Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis.
Highly malignant neuroblastoma tumors generally have defects in differentiation and apoptotic pathways. For a better understanding of these events, we use a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons in response to elevated levels of cAMP. Because one of the main downstream effectors of the cAMP signaling pathway is cAMP-response element binding (CREB), we reasoned that it might affect the expression of genes associated with differentiation and apoptotic events in NBP2 cells. To investigate this, we established tetracycline-regulated expression (TetOff) of VP16CREB, which constitutively transactivates promoters containing the CRE sequence motif. Using this system, we found that inducible expression of VP16CREB in NBP2 cells results in 1) morphological differentiation that is characterized by the formation of neurites and growth cones, 2) reversible cell differentiation unlike cAMP-induced terminal differentiation, 3) cell cycle arrest at G1, 4) no apoptosis in the presence of partial inhibition of proteasome unlike an increase in cAMP levels, and 5) changes in the expression of many genes, including down-regulation of N-myc, cyclin B1, Dickkopf-1, and Mad-2 and up-regulation of tyrosine hydroxylase, c-fos, N10, and ICER genes. Although VP16CREB expression and activation of the cAMP pathway impart many similar effects in NBP2 cells, they also bear some distinct genetic and morphological differences. Our data suggest that increased levels of cAMP function through not only CREB but also other signaling pathways that account for the additional cAMP-induced effects, including irreversible differentiation and onset of apoptosis during partial inhibition of proteasome in NBP2 cells.
Center for Vitamins and Cancer Research, Department of Radiology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Cynthia P
CP
Andreatta
Piruz
P
Nahreini
Amy J
AJ
Hanson
Kedar N
KN
Prasad
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western / Q000379:methods; D003994:Bucladesine / Q000494:pharmacology; D002452:Cell Count / Q000379:methods; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D005822:Genetic Vectors / Q000378:metabolism; D023281:Genomics / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D003994:Bucladesine; C022616:thiazolyl blue