Murine Neuroblastoma Cells Research Articles

Sodium valproate does not augment Prpsc in murine neuroblastoma cells

Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 microM VPA. Higher VPA concentrations (240 and 600 microM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.

CAMP-induced expression of ABCA1 is associated with MAP-kinase-pathway activation

Several lines of evidence suggest that the ATP binding cassette A1 (ABCA1) is also involved in other degenerative processes such as brain neurodegeneration. Cholesterol and cAMP activate ABCA1 in a cell-specific manner. We employed a cell culture model of murine monocytes (P388) and neuroblastoma cells (N2A) and studied the differential induction of the ABCA1-gene product by modifying the cholesterol acceptor and by inhibition of the MAP-kinase pathway. Our study reveals a rise of ABCA1-expression in both N2A and P388 by cAMP. This increase is accompanied by a higher activation of the MAP-kinase-pathway. The inhibition of the MAP-kinase activation disrupts the stimulating effect of cAMP but increases the base line expression of ABCA1. Our data suggest a negative feedback between the MAP-kinase-system and ABCA1. We conclude that the interaction of the MAP-kinase pathway and the ABCA1 system might affect the function of neuronal and microglial cells in the brain.

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na + channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66 kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

Role of increased expression of the proteasome in the protective effects of sulforaphane against hydrogen peroxide-mediated cytotoxicity in murine neuroblastoma cells

The 26S proteasome is responsible for degradation of abnormal proteins and may play a role in cell survival upon oxidative stress. The indirect antioxidant sulforaphane (SFN) protects animal tissues from chemical toxicants by increasing the expression of several families of Nrf2-regulated genes. The role of induction of the 26S proteasome in cytoprotection by SFN was investigated in murine neuroblastoma Neuro2A cells. SFN enhanced the expression of the catalytic subunits of the proteasome, as well as proteasomal peptidase activities in these cells. Such treatment with SFN protected cells from hydrogen peroxide-mediated cytotoxicity in a manner dependent on proteasomal function. Inhibition of proteasome activities using pharmacological interventions significantly attenuated the protective effects of SFN against hydrogen peroxide cytotoxicity, as well as protein oxidation. Moreover, overexpression of the catalytic subunit PSMB5 enhanced proteasome function and led to elevated resistance against hydrogen peroxide toxicity and extent of protein oxidation compared to blank-plasmid-transfected cells. Pretreatment of PSMB5-overexpressing cells with SFN did not further enhance this resistance. Collectively, these results suggest that the cytoprotective effects of SFN against oxidative stress are in part due to up-regulation of the proteasome system. Therefore, inducers of proteasome expression may ameliorate the accumulation of damaged proteins associated with neurodegeneration and other diseases in whose etiologies protein oxidation plays a role.

Neuroprotective effects of pituitary adenylate cyclase–activating polypeptide (PACAP) in MPP+‐induced alteration of translational control in Neuro‐2a neuroblastoma cells

Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra. Although a variety of evidence has shown that degenerative cells have apoptotic features, the role of apoptosis in disease pathology remains controversial. The 1-methyl-4-phenylpyridinium ion (MPP(+)), a metabolite of MPTP, was recently shown to alter the expression of proteins involved in translational control. The initiation step of translational control is regulated by a cascade of phosphorylation affecting proteins of the antiapoptotic way controlled by mammalian target of rapamycin (mTOR) and of the proapoptotic way controlled by double-stranded RNA protein-dependent kinase (PKR). A study showed that MPP(+) induced an increase in eIF2alpha phosphorylation, leading to inhibition of protein synthesis. (1) to assess the effects of MPP(+) toxicity on molecular factors of PKR and mTOR signaling pathways in murine neuroblastoma cells, and (2) to examine the ability of VIP and PACAP peptides to counteract the MPP(+) toxicity. Our findings showed that MPP(+) induced phosphorylation of eIF2alpha and significantly reduced the expression of phosphorylated mTOR, p70S6K, eIF4E, and 4E-BP1, suggesting its toxicity in controlling protein synthesis. Furthermore, the VIP peptide had no effect on either the PKR or the mTOR signaling pathway. On the contrary, the PACAP 27 neuropeptide prevented MPP(+)-induced eIF2alpha phosphorylation and blocked MPP(+) toxicity in molecular factors of the mTOR pathway. And last, PACAP 27 seemed to protect Neuro-2a cells from the apoptotic process as assessed by the decreased nuclear condensation after DAPI staining. These results could open new paths of research of PACAP in PD.

