Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells

As part of investigations on ethylnitrosourea (EtNU)-induced neuro-oncogenesis in the rat, we have produced monoclonal antibodies (Mabs) specific for neural cell surface antigens (NCAs) by immunization with cells of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designated as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and alpha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunoprecipitation and Western blot. The predominant 25-kDa protein was purified from BT4Ca cells by immunoaffinity chromatography with immobilized Mab alpha NCA1 and identified by N-terminal sequencing as the rat homologue of the CD9 antigen. Identification of proline as N-terminal amino acid of the purified protein suggests post-translational modification of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunofluorescence staining of frozen sections. Flow cytometric analyses of isolated fetal rat brain cells (FBC) showed that the proportion and number of NCA/CD9-expressing cells increased during prenatal development. Immunoreactivity of approximately 40% of brain cells isolated 13 days post conception (p.c.) indicated that NCA/CD9 is expressed by neuronal precursors at this stage of development. In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all premature and mature astrocytes, oligodendrocytes, ependymal cells, and microglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons. Furthermore, eight out of ten EtNU-induced malignant neural rat cell lines as well as EtNU-induced tumors of the central and peripheral nervous system exhibited intermediate or strong immunoreactivity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, therefore, characteristic of both normal glial precursor cells and their malignant counterparts in the rat.