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-onc ogenesis in the rat, we have produced monoclonal antibodies (Mabs) spe cific for neural cell surface antigens (NCAs) by immunization with cel ls of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designat ed as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and al pha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunop recipitation and Western blot, The predominant 25-kDa protein was puri fied from BT4Ca cells by immunoaffinity chromatography with immobilize d Mab alpha NCA1 acid identified by N-terminal sequencing as the rat h omologue of the CD9 antigen, Identification of proline as N-terminal a mino acid of the purified protein suggests post-translational modifica tion of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunof luorescence staining of frozen sections, Flow cytometric analyses of i solated fetal rat brain cells (FBC) showed that the proportion and num ber of NCA/CD9-expressing cells increased during prenatal development, Immunoreactivity of similar to 40% of brain cells isolated 13 days po st conception (p.c.) indicated that NCA/CD9 is expressed by neuronal p recursors at this stage of development, In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all prema ture and mature astrocytes, oligodendrocytes, ependymal cells, and mic roglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons, Furthermore, eight out of ten EtNU-induced malignant neu ral rat cell lines as well as EtNU-induced tumors of the central and p eripheral nervous system exhibited intermediate or strong immunoreacti vity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, theref ore, characteristic of both normal glial precursor cells and their mal ignant counterparts in the rat. (C) 1996 Willey-Liss, Inc.