Distinct mode of microtubule-associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108-15.
The properties of microtubule-associated protein-2 (MAP-2) expression were examined in a transformed cell line, and compared to neurons from rodent brain where evidence supports both transcriptional and nontranscriptional regulation of MAP-2 synthesis. A monoclonal antibody that recognizes a common epitope in the adult (HMW MAP-2) and juvenile (MAP-2c) forms was used in an immunoblotting assay to assess the protein levels in actively dividing and differentiated neuroblastoma/glioma (108CC15, also designated NG108-15) cells. Multiply-phosphorylated MAP-2c was the predominant form in actively dividing cells, whereas HMW MAP-2 predominated in differentiated cells, which exhibited several other neuronal-like properties. A progressive increase in the levels of immunoreactive HMW MAP-2 was observed with increasing days of cell differentiation using dBcAMP as the inducing agent. However, the absolute levels of both HMW MAP-2 and MAP-2c in NG108-15 cells were significantly lower (at least 10-fold) than levels measured in rodent brain. To assess whether there are correspondingly lower levels of HMW MAP-2 and MAP-2c mRNAs in NG108-15 cells, relative to rodent brain, a highly sensitive RNA amplification assay (reverse transcription-polymerase chain reaction; RT-PCR) was developed. Oligonucleotide primers were designed to specify either HMW MAP-2 mRNA or MAP-2c mRNA, and whole tissue RNA extracted from adult and neonatal rodent brain was used to verify the reliability of the RT-PCR assay. Accordingly, PCR products of the predicted size, specificity, and abundance were obtained, with similar levels of HMW MAP-2 mRNA and proportionately higher levels of MAP-2c mRNA in neonatal brain, relative to adult brain. MAP-2c mRNA was the predominant transcript in actively dividing NG108-15 cells, and the amount of HMW MAP-2 mRNA gradually increased and became the predominant transcript in cells exposed to dBcAMP for 6-9 days. Thus, the observed changes in MAP-2-specific mRNAs during differentiation paralleled changes in expressed protein, suggesting that MAP-2 synthesis in NG108-15 cells is transcriptionally controlled. However, the levels of both MAP-2 mRNAs in NG108-15 cells were comparable to levels in rodent brain, despite the fact that MAP-2 protein levels are at least 10-fold lower in NG108-15 cells. These data suggest that the low levels of HMW MAP-2 and MAP-2c protein expression in NG108-15 cells are not due to correspondingly lower levels of MAP-2 mRNAs, and that transformed neuronal cell lines demonstrate a unique mode of MAP-2 regulation.
Beaman-Hall
C M
CM
Department of Pharmacology, State University of New York/Health Science Center, Syracuse 13210.
Vallano
M L
ML
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015152:Blotting, Northern; D001932:Brain Neoplasms / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D000939:Epitopes / Q000276:immunology; D005910:Glioma / Q000378:metabolism*; D017403:In Situ Hybridization; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000096:biosynthesis; D051381:Rats; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008869:Microtubule-Associated Proteins; D012334:RNA, Neoplasm; D017239:Paclitaxel