ABSTRACT
OBJECTIVE: Drug-induced differentiation is commonly used as a therapeutic modality for the treatment of neuroblastoma tumors. Increased level of cyclic adenosine 3', 5'-monophosphate (cAMP) mediates terminal differentiation in some neuroblastoma cell lines through activation of several signaling networks, including cAMP response element binding protein (CREB). Objective was to test whether cAMP-induced differentiation in a murine neuroblastoma cell line (NBP2) is partly mediated by CREB. METHODS: Fluorescent microscopy was used to document neuron-like morphological changes imparted by a constitutively active CREB (VP16CREB). Real time PCR (RT-PCR) was performed to verify changes in the expression of cAMP/CREB responsive genes. RESULTS: It was found that transient expression of VP16CREB into NBP2 cells resulted in morphological changes that were characteristics of terminally differentiated neurons. Furthermore, increased expression of cAMP responsive genes was compromised in cells resisting VP16CREB-mediated differentiation. CONCLUSION: A constitutively active CREB induces terminal differentiation in a subset of NBP2 cell population. Altered expression of cAMP responsive genes may account for differentiation resistant phenotype in NBP2 cells.
Subject(s)
Animals , CREB-Binding Protein/genetics , Cell Culture Techniques , Cell Cycle/physiology , Cell Differentiation/genetics , Cell Line, Tumor , Cyclic AMP/genetics , Cyclic AMP Response Element-Binding Protein/genetics , DNA-Binding Proteins , Gene Expression , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Herpes Simplex Virus Protein Vmw65/metabolism , Mice , Neuroblastoma/genetics , Neurons/metabolism , Polymerase Chain Reaction , Receptors, Steroid , Signal Transduction/genetics , Tumor Cells, Cultured/metabolismABSTRACT
Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.