Spontaneous fusion and formation of hybrids between C1300 neuroblastoma cells and lymphoid cells in mixed cultures.

Spleen lymphoid cells from specifically sensitized A/J mice were induced to bind and form rosettes around syngeneic, cloned C1300 neuroblastoma (NB) cells in vitro. Rosette formation usually led to target cell lysis. Electron microscopic evidence suggests, however, that lymphoid cells were sometimes spontaneously incorporated by the target cell and lysed, and their material was probably reutilized by the penetrated cell. When lymphoid cell suspensions were added to cultures of a mutant clone of NB cells (clone NA), which died in HAT medium because of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, HAT medium-resistant clones arose at a frequency of about 5 × 10^?5, at least 200 times the reversion rate of NA. This suggested that correction of the HGPRT deficiency was due to efficient spontaneous fusion. Rosettes were also induced between spleen lymphocytes from allogeneic C₃H/HeJ mice and cloned NB cells. Rosettes were then separated by centrifugation through a discontinous BSA gradient. NB cells were isolated by adherency and were left to grow. Evidence indicated that these resulting NB cultures contained hybrids and that lymphocytes introduced new genes into NB cells with detectable frequency. Cells synthesized in culture a heteropolymer of glucose phosphate isomerase, while lymphocytes and original neuroblastoma cells alone supplied, respectively, only a fast and a slow form of the isozyme, as shown by electrophoretic assay. Furthermore, expression of hybrid surface markers, changes in lymphocyte recognition capacity in in vitro mixed cell culture assays, and delayed malignancy in A/J mice proved somatic cell hybrid formation.     

Release of adenosine by C1300 neuroblastoma cells in tissue culture

Previous work in our laboratory led us to postulate that N2a cells release adenosine into growth medium, where it acts at the extracellular adenosine receptors to modulate the sensitivity of the cells to the cyclic AMP-elevating effect of adenosine [Green, RD, J Pharmacol Exp Ther 201:610, 1977]. We have now devised a high-performance liquid chromatographic (HPLC) procedure capable of quantitating the concentrations of adenosine in cells and tissue culture media. Growth media of N2a cells and a variant of N2a cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT^?) contain 10–20 nM adenosine, while that of a variant deficient in adenosine kinase (AK^?) is elevated severalfold. It appears that the concentration of adenosine in growth media is determined by both the rate at which it is released by cells into the medium and the rate at which it is metabolized by adenosine deaminase present in the serum in the growth medium. Both N2a and AK^? cells release considerable amounts of adenosine into serum-free medium (SFM) over a short period. Adenosine release is greater from AK^? cells and is accelerated by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), a potent adenosine deaminase inhibitor. This accelerated release is retarded by dipyridamole and homocysteine. Surprisingly, dipyridamole and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20 1724), a potent phosphodiesterase inhibitor, stimulate basal adenosine release from N2a but not from AK^? cells. It remains to be determined if this is due to an effect of these compounds on adenosine kinase. These results give further support for the hypothesis that adenosine in growth medium modulates the sensitivity of the cells to the cyclic AMP-elevating affect of adenosine, and furthermore they suggest that adenosine in growth media may tonically stimulate adenylate cyclase and affect processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.     

Interaction of neuroblastoma C 1300 N18 cells with liposomes and intermembrane metabolism of lipids

The methods of isotopic and fluorescent labels have shown that interaction of cells of neuroblastoma S 1300 N 18 with small one-layer neutral liposomes prepared of the egg phosphatidyl choline with the addition of different amounts of cholesterol is realized by two mechanisms: the transmembrane transfer of cholesterol by the concentration gradient and membrane lipid metabolism proper, the ratios of cholesterol phospholipids in the biological and artificial membranes being equal. A dependence is established of the neuroblastoma cell viability on the activity of the cholesterol membrane metabolism. A problem on the mechanism which causes the death of cells during the interaction with phosphatidyl-cholesterol liposomes is under discussion.     

