-gated cationic currents was significantly impaired when tamoxifen was applied in a serum-containing solution. The steroidal antioestrogen ICI 182,780 did not inhibit any of the currents at 10 μM.

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma.

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.