NUB-7: a stable I-type human neuroblastoma cell line inducible along N- and S-type cell lineages.

Human NB cell lines express features of one or more of three recognizable phenotypes that include N-type (neuroblastic), S-type (Schwannian), and I-type (intermediate phenotype) cells. The I-type cell, which shares properties of both N- and S-type cells, is thought to represent the progenitor cell from which the other two cell types are derived. The MYCN amplified NB cell line NUB-7, established in our laboratory, is now shown to be composed principally of I-type cells. The observed phenotype was stable in culture and was representative of the original surgically resected tumor. The I-type cell designation was established based on morphological characteristics, the coexpression of various N-type (neurofilaments, peripherin, GAP-43, NCAM, MYCN) and S-type cell (vimentin, laminin, fibronectin) markers, and the relatively high level of expression of these markers in comparison to five predominantly N-type, one S-type, and one N/S mixed NB cell lines. Dibutyryl cyclic AMP and retinoic acid induced enhanced expression of N- and S-type phenotypes, respectively, in NUB-7 as supported by specific morphological changes, reduced growth, and changes in the levels of expression of both N- and S-type markers. Our studies with the NUB-7 cell line have now provided convincing evidence for the existence of a bipotential progenitor of N- and S-type cells in NB. As well, the NUB-7 cell line may also represent the tumor counterpart of a sympathetic ganglion progenitor cell.

Importance of phenotypic and molecular characterization for identification of a neuroepithelioma tumor cell line, NUB-20.

A neuroblastic-like cell line (NUB-20) was derived from a case of histopathologically diagnosed metastatic neuroblastoma. The metastatic tumor and nude mouse heterotransplant resembled neuroblastoma by histological criteria, in contrast to the primary tumor, which was differentially classified as Ewing's sarcoma. However, the cell line demonstrated a unique phenotype in culture with respect to morphology, immunohistochemical markers, and sensitivity to a battery of differentiation modulators. These characteristics, together with the presence of a chromosomal translocation (11;22),(q24;q12) and amplification with enhanced expression of the c-myc protooncogene rather than N-myc, established this tumor as neuroepithelioma. Neuroepithelioma is a tumor type distinct from, but related to, neuroblastoma in its development from the neural crest lineage. These results emphasize the growing importance of cytogenetic and molecular markers in the classification and characterization of human tumors.

Phenotypic and molecular characterization of inducible human neuroblastoma cell lines.

Two new neuroblastoma (NB) cell lines, NUB-6 and NUB-7, were established from recurrent and primary NB tumours respectively and identified conclusively as NB by their phenotypic characteristics, catecholamine production and N-myc amplification. The cell lines could be distinguished on the bases of distinctive growth patterns in monolayer culture and semi-solid media (collagen gel and agarose), neurite formation and their response to four classes of growth and differentiation modulators. The NUB-6 cell line consisted of two distinct cell subtypes, small typical neuroblasts and larger spheroid-forming cells, while NUB-7 was homogeneously neuroblastic. Class-I agents (dibutyrl cyclic AMP [dbcAMP], butyrate, and papaverine) inhibited growth of both cell lines, while only dbcAMP stimulated the formation of short neurites by NUB-6 neuroblast cells in monolayer culture and collagen. Of the class-II agents (vitamins), retinoic acid inhibited growth of both cell lines and stimulated formation of long neurites by NUB-6 cells and NUB-7 cells in later passages. In contrast, vitamin E inhibited growth of NUB-6 and late-passage NUB-7, but stimulated early passage NUB-7. The class III agent (nerve growth factor) resembled vitamin E. The class-IV agents (interferons; rIFN-alpha 2a and rIFN-gamma 1) inhibited growth of both cell lines in monolayer culture and agarose, but stimulated NUB-6 neuroblasts and early passage NUB-7 cells to form long neurites. Thus phenotypically distinct NB cell lines were established in vitro and shown to be differentially influenced by various growth and differentiation modulators. The potent effect of IFN suggests a role for these modulators in NB behaviour in vivo.

Selective protection of tubercidin toxicity by nitrobenzyl thioinosine in normal tissues but not in human neuroblastoma cells.

Tubercidin, an adenosine analogue, is toxic to human neuroblastoma cell lines, to peripheral blood mononuclear cells (PBMCs), and to myeloid colony-forming cells (CFU-C) as tested by a short-term labeled precursor uptake and by a clonogenic assay. When it was co-administered with a potent purine transport inhibitor, nitrobenzyl thioinosine (NBTI), the cytotoxic effect of tubercidin was abolished in PBMCs but not in neuroblastoma cells. Studies of nucleoside transport in neuroblastoma cells demonstrate that although [3H]NBTI binds to the plasma membrane of these cells, the transport of thymidine into the cells is only partially inhibited in the presence of excess NBTI. These data imply that neuroblastoma cells contain a nucleoside transport mechanism which is insensitive to NBTI. "Host protection" with a nucleoside transport inhibitor such as NBTI, may allow effective therapy with otherwise toxic dosages of tubercidin and other cytotoxic nucleosides in patients with neuroblastoma.

Relationship of histology of Wilms' tumor to growth characteristics of nude mouse heterotransplants.

Eighteen Wilms' tumors (WIT), including classical triphasic WIT (blastema, tubules, and mesenchyme) and WIT variants (blastema and tubules, monomorphous tubules, multiloculated cysts, rhabdomyomatous WIT, and clear cell sarcoma), were heterotransplanted in nude mice. Ten (56%) tumors grew and were serially passaged. With two exceptions, the histology of the surgically resected tumors and heterotransplants was found to be similar. Tumors showing a prominent blastema component grew rapidly, whereas those with tubular epithelial or mesenchymal differentiation grew more slowly. Tumors injected s.c. consisted almost entirely of blastema, while tumors injected i.p. consisted of blastema with large areas of tubular epithelium. These results demonstrate that nude mouse heterotransplants of WIT closely resemble the surgically resected tumors from which they derive, that growth rates of WIT heterotransplants depend on the identity of the tumor cells, and that differentiation of WIT heterotransplants can be modulated, depending on the route of administration of tumor cells.

Histochemical and immunohistochemical characterization of surgically resected and heterotransplanted Wilms' tumor.

Nine surgically resected Wilms' tumors (WIT) and nude mouse heterotransplants from one WIT were studied by histochemistry and immunohistochemistry. Histochemistry showed acid phosphatase in all cells, while alkaline phosphatase and gamma-glutamyl transpeptidase were present in only some tubules. Using immunohistochemistry, antibodies to the intermediate filaments cytokeratin and vimentin distinguished tubular epithelium and mesenchyme, respectively. WIT tubules were also identified using antibody against a structural component (epithelial membrane antigen) and a secretory product (uromucoid) associated with distal convoluted tubules of normal kidney. Basement membrane surrounding the tubules of WIT was demonstrated using antibody to type IV collagen plus laminin. Different blastema subpopulations were negative or stained positively with antibodies to cytokeratin and vimentin. Production of basement membrane by blastema was also shown. Fetal antigen expression in WIT was examined using the monoclonal PI 153/3 and J5 antibodies. The blastema and tubules of WIT were strongly stained by PI 153/3, which did not label normal adult kidney, and weakly stained by J5, which strongly labeled glomeruli and proximal convoluted tubules of normal kidney. These studies show that WIT blastema is heterogeneous in intermediate filament subtypes, while WIT tubules more closely resemble distal than proximal convoluted tubules of adult kidneys but also retain expression of fetal antigens.