The anti-RET antibodies were reactive with 64-kDa (p64ptc-1) and 81-kDa (p81ptc-2) proteins from lysates of ptc-1 and ptc-2 transformed cells, respectively, and identified two proteins of 140 kDa and 160 kDa from extracts of SK-N-SH, a neuroblastoma cell line previously shown to express two differently glycosylated forms of the normal RET product.
We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos.
We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation.
NB-39-nu neuroblastoma cells show high expression and elevated tyrosine phosphorylation of RET, although short interfering RNA against RET (RET siRNA) did not significantly inhibit cell proliferation or suppression of basal levels of phosphorylation of extracellular regulated kinase (ERK)1/2 or protein kinase B (AKT).
The level of tyrosine phosphorylation of the 150 kDa proto-Ret protein was approximately 10-fold higher than that of the 170 kDa proto-Ret protein, although both proteins were expressed at similar levels in neuroblastoma cells.
Transfection experiments using human neuroblastoma cells showed that the mutant RET, unlike the wild-type receptor, is constitutively phosphorylated in the absence of ligand, and thus resembles other previously characterized MEN 2A mutations.
Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.
Furthermore, the antibodies detected a unique 190 kd protein as well as 150 kd protein in a cell lysate from THP-1 human monocytic leukemia cell line, suggesting that glycosylated forms of the c-ret protein are different between neuroblastoma and leukemia cells.
These results indicate that the positive transcriptional regulation of RET is closely associated with early neuronal differentiation and suggest that a negative regulatory factor/s controls RET transcription in neuroblastoma cells.
To better understand the role of the activated RET oncogene in neural crest cells, we transfected two adherent human neuroblastoma tumor cell lines with oncogenic MEN2 mutant RET cDNAs.
RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up-regulates RET expression in neuroblastoma cell lines.
In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.
Identification and analysis of the ret proto-oncogene promoter region in neuroblastoma cell lines and medullary thyroid carcinomas from MEN2A patients.
Furthermore, there is strong evidence against linkage to two Hirschsprung disease (a condition that can cosegregate with neuroblastoma) susceptibility genes, RET and EDNRB.
Consistent with this result, ZAR1-depleted NB cells showed well-differentiated phenotypes with elongated neurites and upregulated expression of TRKA and RET, which are markers for differentiated NB.
In the present work, we have studied the ability of GDNF proteins, each bearing one of the reported mutations, to activate RET by performing a functional test in cultured neuroblastoma cells.
These analyses were performed using both RET cDNA cloned from a pheochromocytoma library and reverse transcriptase PCR products generated using RNA from a neuroblastoma cell line (LA-N-2).
Taken together, these results indicate that GDNF up-regulates the expression of the TH gene by promoting the transcription of the TH gene and the stability of TH mRNA with the Ret receptor dependency in some neuroblastoma cell lines.