Morphological differentiation and change in the lipid composition of neuroblastoma C1300 cells treated with gangliosides

The effect of exogenous gangliosides on the morphological differentiation of neuroblastoma 1300 N18 cells was studied Simultaneously the content of gangliosides and lipid composition of the cells was investigated. Gangliosides were shown to increase the quantity of cells with long neurites. This effect depended on the dose of gangliosides. The addition of 50 and 100 micrograms of gangliosides per 4 ml of serum-free culture medium increased the quantity of cells with neurites by 38 and 63.4%, respectively. The level of morphological differentiation in cells cultivated with gangliosides was higher than in cells incubated with 5'-bromodeoxyuridine. Noticeable quantities of lysophosphatidylcholine (absent in the control) appeared in ganglioside-treated cells and the level of cholesterol increased. The amount of other lipid compounds in cells differentiated in the presence of gangliosides was similar, but not identical to the quantity of lipid compounds in cells differentiated by 5'-bromodeoxyuridine and by the serum-free medium.     

Ultrastructural studies of C1300 neuroblastoma cell interaction with syngeneic spleen lymphoid cells.

Sensitized and unsensitized spleen lymphoid cells from A/J mice were induced to form rosettes with cells of clone NB6R of syngeneic C1300 neuroblastoma cells. Light and transmission electron microscopy were applied in combination with ⁵¹Cr release experiments to follow the time course of reaction after rosette formation. With unsensitized lymphoid cells, rosettes formed but target cell morphology in general remained unchanged. With sensitized lymphoid cells a progressive series of morphological changes in the target cells was seen, initially in the mitochondria and, later, when specific ⁵¹Cr release became significant, in the formation of large surface blebs and protrusions. Our data also show another phenomenon occasionally following rosette formation. Lymphocytes were seen within the target cell; these either apparently transformed to lymphoblasts and killed the target cell from the inside or alternatively were destroyed by the host cell and their material was reutilized.     

Transmembrane lipid transport between lecithin liposomes and neuroblastoma C1300 N18 cells

The method of double isotopic labels was used to study dynamics of lipid metabolism between neuroblastoma C 1300 N 18 A 1 cells and lecithin liposomes which contained 4.5-5 mumol of lecithin in 1 ml of the suspension. The cell lipids were labelled by radioactive carbon and cultivated on the medium with [1-14C] sodium acetate, phosphatidylcholine of liposomes was labelled by tritium. It is shown that 15-30 min long incubation with liposomes causes a sharp decrease of the cholesterol esters amount with a simultaneous fall of the free cholesterol level. The total content of phospholipids in this case remains unchanged though there occurs the noticeable exchange of labelled phospholipids between cells and liposomes. The cholesterol content in the plasma membranes of cells lowers sharply. The neuroblastoma cells are able to compensate arising changes in the cholesterol level for 45-60 min after which they progressively die. 90 min later only an insignificant part of the population (about 10% of cells) is retained.     

Aging and the immune response. I. Antibody formation and chronic infection in Toxoplasma gondii-infected mice

Studies were performed to determine the effect of aging on the antibody response and cyst formations after infection with a relatively avirulent strain of the intracellular parasite Toxoplasma gondii. When compared with young mice (4 months), aged mice showed a significant decrease in the magnitude of humoral immune response to infection. This decrease was observed at the peak of the acute infection and also during chronic infection. Evaluation of the presence of Toxoplasma cysts, a measure of the latent infection, revealed that the numbers of tissue cysts present 11 weeks after infection increased with the age of the mice at time of infection. The larger numbers of cysts in older mice which had received the same inoculum size of T. gondii as young mice, together with our previous observations of increased susceptibility of these older mice to T. gondii, suggest that an age-related decrease in the early immune response to this infection allows an increased multiplication of the organism in vivo, leading to increased cyst numbers or death.     

Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice

 In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86⁺C1300, expressing a high level of mouse CD86 on the cell surface. While CD86⁺C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86⁺C1300-rejecting mice with CD86⁺C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86⁺C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86⁺C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86⁺C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors. Received: 18 November 1996 / Accepted: 3 March 1997     

Unmasking of nerve growth factor membrane-specific binding sites in synchronized murine C 1300 neuroblastoma cells

The resistance to actinomycin D (AD) of a clone of SV40 virus-transformed hamster cells (TSV5 C12 A^R) was investigated. This line was found to be much less permeable to the antibiotic. Penetration of proflavine and puromycin is also greatly reduced without any prior selection for these drugs. The AD that enters the cells is capable of inhibiting RNA synthesis. Thus the resistance of these cells to AD seems to be due only to the decreased permeability of the drugs possibly resulting from an alteration of the cell membrane.     

Adenosine transport by a variant of C1300 murine neuroblastoma cells deficient in adenosine kinase

The uptake of adenosine by an adenosine kinase deficient variant of C1300 murine neuroblastoma cells has been studied in the absence and in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenine deaminase inhibitor. Although 100 micro M inhibitor completely blocks the metabolism of adenosine under the conditions studied, the uptake of adenosine is concentrative, i.e., the intracellular adenosine concentration exceeds the extracellular concentration. This concentrative effect decreases as the concentration of adenosine increases and is hypothesized to be due to the binding of adenosine to an intracellular component. Despite this concentrative effect, we believe that the kinetics of uptake, as determined in experiments with short (10-20 s) uptake periods, reflect the kinetics of adenosine transport by a facilitated diffusion process. This nucleoside transport system appears to be nonspecific in that the transport of adenosine is competitively antagonized by thymidine. It does not appear to be necessary to inhibit adenosine deaminase in order to study transport in these cells as the Km for transport is not affected by the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. However, erythro-9-(2-hydroxy-3-nonyl)adenine does depress the V for transport. This effect of the inhibitor is probably not due to the inhibition of adenosine deaminase as the transport of thymidine is similarly affected.     

Rapid simultaneous isolation of microsomes and plasma membranes from neuroblastoma C 1300 N 18 cells

The method is suggested to isolate simultaneously microsomes and plasma membranes of neuroblastoma S 1300 N 18 cells by means of differential centrifugation in the step density gradient of Percoll/Ficoll with a high degree of purification determined from the activity of marker enzymes (acetyl cholinesterase Na+,K+-ATPase, alkali phosphatase, glucose-6-phosphatase, succinate-dehydrogenase, acid phosphatase) as well as from the content of DNA and RNA and with a sufficiently high protein yield. The purified fractions of microsomes and plasma membranes are established to contain no phosphatidyl glycerol and cardiolipin--safety markers of mitochondrial membrane purification. A degree of separation of microsomes, plasma membranes and proteins dissolved in cytosol may be estimated by the activity of the cholesterol-synthesizing system of enzymes with the use of sterol-transferring protein.     

Identification and properties of insulin receptors in neuroblastoma C 1300 N 18 cells in various functional states

The presence of insulin receptors in neuroblastoma C 1300 N18 cells was shown by the method of the [125I]insulin binding with cells as well as by the electron-cytochemical methods based on the analysis of the binding of colloid gold-labelled insulin and of agglutinin of wheat embryos with the surface of plasma membrane. It is established that the number and distribution of insulin receptors depend on the functional state of cells. Expression of receptors on the membrane increases after 5'-deoxyuridine-induced differentiation of cells. Due to changes in the lipid composition of cells caused by the incubation with lecithin-cholesterol liposomes (an increase in the content of cholesterol and its esters, as well as of unsaturated fatty acids and appearance of lysolecithin) the quantity of insulin receptors decreases and cell membranes are damaged. The lowering insulin regulation is not observed for insulin receptors of the neuroblastoma C 1300 N18 cells.     

Changes in the lipid composition of murine neuroblastoma C 1300 cells during the process of differentiation

The paper deals with the lipid composition of nondifferentiated cells at the logarithmic and stationary growth phases as well as with differentiated cells of C 1300 neuroblastoma adopted to the Eagle medium. It is shown that phospholipids and cholesterol dominate at the studied growth phases among lipids. Their amount in the differentiation process is thrice as high calculating per cells. The quantity of individual phospholipids changes in different manner during differentiation. The phosphatidylinosite level is higher than of other substances.