We postulate that neuronal cell differentiation is regulated by extrinsic factors such as components of the extracellular matrix (ECM) and by intrinsic factors such as levels of N-myc protein. These control mechanisms are lost upon transformation. We have chosen neuroblastoma cells (NB) in culture to test our hypothesis. NB is a highly malignant childhood tumor of meuroectodermal origin. Most NB progress and metastasize rapidly. Tumors show occasional differentiation and regression may occur. The pattern of expression and amplification of the N- myc oncogene was found to correlate positively with the extent of the disease suggesting a role of the N-myc gene in rumor progression. We have collected and viably frozen more than 30 NB specimens from patients with different stage disease. A novel NB cell line (HTLA 230) was isolated which demonstrates morphological and molecular differentiation upon stimulation with retinoid acid (RA), has elevated N-myc gene expression and forms tumors in the nude mouse. These specimens will be used to study regulation of NB cell differentiation. HTLA 230 cells will be grown on complete or enzymatically modified ECM's. Retinoid acid induced NB cell differentiation will be studied molecularly and the role of the ECM in modulation of differentiation determined biochemically. The role of the N-myc gene in NB cell differentiation will be studied by up or down regulation of cellular N-myc protein levels. This will be accomplished by introducing retroviral constructs into NB cells from which sense and antisense N-myc RNA will be transcribed. Such vectors will be constructed containing the full length N-myc cDNA under the control of inducible promotors. HTLA 230 cells express a human homeotic gene (Hu-l) upon induction of differentiation with RA. We will isolate a full length Hu-l cDNA clone from a fetal cDNA library and introduce it into NB cells by retroviral infection. The effect of an inducible homeotic gene on the biology of NB cells will be studied. These experiments attempt to determine control mechanisms of neuronal cell differentiation and the role of the N-myc gene in this process. We will attempt to study a link between mechanisms controlling development and genetic elements with transforming potential.
