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Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.

When established in culture, human neuroblastoma cell lines typically are comprised of heterogeneous cellular subpopulations, including neuroblastic (N-type), substrate-adherent (S-type), and intermediate (I-type) cells that can be distinguished by their characteristic morphologies and expression of differentiation-associated antigens. Here we examined the relative levels of the Bcl-2 oncoprotein in 15 clones derived from four different neuroblastoma cell lines. Among six clones isolated from the SK-N-SH line, levels of p26-Bcl-2 correlated with morphology and differentiation markers with the hierarchy of bcl-2 expression being: N-type cells > N/I-type > I-type > S-type. Furthermore, stimulation of one of the N-type clones, SH-SY5Y, with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induced differentiation toward a more neuronal-like phenotype and resulted in a 5- to 10-fold elevation in the relative levels of Bcl-2 protein. High relative amounts of p26-Bcl-2 protein were also found in an N-type clone derived from the SMS-KCN line. In two N-type clones derived from the LA-N-1 line, however, levels of Bcl-2 protein were only moderately elevated, and in one N-type clone from the SK-N-BE(2) line the levels of Bcl-2 protein were low. Thus, high relative levels of Bcl-2 oncoprotein are not a universal feature of N-type cells (three of six clones tested). In contrast, all 5 of the S-type clones evaluated contained relatively low levels of Bcl-2 protein, suggesting that these cells (which may represent embryonic precursors of Schwann, glial, and melanocytic cells) do not typically express the bcl-2 gene at high levels. Consistent with this inverse correlation between Bcl-2 protein levels and S-type characteristics, stimulation of an I-type clone derived from the SK-N-BE(2) line with 5-bromodeoxyuridine was accompanied by an accumulation of S-type cells in these cultures, decreased Bcl-2 protein, diminutions in the neuronal markers neurofilament-M and neuron-specific enolase, and an increase in the relative levels of the S-type marker proteins vimentin and beta-2-microglobulin. Conversely, stimulation of this I-type clone with retinoic acid resulted in an accumulation of N-type cells (which are thought to represent embryonic precursors of sympathetic neurons), decreased vimentin and beta-2-microglobulin, increased neurofilament-M, and a marked elevation in p26-Bcl-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Little is known about the prevalence and significance of ras gene activation in neural crest tumors such as neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs). Therefore, we analyzed DNA from 10 human neuroblastoma cell lines and 10 primary human pheochromocytomas for activating mutations in N-ras, H-ras, and K-ras. We also studied DNA from 24 primary neuroblastomas and 10 MTCs for N-ras mutations. ras genes were analyzed by direct sequencing of specific DNA fragments amplified by the polymerase chain reaction. With the exception of the SK-N-SH cell line, the examined ras gene sequences were normal in all the neuroblastomas, pheochromocytomas, and MTCs tested. A single point mutation was identified at codon 59 (GCT(ala)----ACT(thr)) in one N-ras allele in an SK-N-SH subline. Interestingly, this mutation is different from the activating codon 61 mutation which resulted in the initial identification of N-ras from SK-N-SH DNA. Therefore, we analyzed the sequences of earlier passages and sublines of the SK-N-SH cell line, but mutations at codon 59 or 61 were not detected, suggesting that neither mutation was present in the primary tumor. Our results indicate that N-ras mutations may occur spontaneously during in vitro passage of cell lines but rarely, if ever, occur in primary neuroblastomas, pheochromocytomas, and MTCs. In addition, we have not found H-ras or K-ras mutations in any neuroblastoma cell line or primary pheochromocytoma.

At least five gene classes are amplified in the multidrug-resistant CHO cell line CHRC5. Protein products have been identified for two classes; class 2 codes for the large membrane P-glycoprotein, whereas class 4 encodes the small cytoplasmic calcium-binding protein sorcin (V19). By DNA analysis we have shown previously that these five genes are linked in two groups: class 1 + 2 + 3; and class 4 + 5. By use of in situ hybridization with complementary DNAs derived from the resistant cell line we demonstrate here that genes from both linkage groups are amplified and situated together in each of two different chromosomal regions of the resistant Chinese hamster cell line. The positions of the amplicons correspond to cytogenetically identified homogeneously staining regions in an altered 7q+ chromosome and in a rearranged Z-7 [t(3;4)] chromosome. The native genes were mapped both in the CHRC5 line and in a normal diploid Chinese hamster cell strain, CHNF 86. We confirm the position of the class 2 gene on 1q26 and we show that class 4 and 5 genes are located in the same region of 1q. We conclude that the gene classes 2, 4, and 5 are closely juxtaposed in the normal Chinese hamster genome and comprise one amplicon in resistant cells. Our results are compatible with the hypothesis that multidrug resistance is due to overexpression of P-glycoprotein genes and that the other genes amplified in the CHRC5 line are coamplified because they happen to lie close to the P-glycoprotein genes.

Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes. One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes. Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific. Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells. Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification. Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes. However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.

Two morphologically distinct types of cells have been observed in cultures of human neuroblastoma cells. One, designated N-type, is composed of small neuroblast-like cells; the other, designated S-type, is composed of large, substrate-adherent cells. To obtain additional information on the nature of these two phenotypes, we have investigated the collagen biosynthesis in several clones of human neuroblastoma cells with N-type, S-type, or mixed morphology, using acrylamide gel electrophoresis, ion exchange chromatography, and Northern and Southern blots. A direct correlation between the proportion of cells with S-type morphology and the amount of collagen secreted was observed, with the largest amount of collagen being produced by clones composed exclusively of S-type cells. Type I trimer and type III collagens were the two major isotypes secreted by these cells. The absence of secretion of the alpha 2(I) collagen chain was associated with absence of RNA coding for this protein chain. The pro-alpha 2 gene of type I collagen was found highly methylated in these cells. Our data indicate that the presence of S-type cells in neuroblastoma cultures represents a differentiation of these neural crest-derived neuroblastic cells into glial cells such as Schwann cells.

Human neuroblastoma cells growing in culture offer a unique opportunity to study proliferating human cells with a neuronal phenotype. We have previously identified several neuroblastoma cell lines which show spontaneous conversion (N/S interconversion) between two morphologically distinct cell types: neuroblastic (N-type) cells and variant, substrate-adherent (S-type) cells resembling cultured glial or mesenchymal cells. In the present study, we have used molecular markers to confirm the neuronal phenotype of N-type cells and to demonstrate that S-type cells have a nonneuronal phenotype. Furthermore, we have used these markers, including a series of cell surface differentiation antigens, to compare S-type neuroblastoma cells with a wide range of cultured epithelial, mesenchymal, and neuroectodermal cells. The results suggest that N/S interconversion represents an ordered transition between two neuroectodermal differentiation programs rather than random phenotypic instability of cultured cells; S-type variant cells show a molecular phenotype most closely resembling the phenotype of cultured ectomesenchymal cells; and in vitro variant formation of human neuroblastomas may provide an experimental model for the observed in vivo transition of some malignant neuroblastomas into benign ganglioneuromas.

Previous studies of the human neuroblastoma cell line SK-N-SH had demonstrated the presence of and phenotypic interconversion (transdifferentiation) between two morphologically and biochemically distinct cell types: N (neuroblastic) cells with properties of noradrenergic neurons and S (substrate-adherent) cells with properties of melanocytes. Current studies have sought to test the generality of these findings among other cultured human neuroblastoma cell lines and to define further the S-cell phenotype and that of a newly identified, morphologically intermediate, I-type cell. Morphologically homogeneous populations (clonal sublines or subpopulations) of N, S, and I cells were isolated from five additional neuroblastoma cell lines and analyzed biochemically for neuronal, glial, and melanocytic marker enzyme activities and norepinephrine uptake. Immunoblot techniques were used to detect intermediate filament proteins (neurofilament protein, vimentin, glial fibrillary acidic protein) and fibronectin. All N-type cells exhibited neuronal marker enzyme activities, specific uptake of norepinephrine, and presence of one or more neurofilament proteins. S-type cells generally lacked neuronal characteristics but contained, instead, tyrosinase activity (a melanocytic marker enzyme), vimentin, and fibronectin. This combination of attributes is suggestive of a multipotent embryonal precursor cell of the neural crest. I-type cells differentially expressed both S- and N-cell properties and could represent either a stem cell or an intermediate in the transdifferentiation process. Studies of the biological significance of human neuroblastoma cell transdifferentiation and the molecular mechanisms underlying this process may be of relevance to the biological and clinical behavior of this tumor in the patient.

The lymphoblastic leukemia ERLD, induced by radiation in a C57BL/6 mouse, was established in culture. Three cell lines, ERLD/Y3, ERLD/T ERLD/Two, have been in culture for nearly three years. Their isolation and growth depended upon the presence of 2-mercaptoethanol, glutamine, and asparagine in the medium. The cell lines, except ERLD/T, possess the TL antigen, a characteristic of ERLD and of other murine leukemia cells in vivo and of normal thymus cells of certain mouse strains, but not of C57BL/6. A distinctive submetacentric marker chromosome is also common to ERLD and the derived cell lines. The successful establishment of ERLD in culture provides a malignant thymocyte-related cell system for studies in nutrition and immunobiology.