ABSTRACT
BACKGROUND: The neuropeptide contents in neuroblastomas were quantified by radioimmunoassay (RIA) to assess their possible biologic significance. METHODS: Neuroblastoma tumor tissue was obtained from the primary tumor site before therapy in 16 patients with newly diagnosed neuroblastoma and in 7 patients with central nervous system medulloblastomas or gliomas. RESULTS: The tumor tissue was assayed for vasoactive intestinal peptide (VIP), somatostatin (SRIF), substance P, and neurotensin by both immunostaining and RIA techniques. Increased VIP levels of 1.2 pg/micrograms DNA or more correlated significantly with cellular differentiation (P = 0.003) and favorable disease stage (P = 0.002) in neuroblastomas. Increased SRIF contents (greater than 1.3 pg/micrograms DNA) correlated with differentiation of the tumor (P = 0.002). Increased VIP and SRIF content did not correlate with N-myc oncogene expression or ras oncogene protein immunostaining. No VIP was detectable in brain tumors, and other neuropeptides were variable in content. CONCLUSIONS: RIA of VIP and SRIF levels in primary tumor tissue may offer an independent objective assay of biologic behavior in neuroblastoma biopsy specimens.
Subject(s)
Neuroblastoma/chemistry , Neuropeptides/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Ganglioneuroma , Genes, myc , Genes, ras , Humans , Immunoenzyme Techniques , Male , Neuroblastoma/genetics , Neuroblastoma/pathology , Prognosis , Prospective Studies , Radioimmunoassay , Somatostatin/analysis , Substance P/analysis , Vasoactive Intestinal Peptide/analysisABSTRACT
Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies (KD = 1.3-12.4 nM) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression. Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors (KD = 4 nM and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10(6) cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10(6) cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.
Subject(s)
Neuroblastoma/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Vasoactive Intestinal Peptide/metabolism , Adenylyl Cyclases/metabolism , Binding, Competitive , Cell Division/drug effects , Cell Survival/drug effects , Humans , Neuroblastoma/pathology , Radioimmunoassay , Receptors, Vasoactive Intestinal Peptide , Tumor Cells, Cultured , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.