Agonist selective modulation of tyrosine hydroxylase expression by cannabinoid ligands in a murine neuroblastoma cell line

Functional interactions between catecholamines and cannabinoid transmission systems could explain the influence of Delta(9)-tetrahydrocannabinol on several central activities. Hence, the presence of cannabinoid CB(1) receptors in tyrosine hydroxylase (TH) containing cells has been suggested, providing clue for a direct control of catecholamines synthesis. In the present study, we evidenced the constitutive expression of functional cannabinoid CB(1) receptors in N1E-115 neuroblastoma and reported on the use of this model to examine the influence of diverse cannabinoid ligands on TH expression. Exposure of the cells to the high-affinity agonist HU 210 (5 h) resulted in a significant decrease in TH content (pEC(50): 6.40). In contrast, no change was observed after a similar treatment with the structurally unrelated agonist CP 55,940. Besides, the use of a luciferase reporter assay revealed that these two agonists showed opposite influences on TH gene promoter activity. Thus, in cells expressing pTH-luc constructs, inhibition and induction of luciferase activity were respectively observed with HU 210 (pEC(50): 8.95) and CP 55,940 (pEC(50): 9.09). Pharmacological characterisation revealed that these reciprocal responses were both related to the specific activation of cannabinoid CB(1) receptor, suggesting an agonist-dependent modulation of distinct signalling pathways. While these data points out the possible pharmacological manipulation of TH expression by cannabinoid ligands, such approach should take into account the existence of agonist selective trafficking of cannabinoid CB(1) receptor signalling.

Tissue specific increase of the catalytic subunits of the 26S proteasome by indirect antioxidant dithiolethione in mice: Enhanced activity for degradation of abnormal protein

Decreases in the 26S proteasome are related to the toxicities of abnormal protein aggregates and may contribute to pathogenesis of degenerative diseases. Therefore, maintenance of proteasome function can be a novel strategy to protect cells against abnormal protein-mediated toxicity. In the present study, we have demonstrated the tissue specific increase of the catalytic subunits of the proteasome in mice following oral administration of 3 H-1,2-dithiole-3-thione (D3T, 0.5 mmol/kg), which functions as a cancer preventive agent in animal and human studies. Expression of the 20S catalytic core subunits PSMB5, PSMB6, and PSMB7 were increased in liver, lung, small intestine, and colon of mice at 24 h after D3T treatment. Elevated expression of proteasome catalytic subunits led to increases in proteasomal peptidase activities in these tissues. Oral administration of D3T also exerted a pharmacodynamic action in some brain regions of these mice and proteasomal peptidase activities were significantly elevated in the cerebral cortex–hippocampus. Moreover, tissue extracts from D3T-treated mice and cell lysates obtained from D3T-incubated murine neuroblastoma cells exhibited the enhanced capacity to degrade mutant human SOD1G93A protein. These results indicate that the catalytic subunits of the 26S proteasome are inducible in multiple tissues of mouse including brain by exogenous chemical treatment. Increased proteasome expression by inducers may have a role in protection/attenuation of protein aggregate-mediated disorders.

Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen

BackgroundWe have developed a cell-based vaccine that features the expression of both CD80 and CD86 on the surface of a murine neuroblastoma cell line. The cellular immunity induced by this vaccine is enhanced by treatment with antibody that interferes with T-regulatory cell (Treg) function and we report here that immunization combined with interfering with Treg function also produces a profound serological effect. Serum from mice immunized with our cell-based vaccine in the context of Treg blockade was used to screen a cDNA expression library constructed from the parental neuroblastoma tumor cell line, AGN2a.ResultsSerum from mice vaccinated in the context of Treg blockade identified a number of potentially oncogenic transcripts that may serve as important immune targets in a tumor-derived cDNA library screen. This novel approach identified far more candidates than could be seen with serum derived from vaccine-treated only, Treg-depleted only, or tumor-bearing mice. The most commonly identified tumor-associated antigen, using serum from immunized and Treg-depleted mice, was the DEK oncogene. Altered expression of the DEK oncogene has been implicated in a number of human cancers. Importantly, we were able to demonstrate that the DEK oncogene also induces a T cell response.ConclusionThe use of post-vaccine immune serum in this report differs from previous approaches where serum collected at the time of cancer onset or diagnosis and was used for tumor antigen identification. We hypothesize that the use of diagnostic serum samples may be inadequate for the clinical translation of this approach, and that identification of protective immunogenic tumor antigens may require the use of serum from post-treatment or vaccinated subjects. The identification of DEK as a tumor-associated antigen capable of eliciting a T cell response validates our experimental approach and argues for the antigens we have identified here to be evaluated as targets of effector immunity and as vaccine candidates.

Mangiferin protects against 1-methyl-4-phenylpyridinium toxicity mediated by oxidative stress in N2A cells

1-Methyl-4-phenyl-pyridine ion (MPP +), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a Parkinsonian syndrome in humans and animals, a neurotoxic effect postulated to derive from oxidative stress. We report here the first investigation of MPP +-induced oxidative stress in the murine neuroblastoma cell line N2A. Significant cell death was observed following exposure to 0.25 mM MPP +. Markers of oxidative stress included decreased intracellular levels of GSH after 48 h of exposure (85% depletion) as well as an increase in GSSG. Expression of both superoxide dismutase 1 ( sod1) and catalase ( cat) mRNA was increased, as well the activity of catalase. These cellular effects were, at least partially, reversed by treatment with the natural polyphenol mangiferin. Administration of mangiferin protected N2A cells against MPP +-induced cytotoxicity, restored the GSH content (to 60% of control levels), and down-regulated both sod1 and cat mRNA expression. Together, these results suggest that the protective effect of mangiferin in N2A cells is mediated by the quenching of reactive oxygen intermediates. Therefore, mangiferin could be a useful compound in therapies for degenerative diseases, including Parkinson's disease, in which oxidative stress plays a crucial role.

Assessment of Metal Nanoparticle Agglomeration, Uptake, and Interaction Using High-Illuminating System

In the present study, an ultrahigh-resolution system was applied as a simple and convenient technique to characterize the extent of metal nanoparticle agglomeration in solution and to visualize nanoparticle agglomeration, uptake, and surface interaction in three cell phenotypes under normal culture conditions. The experimental results demonstrated that silver (25, 80, 130 nm); aluminum (80 nm); and manganese (40 nm) particles and agglomerates were effectively internalized by rat liver cells (BRL 3A), rat alveolar macrophages (MACs), and rat neuroendocrine cells (PC-12). Individual and agglomerated nanoparticles were observed within the cells and agglomerates were observed on the cell surface membranes. The particles were initially dispersed in aqueous or physiological balanced salt solutions and agglomeration was observed using the Ultra Resolution Imaging (URI) system. Different methods, such as sonication and addition of surfactant (0.1% sodium dodecyl sulfate [SDS]) reduced agglomeration. Due to effects of SDS itself on cell viability, the surfactant could not be directly applied during cell exposure. Therefore, following addition of 0.1% SDS, the particles were washed twice with ultrapure water, which reduced agglomeration even further. Reducing the agglomeration of the nanoparticles is important for studying their uptake and in applications that benefit from individual nanoparticles such as diagnostics. In summary, this study demonstrates a simple technique to characterize the extent of nanoparticle agglomeration in solution and visualize nanoparticle (40 nm and larger) uptake and interaction with cells. Additionally, an example application of nanoparticle labeling onto the surface and neurite extensions of murine neuroblastoma cells (N2A) is presented as a potential imaging tool.

Anandamide Protects from Low Serum-induced Apoptosis via Its Degradation to Ethanolamine

Anandamide (AEA) is a lipid molecule belonging to the family of endocannabinoids. Various studies report neuroprotective activity of AEA against toxic insults, such as ischemic conditions and excitotoxicity, whereas some show that AEA has pro-apoptotic effects. Here we have shown that AEA confers a protective activity in N18TG2 murine neuroblastoma cells subjected to low serum-induced apoptosis. We have demonstrated that the protection from apoptosis by AEA is not mediated via the CB1 receptor, the CB2 receptor, or the vanilloid receptor 1. Interestingly, breakdown of AEA by fatty acid amide hydrolase is required for the protective effect of AEA. Furthermore, the ethanolamine (EA) generated in this reaction is the metabolite responsible for the protective response. The elevation in the levels of reactive oxygen species during low serum-induced apoptosis is not affected by AEA or EA. On the other hand, AEA and EA reduce caspase 3/7 activity, and AEA attenuates the cleavage of PARP-1. Taken together, our results demonstrate a role for AEA and EA in the protection against low serum-induced apoptosis.

생쥐 신경교세포 유래 신경영양인자 유도성 전사인자 (mGIF) 유전자의 유전체 구조 및 프로모터 특성 분석

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was isolated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Sp1. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

Prion Infection of Muscle Cells In Vitro

The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrP(Sc). The amount of scrapie infectivity or PrP(Sc) in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrP(Sc) in myotubes, and the C-terminal C2 polypeptide fragment of PrP(Sc) was found based on deglycosylation and PrP(Sc)-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Proteomics Analysis of the Expression of Neurogranin in Murine Neuroblastoma (Neuro-2a) Cells Reveals Its Involvement for Cell Differentiation

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Role of induction of the 26S proteasome in protective effects of sulforaphane against hydrogen peroxide‐mediated cytotoxicity in murine neuroblastoma cells

The 26S proteasome is responsible for degradation of cellular abnormal proteins and may play a role in cell survival upon oxidative stress. Indirect antioxidant sulforaphane found to protect animal tissues from chemical toxicants by increasing Nrf2-regulated antioxidative genes. Cytoprotective effects of sulforaphane through an induction of the 26S proteasome were investigated in murine neuroblastoma Neuro2A cells. Sulforaphane enhanced expression of catalytic subunits of the proteasome, as well as proteasomal peptidase activities in these cells. Treatment with sulforaphane protected cells from cytotoxicity mediated by hydrogen peroxide and these protective effects were proteasomal function dependent. Inhibition of proteasome activities using pharmacological interventions significantly attenuated protective effects of sulforaphane against hydrogen peroxide, as well as reductive effects against hydrogen peroxide induced protein oxidation. While, enhanced proteasome function obtained by overexpressing the catalytic subunit PSMB5 demonstrated an elevated resistance against hydrogen peroxide treatment with reduced levels of oxidized proteins compared to those in blank plasmid transfected cells. Collectively, these results are suggesting that cytoprotective effects of sulforaphane against oxidative stress are in part due to an induction of the proteasome system. Therefore, application of proteasome inducers may provide a potential benefit in preventing oxidative stress associated damages in neuronal cells.

Induction of 26S proteasome subunit PSMB5 by the bifunctional inducers through the Nrf2‐ARE pathway

The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the Xenobiotic Response Elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

Induction of axonal differentiation by silencing plasma membrane‐associated sialidase Neu3 in neuroblastoma cells

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.

Amphiphilic Poly(vinyl alcohol) Derivatives as Complexing Agents for Fenretinide

Poly(vinyl alcohol) (PVA) substituted with oleyl chains and tetraethyleneglycol monoethyl ether chains (TEGMEE) at 1.5% and 1% degrees of substitution respectively (mol of substituent to mol of hydroxyvinyl monomer) has previously been shown to self-assemble in water, providing aggregates selectively cytotoxic toward tumor cells vs normal cells. These polymers have also been shown to increase the long-term survival of nude mice injected with both human and murine neuroblastoma cell lines. In the present work, we changed the substitution degree of the oleyl chains on the poly(vinyl alcohol) backbone and maintained constant at 1% the degree of TEGMEE substitution. We evaluated the main physicochemical characteristics of the final polymers, their cytotoxicity toward tumor cells, and their complexing ability for hydrophobic molecules. The aim was to investigate the possibility of improving intrinsic antitumor efficacy of the polymer by changing the degree of oleyl chain substitution and further increase activity by complexation with antitumor drugs. The polymers were prepared at oleyl chain substitution degrees ranging from 0.5 to 3% (mol of substituent to mol of hydroxyvinyl monomer). The most active was again the 1.5% substituted polymer. It was further characterized by exhibiting the highest complexing ability toward hydrophobic molecules allowing the formation of a complex with fenretinide (HPR). The polymer-HPR complex was stable in aqueous environment and released the free drug prevalently in the presence of fluid hydrophobic phases. It was cytotoxic toward tumor cells with minimal activity toward normal cells. Antitumor activity exceeded that of the separate complex components resulting from the concomitant effect of the polymer and the HPR solubilized by complexation.

Enriching suicide gene bearing tumor cells for an increased bystander effect

The success of cancer gene therapies requiring in vivo gene transfer is severely hampered by the low efficacy of gene transfer, which has been difficult to improve. We therefore established a novel strategy to increase the share of transduced cells post gene transfer. We hypothesized that in vivo selection of tumor cells transduced with a suicide gene effectively enriches these cells within a tumor, thus allowing for an increased bystander effect after the prodrug is given, leading to enhanced eradication of tumor cells. We reasoned that in vivo enrichment should be achieved by exploiting the metabolism of the suicide gene product. For this 'enrichment-eradication' strategy we chose a fusion gene of cytosine deaminase and uracil phosphoribosyl transferase. Positive selection (enrichment) was to be achieved by concurrently giving N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis, which leads to pyrimidine depletion-mediated death of non-transduced cells, and cytosine, to rescue fusion gene expressing cells via the pyrimidine salvage pathway. Negative selection (eradication) was to be induced by giving the prodrug 5-fluorocytosine. Indeed, murine NXS2 neuroblastoma cells transduced with the fusion gene were effectively enriched in vitro, leading to a near-complete bystander effect. In vivo enrichment-eradication of NXS2 cells led to decreased tumor growth. This proof-of-principle study shows that enrichment-eradication may compensate the effects of low in vivo gene transfer efficacy, a major obstacle in cancer gene therapy.