agar and Neuroblastoma

High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation.. Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins.. Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability.. Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.

Topics: Agar; Animals; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Histone Demethylases; Humans; Mice; Mice, Knockout; Mice, Nude; Mice, Transgenic; Neuroblastoma; Tretinoin

ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and -independent functions of ShcC.

Topics: Agar; Animals; Apoptosis; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Genes, Dominant; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Neuroblastoma; Neuropeptides; Phosphorylation; Phosphotyrosine; Plasmids; Protein Binding; Protein Structure, Tertiary; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 3; src Homology Domains; Thymidine; Time Factors; Transfection; Tretinoin; Wound Healing

A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.

Topics: Agar; Biomarkers, Tumor; Blotting, Northern; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Transformation, Neoplastic; Cloning, Molecular; Down-Regulation; Gene Expression Profiling; Glutathione Transferase; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Prognosis; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; RNA, Messenger; Sequence Analysis, DNA; Time Factors

Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wildtype but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.

Topics: Agar; Apoptosis; Blotting, Western; Cell Adhesion; Cell Differentiation; Cell Division; DNA; Down-Regulation; Fibroblasts; Fluorescent Antibody Technique, Indirect; Humans; Neuroblastoma; Neurofilament Proteins; Oncogene Proteins, Viral; Phenotype; Plasmids; Radiation, Ionizing; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection; Tretinoin; Tubulin; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vimentin

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5.. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar.. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Topics: Agar; Animals; Cell Adhesion; Cell Differentiation; Collagen; Culture Media; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Intracellular Signaling Peptides and Proteins; Isoenzymes; Mice; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; Neoplasm Proteins; Neurites; Neuroblastoma; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phenotype; Recombinant Fusion Proteins; Transcription Factors; Transfection; Tumor Cells, Cultured; Vimentin

Tumor-specific screening of investigational anticancer drugs is a new approach to cytotoxic drug screening. A tumor-specific drug screening model has been designed using established human neuroblastoma cell lines. In vitro drug sensitivity was studied using a modification of the human tumor stem cell assay. Eleven cell lines were tested initially for colony formation in soft agar. Four cell lines formed only small cell clusters, while three cell lines formed colonies at a very low rate. The four remaining lines formed colonies with satisfactory efficiency. These cell lines were exposed to three concentrations each of cis-platinum, vincristine, doxorubicin, L-phenylalanine mustard, and ethidium chloride and suspended in soft agar. Colony formation was tabulated and sensitivities determined for each cell line. A heterogeneous pattern of in vitro sensitivity was observed. Limitations and pitfalls as well as potential advantages of using such a tumor-specific drug screening system are discussed.

Topics: Agar; Antineoplastic Agents; Cell Line; Clone Cells; Culture Media; Drug Evaluation, Preclinical; Humans; In Vitro Techniques; Neuroblastoma

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.

Topics: Agar; Animals; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Kidney; Mice; Neuroblastoma; Peptides; Phenotype; Rats; Receptors, Cell Surface; Transforming Growth Factors

Topics: Agar; Bone Marrow; Carcinoma, Squamous Cell; Clone Cells; Cytoplasm; Desmosomes; Head and Neck Neoplasms; Humans; Melanoma; Microscopy, Electron; Neuroblastoma; Skin Neoplasms

An in vitro soft agar technique was used in an attempt to culture neuroblastoma cells from 71 bone marrow, 3 lymph node, and 2 solid tumor specimens from 18 patients with neuroblastoma. One-half of each specimen was sent for routine pathology studies and one-half was cultured in the soft agar system. Colonies appeared within 10 days in histologically positive bone marrows. Light microscopy, electron microscopy, catecholamine secretion, and karyology provided evidence that the colonies were composed of neuroblastoma cells. There were 38 instances in which histological study of the specimen demonstrated neuroblastoma cells. The soft agar system showed colony growth in 30 of these samples (79%). There were a total of 38 specimens that were histologically negative for neuroblastoma. Thirty of these 38 specimens showed no growth in the stem cell assay. Eight histologically negative specimens from 6 patients formed colonies in the soft agar system. Five of these 6 patients showed tumor histologically on prior or subsequent marrow examinations. In addition to a significant correlation between histological and soft agar culture results (p < 0.001), there exists a highly significant positive correlation between the number of colonies per plate and the histological status of the specimen (p < 0.005). Serial marrow samples were cultured on 7 patients. There appears to be an association between the number of colonies that develop in the plate and the clinical course and prognosis of the patient. Decreasing plating efficiencies (number of colonies per number of cells plated) correlated with tumor response. Increasing plating efficiencies indicated tumor relapse. A plating efficiency of greater than or equal to 0.1% portended a particularly poor prognosis. Neuroblastoma grows well in this soft agar culture system. This excellent growth provides a good model for both clinical and basic science studies of neuroblastoma.

Topics: Abdominal Neoplasms; Agar; Bone Marrow; Cells, Cultured; Child; Child, Preschool; Clone Cells; Colony-Forming Units Assay; Cytological Techniques; Female; Humans; Infant; Lymph Nodes; Male; Microscopy, Electron; Neuroblastoma; Prognosis

Topics: Agar; Bone Marrow; Cell Division; Cells, Cultured; Clone Cells; Humans; Karyotyping; Lymph Nodes; Neuroblastoma

Topics: Agar; Biopsy; Carcinoma, Squamous Cell; Cell Division; Cell Line; Clone Cells; Colonic Neoplasms; Head and Neck Neoplasms; Humans; Intestinal Neoplasms; Neuroblastoma; Rectal Neoplasms