Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis.
DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.
Negroni
A
A
Section of Toxicology and Biomedical Sciences, Ente Nuove Tecnologie e Ambiente (ENEA) Via Anguillarese 301, 00060 Rome, Italy.
Venturelli
D
D
Tanno
B
B
Amendola
R
R
Ransac
S
S
Cesi
V
V
Calabretta
B
B
Raschellà
G
G
D000818:Animals; D017209:Apoptosis; D020134:Catalytic Domain; D002454:Cell Differentiation / Q000235:genetics; D002458:Cell Fractionation; D048429:Cell Size; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000378:metabolism; D017930:Genes, Reporter; D016259:Genes, myc; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054778:NM23 Nucleoside Diphosphate Kinases; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D061107:Nucleoside Diphosphate Kinase D; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; C517939:Nme1 protein, mouse; C517947:Nme4 protein, mouse; D061107:Nucleoside Diphosphate Kinase D; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins