C1300 Neuroblastoma Cells Research Articles

Modulation of drug resistance in homoharringtonine‐resistant C‐1300 neuroblastoma cells with cyclosporine A and dipyridamole

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.

Neuroblastoma grafts are noninvasively removed within mouse neocortex by selective laser activation of intracellular photolytic chromophore

Studies of neural cell transplantation would be aided by the ability to damage or destroy, noninvasively and extremely selectively, grafted cells at defined times following their initial implantation. Mechanisms of graft integration and performance could be investigated, also providing insight into natural injury and repair mechanisms. At long wavelengths between 650 and 850 nm, laser energy can penetrate several millimeters of brain tissue without absorption or damage to the unpigmented tissue. However, targeted cells are selectively damaged by illumination at these long wavelengths if they contain latex nanospheres with incorporated cytolytic chromophores (e.g., chlorin e6). Light penetration allows many thousands of cells to be lesioned simultaneously, noninvasively, and deep within a surrounding matrix of other tissue. Such laser-activated damage has been termed laser photolysis (PL). We studied damage to C1300 neuroblastoma (NB) cells grafted into mouse neocortex in vivo by this process of PL. NB cells provided a simple and reproducible model of neural grafting, allowing direct histologic assessment of cellular growth and viability by distinct morphologic and mitotic criteria. Cells were cultured by standard methods, labeled in vitro by brief exposure to nanospheres containing chlorin e6, and grafted to sites within deep layers of mouse neocortex. Mice were exposed to transcranial, fractionated, unfocused pulses of 670-nm-wavelength energy totaling 90-120 J/cm2. We histologically assessed graft growth and cellular viability over a period from 2 d to 4 weeks, measured graft volumes quantitatively during the period of early rapid growth in controls (2 and 7 d), and generated 3-D reconstructions from serial sections to assist in visual analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The herpes simplex virus protein Vmw65 can trans-activate both viral and cellular promoters in neuronal cells

Transcription of the herpes simplex virus (HSV) immediate-early (IE) genes in lytic infection is dependent upon the formation of a complex between the cellular transcription factor Oct-1 and the HSV virion protein Vmw65. This complex then binds to the TAATGARAT sequence in the IE promoters and trans-activates the IE genes. Following infection of neuronal cells such as the C1300 neuroblastoma cell line, however, the viral (IE) genes are not transcribed and the lytic cycle is aborted at an early stage. We show here that the cellular factors necessary to form a trans-activating complex with Vmw65 are present in C1300 cells and that trans-activation of both viral and cellular promoters by Vmw65 can be observed in these cells. In contrast with permissive cells, however, trans-activation is only observed in C1300 cells at a high concentration of the target viral promoter and not at a low concentration of the target promoter, regardless of the amount of Vmw65 transfected. The significance of these effects for the regulation of latent infection and cellular gene expression in neuronal cells is discussed.

Exogenous expression of mouse interferon gamma cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity.

To examine the influence of interferon gamma (IFN-gamma) on tumorigenicity, we established constitutively IFN-gamma-producing cell lines from a malignant mouse neuroblastoma, C1300, by retroviral transfer of a mouse IFN-gamma cDNA. The gene-transferred cells generally showed an enhanced high-level expression of the major histocompatibility complex class I antigens at the cell surface and the transcription levels, irrespective of their IFN-gamma-producing potential. Although in vitro cell growth of these cells was unaffected by the IFN-gamma production, their s.c. tumor growth in syngeneic A/J mice was dependent upon levels of IFN-gamma production; tumors induced by a low-producer line grew well at a rate similar to those induced by the parental one, but tumor growth of a high-producer line was strongly suppressed. This apparent tumor suppression was abolished by simultaneous i.p. injection of anti-Lyt2.2 and/or anti-IFN-gamma monoclonal antibodies, and subsequently large tumors of the high producer were generated. Anti-asialoganglioside GM1 antibodies allowed the high-producer line to induce a substantial but only transient tumor growth, whereas other antibodies, such as anti-Lyt2.1, anti-IFN-beta, and anti-activated macrophage, had no such effect. The mice immunized with the high-producer line were resistant to tumor growth of the parental cells but permitted another kind of A/J tumor line, Sa-1, to induce remarkable tumors. These results indicate that the reduced tumorigenicity of the IFN-gamma high-producer line was due to the augmented specific anti-tumor immunity, in which cytotoxic T lymphocytes seemed to play a decisive role, probably as a result of the immunomodulatory effects of the IFN-gamma derived from the tumor.

Regulated transcription of herpes simplex virus immediate-early genes in neuroblastoma cells

C1300 neuroblastoma cells are nonpermissive for infection with herpes simplex virus but can be rendered permissive by pretreatment with sodium butyrate. This increased permissivity which is specific for HSV is caused by increased transcription of the viral immediate-early genes following infection of butyrate-treated cells and can be observed for at least 24 hr following withdrawal of butyrate. The use of C1300 cells as a model system for studying the regulation of immediate-early gene expression in neuronal cells in vitro and its possible relevance to the study of the processes regulating latent infection in vivo is discussed.

Lack of effect of the opioid antagonist, naltrexone, on the [formula omitted] growth of C-1300, NIE-115 and NS206 murine neuroblastoma tumor cell lines

Attempts have been made to confirm previously reported results which demonstrated that the opioid antagonist, naltrexone, altered the in situ growth of murine neuroblastoma tumors. Adult male A/J mice were injected with tumor cells from three different cell lines of murine neuroblastoma; the spontaneously arising C-1300 line, the adrenergic clonal line NIE-115, and the cholinergic clonal line NS20Y. Naltrexone was administered daily in doses of 0.1, 0.4, or 10.0 mg/kg subcutaneously, to replicate the reported experimental design. In contrast to previous studies, we were unable to demonstrate any effect of naltrexone on in situ growth or other characteristics of tumors produced by the C-1300, NIE-115 or NS20Y murine neuroblastoma cell lines. Ligand binding studies have demonstrated the presence of high levels of opiate binding sites on membranes prepared from the NS20Y clonal cell line and low levels on the membranes of the C1300 tumor line.

Effects of ultrasound exposure in vitro on neuroblastoma cell membranes

Mechanisms of action of ultrasound on cell membranes were studied on two murine C1300 neuroblastoma cell lines of minimum genetic diversity. Cavitation was established in rotating polystyrene centrifuge tubes by 1 MHz cw ultrasound; exposure time was 5 minutes. NS20Y and N2A cells exposed in suspension responded similarly by 86Rb+ transport and Na+-K+-ATPase activity assays, but differently by trypan blue dye exclusion and lysis assays. This indicates similar overall damage to the cell membranes, despite use of trypsin to release N2As only. Primary evidence of damage was lysis of NS20Ys and permeabilization of N2As. These results indicate that the same ultrasound exposure conditions can produce different effects in cells that differ in their membrane properties.

Surface charge at the cytoplasmic membrane of murine C1300 neuroblastoma cells

“Inside-out” vesicles (IV, mainly with the cytoplasmic side outermost) were obtained from the plasma membranes of neuroblastoma cells from strain C1300 mice, clone N18. These served as a convenient model for investigating the surface charge of the cytoplasmic side of the cell membrane using microelectrophoretic techniques. Electrophoretic mobility (EM) at a neutral pH and with an external medium of the same ionic strength was found to be 2.7-fold less than that of “right-side-out” vesicles (RV). Processing vesicles with neuraminidase reduced EM in RV but not in IV, while trypsin did so in both. Treatment with phospholipase C produced the same effect in IV but none in RV. Phospholipase D increases the EM of both types of vesicles. Charged groups at the surface of IV are titrated in relation to a pK of 3.5. The EM of IV is dependent on the Ca2+ concentration of the external medium. On the cytoplasmic side of the membrane, Ca2+ forms a 1∶1 complex with negatively charged protein and lipid molecules as well as those lipid molecules found with a neutral pH, in the form of a zwitterion with binding constants of 50, 12, and 25 liter/mole respectively.

Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors.

Dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting step in pyrimidine degradation, was studied in two cell lines of murine neuroblastoma (MNB-T1 and MNB-T2) that were derived from C-1300 MNB tumor carried in A/J mice. The MNB-T2 (low malignancy) cell line was originally derived from the in situ tumor and carried in tissue culture for more than 100 passages; the MNB-T1 (high malignancy) line consisted of a new sub-culture that was also established from the in situ MNB tumor. DPD activity was determined in cytosolic preparations of MNB utilizing high performance liquid chromatography to separate the radiolabeled substrate ([2-14C]thymine) from [2-14C]dihydrothymine. The apparent affinity of DPD for NADPH in MNB cells (Km approximately 0.08 mM) was identical to that of A/J mouse brain and liver. The DPD activity of the high malignancy (MNB-T1) cell line was 14.3% of that observed in the low malignancy (MNB-T2) line. In situ tumors formed after implantation of high malignancy (MNB-T1) cells into A/J mice had only 25.2% of the DPD activity observed in tumors derived from low malignancy (MNB-T2) cells. When MNB-T2 cells were injected into naive A/J mice, tumors developed in only 68% of animals, the tumor growth rate was slow and a mortality of 20% was observed. In contrast, tumors derived from injected MNB-T1 cells showed a faster growth rate and 100% mortality. Most MNB-T2 derived tumors were not lethal and ultimately resolved while the MNB-T1 derived tumors were invariably lethal. These studies support the concept that the levels of DPD activity in neoplastic cells are inversely related to their malignant expression and also provide a model to study differences between neuroblastoma cell lines derived from the same in situ tumor but which manifest different neoplastic behavior.

Protein synthesis by neuroblastoma cells is enhanced by exposure to burst-mode ultrasound cavitation.

We observed enhancement of protein synthesis, compared to control cells, 24 h after exposing C1300 neuroblastoma cells (N2A) in rotating tubes at 37 degrees C to 1-MHz ultrasound tone bursts [1:1; durations from 6 to 600 ms; 3.4 W/cm2 spatial-peak burst-average intensity; 5 min total treatment duration (on + off periods)]. Protein synthesis was measured by uptake of 3H-leucine and normalized for cell proliferation by measured uptake of 14C thymidine. The similarity between results for enhanced cavitation-stimulated iodine-release reported by Ciaravino and our results for cellular protein synthesis suggests that cavitation causes this biological effect.

Transport of leucine, lysine, glycine and aspartate in neuroblastoma C1300 and glioma C6 cells

Non-saturable penetration and the V and Km constants of saturable influx of leucine, lysine and glycine were always greater in cultured neuroblastoma (C1300) than in glioma (C6) cells. Aspartate uptake was detected only in glioma cells. Unstimulated efflux of the amino acids was initially fast in both cell types but soon slowed down. The efflux of glycine and aspartate exhibited no heteroexchange, the efflux of lysine was stimulated by extracellular leucine and that of leucine slightly by lysine and glycine but only in glioma cells.

A herpes simplex virus mutant is temperature sensitive for reactivation from the latent state: Evidence for selective restriction in neuronal cells

When the replicative defect in the HSV-1 temperature sensitive mutant tsI was repaired, the agent derived (RI-1) was found to possess an additional temperature sensitive lesion limiting its reactivation from the latent state. Thus, when spinal ganglia from latently infected mice were scored for reactivation by cocultivating them with indicator cells in vitro, significantly more were found to be positive at 31° than at 38.5°. To assess a possible relationship between reactivation and replication in neurons, the replication of RI-1 in murine C1300 neuroblastoma cells was studied. In these cells, RI-1 was severely restricted, and viral replication was delayed at 38.5°. Serial passage of RI-1 in neuroblastoma cells at the restrictive temperature resulted in selection of an agent which gained both the capacity to replicate efficiently in neuroblastoma cells and reactivate from the latent state at 38.5°. However, the replication pattern of this neuron adapted virus in mouse embryo fibroblasts remained unchanged from the parental RI-1. Taken together, these results indicate that RI-1 possesses a neuron specific temperature sensitive replicative lesion which is also manifest during reactivation from the latent state.

Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system.

After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection. Previous work from this laboratory focused on the structure of the viral DNA in this system. We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion. Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected. A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line).

A multiple cell-culture toxicity test system based on neuroblastoma C1300 cells and differentiated primary cultures of brain, muscle, heart and liver cells

An experimental substance ( H200 68 ) was tested for its cytostatic and general cytotoxic properties in a set of in vitro toxicity tests. H200 60 (5 × 10 −5 m ) was found to be more toxic to growing than to stationary neuroblastoma C1300 cells. In the presence of induced chick hepatocytes the substance was detoxified. In glial, muscle and heart cells, H200 68 slightly reduced protein synthesis, whereas liver cells remained unaffected. In primary cultures of chick-brain neurons, protein synthesis was considerably reduced.

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development.

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N-CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice.

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.

Effects of toxic chemicals on the respiratory activity of cultured mouse neuroblastoma cells.

Twenty common toxic chemicals were tested for their ability to inhibit respiratory activity in cultured mouse neuroblastoma C1300 cells, clone 41A3. Pentachlorophenol and hexachlorophene exhibited the properties of uncouplers of oxidative phosphorylation, whereas for KCN, pyridine, 2,5-hexandione, NaAsO2, K2Cr2O7, HgCl2, methylmercury and triethyltin more simple time-courses of inhibition were obtained. Ethanol, methanol, dimethyl sulphoxide, benzidine, nickel acetate, MnCl2, phenol, CoCl2, Na2SeO3 and CdCl2 did not cause any significant changes in respiratory activity. Among the effective compounds, those with well-known neurotoxic properties were the most potent in inhibiting respiration in 41A3 cells.

Effect of dihydroergocristine on energy metabolism studied in the isolated perfused rat brain affected by ischemia and in neuroblastoma cells deprived of oxygen and glucose.

The effect of dihydroergocristine on energy metabolism was studied in the isolated perfused rat brain affected by ischemia and in cultivated C-1300 neuroblastoma cells deprived of oxygen and glucose. Creatine phosphate, ATP, ADP, AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate were measured enzymatically. After a perfusion period of 30 min, the cortex of the isolated perfused rat brain exhibited an energy state not different from that in vivo. Dihydroergocristine added to the perfusion medium (5 mumol/L) did not influence these substrate levels under normal perfusion conditions. However, this drug was able to retard the breakdown of high-energy phosphates during ischemia and to accelerate the restoration of the energy state during the postischemic reperfusion period. The perfusion rate was not changed by the drug, and therefore it was assumed that dihydroergocristine could act directly on cell metabolism. This view was supported by the results obtained from experiments using cultivated N-2a neuroblastoma cells. These cells were incubated in a buffered salt solution deprived of glucose and oxygen for 15 min. Under these conditions, dihydroergocristine (2 mumol/L) added to the incubation medium caused changes in the concentrations or the high-energy phosphates similar to those in the isolated brain preparation: It increased the ATP concentration and decreased the ADP concentration significantly.

Taurine and hypotaurine transport by a single system in cultured neuroblastoma cells.

The kinetics of mutual inhibition of taurine and hypotaurine uptake were studied using neuroblastoma C1300 cells as neuronal model. Hypotaurine and GABA inhibited taurine uptake competitively, increasing the apparent Km. High-affinity uptake of hypotaurine was completely abolished and the low-affinity component competitively inhibited by taurine. GABA affected noncompetitively low-affinity hypotaurine uptake, whereas the effect on high-affinity uptake was competitive, with an increase in the apparent Km. All structural analogues tested inhibited taurine and hypotaurine uptakes similarly. The most potent inhibitors were beta-alanine and 2-guanidinoethanesulphonic acid. The mutual inhibition and similar specificity profiles of taurine and hypotaurine uptakes showed that these amino acids employ a single transport system in neuroblastoma cells. Competitive inhibition by GABA of the high-affinity uptake of taurine and hypotaurine further suggests that also GABA uses the same carrier system.

On the application of cultured neuroblastoma cells in chemical toxicity screening.

The acute toxic action of a number of common chemicals was tested by their ability to cause detachment of cultured mouse neuroblastoma C1300 cells. A TD25 value was obtained by graphic estimation of the concentration needed to cause 25% of the total cell number to detach. These TD25 values were compared with LD50 values obtained from the literature, and they were found to correlate with a coefficient of 0.86. For six of the tested substances-diuron, butylated hydroxytoluene, benzidine, cyclophosphamide, Na2SeO3, and KCN-a very poor correlation was obtained. These diverging results could be ascribed to deficiencies in the neuroblastoma cell detachment test and emphasize the necessity for combined in vitro test procedures.