Distinct mode of microtubule‐associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108‐15

Abstract

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.

The angiotensin type 2 receptor of angiotensin II and neuronal differentiation: from observations to mechanisms

“…Furthermore, addition of H-89, an inhibitor of PKA, or Rp-8-Br-cAMPS, an inactive cAMP analog, failed to impair p42/p44 mapk activity and neurite outgrowth induced by Ang II. These observations indicate that the AT 2 signaling is independent of cAMP, and further indicate that neurite elongation induced by cAMP did not involve the Rap1/B-Raf pathway of signaling, at least in the NG108-15 cells (Hamprecht et al 1985, Beaman-Hall & Vallano 1993, Cowley et al 1994, Laflamme et al 1996. The mechanisms involved in the AT 2 -induced Rap1/B-Raf activation remain unclear but it could be hypothesized that the AT 2 receptor transactivates other receptors, such as growth factor receptors.…”

Cyclic AMP-independent Involvement of Rap1/B-Raf in the Angiotensin II AT2 Receptor Signaling Pathway in NG108-15 Cells

“…In several models, cAMP and cAMP-activated proteins appeared to be of great importance, and many studies have reported the requirement of PKA for NGF activation of Rap1 as well as for p42/p44 mapk (47). Indeed, several studies indicate that treatment of cells with cAMP or analogs induced neurite outgrowth, both in PC12 (44) and in NG108-15 cells (24,42,43). Activation of Rap1 by cAMP favored its association with Raf-1, consequently inhibiting the transient p21 ras -dependent activation of p42/p44 mapk (66).…”

“…Moreover, NGF stimulated Rap1 activity via the exchange factor Crk/C3G and was also shown to activate this pathway through a cAMP-dependent mechanism, involving (51) or not involving protein kinase A (52). Similar mechanisms could also be considered for NG108-15 cells because chronic treatment with cAMP analogs is a well known differentiating factor for these cells (42,43). The aim of the present study was to investigate whether the Rap1/B-Raf pathway could be involved in the Ang II-induced sustained increase in p42/p44 mapk activity participating in the morphological differentiation of the NG108-15 cells and to verify whether cAMP could be involved in the signaling pathway of the AT 2 receptor.…”

“…In their undifferentiated state, neuroblastoma ϫ glioma hybrid NG108-15 cells have a rounded shape and divide actively. It is now well documented that chronic exposure to dibutyryl cAMP induces a process resulting in both morphological and functional differentiation (42,43). We have shown previously that these cells express only the AT 2 receptor of Ang II (6,24) and that a 3-day treatment with Ang II or CGP42112 induced neurite outgrowth (24), through a mechanism involving p21 ras inhibition, and a sustained increase in p42/p44 mapk activity (41).…”

Effects of dibutyryl cAMP on stanniocalcin and stanniocalcin-related protein mRNA expression in neuroblastoma cells

“…The primers were designed on the basis of the published cDNA sequence of mouse STC: ATGTCCA AAACTCAGCAGTGATTC-forward and ACACTCA AAGTTGGTGTG-reverse ; STCrP: AGAATCCAGGTCTCCAGGCG-forward and TGT GACATCCCCCCTCTTTAATGT-reverse (Chang & Reddel 1998, Ishibashi et al 1998; G3PDH: ATGGT GAAG GTCGTGTGAAC-forward and TTCAAGAGA GTAGGGAGGGC-reverse; MAP-2c: AACGGAGAG CTGACCTCAGCTGACA-forward and TCCGTC CTGAAGAGATGGAGCT-reverse (Beaman-Hall & Vallano 1993). The PCR was run for 35 cycles with a 56 C or 60 C (MAP-2c) annealing cycle (1 min), a 72 C extension cycle (3 min), and a 95 C denaturing cycle (50 s), plus final incubation at 72 C for 10 min.…”