RESUMO
Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined.
Assuntos
Antígenos de Neoplasias/imunologia , Apoptose/efeitos dos fármacos , Imunoglobulina M/farmacologia , Neuroblastoma/patologia , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Neoplasias/fisiologia , Neoplasias Ósseas/patologia , Galinhas , Fragmentação do DNA , Citometria de Fluxo , Cabras , Humanos , Imunização Passiva , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Imunoglobulina M/uso terapêutico , Imunoglobulinas/imunologia , Masculino , Melanoma/patologia , Camundongos , Peso Molecular , Transplante de Neoplasias , Neuroblastoma/imunologia , Neuroblastoma/terapia , Osteossarcoma/patologia , Ratos , Ratos Nus , Rabdomiossarcoma/patologia , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Cerulenin, a fungal metabolite, is known to be a specific inhibitor of fatty acid synthase. Here we report that cerulenin is an effective inducer of apoptosis in different wild-type p53 and mutant p53 tumor cell lines, whereas normal human keratinocytes and fibroblasts are resistant to the apoptotic effect. To get more insight into the mechanisms of how cerulenin induces apoptosis we investigated several signal transduction molecules, including p53, p73, p21/WAF1, Bax, cytochrome c, and caspases 3 and 9. Our data strongly indicate that mitochondria play a key role in the cerulenin-mediated pathway. Bax overexpression correlated with the extent of apoptosis and appears to be regulated in a p53-independent manner. The significance of the mitochondrial pathway for the cerulenin-mediated apoptosis was confirmed by the rapid mitochondrial release of cytochrome c both in wild-type p53 and mutant cell lines. Interestingly, the rapid release of cytochrome c was not accompanied by a breakdown of the mitochondrial potential. Instead, the complete disruption of the mitochondrial function coincided with the appearance of a p18 kDa cleavage product of Bax.
Assuntos
Apoptose/fisiologia , Cerulenina/farmacologia , Citotoxinas/farmacologia , Células Eucarióticas/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Células Tumorais Cultivadas/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 9 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/efeitos dos fármacos , Ciclinas/metabolismo , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/efeitos dos fármacos , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Genes Supressores de Tumor , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mutação/efeitos dos fármacos , Mutação/genética , Neoplasias/tratamento farmacológico , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Proteína X Associada a bcl-2RESUMO
BACKGROUND: Human neuroblastoma (NB) cells contain a 260 kDa surface antigen (NB-p260), which serves as receptor for natural human IgM antibodies (anti-NB IgM). Upon binding to NB-p260, these antibodies induce apoptosis in human NB cells. PROCEDURE AND RESULTS: In this study, we purified NB-p260 to homogeneity from human LA-N-1 NB cells by sequential ion exchange chromatography followed by preparative SDS gel electrophoresis. Purified NB-p260 exhibited rapid autodegradation despite the presence of various protease inhibitors. The autodegradation process precluded extensive N-terminal sequencing. However, from repeat N-terminal sequence analysis, a consensus sequence of seven amino acid residues emerged that exhibited significant homology to the subunit c of the human mitochondrial ATP synthase, a hydrophobic membrane protein of 7.6 kDa. Western blot analyses demonstrated that purified NB-p260 was recognized by polyclonal antibodies raised against both subunit c-containing storage bodies and a synthetic peptide consisting of amino acid residues 32-45 of subunit c. In addition to peptide sequences related to subunit c, NB-p260 also contained epitopes related to the human heat shock protein HSP90. In Western blots, a monoclonal anti-HSP90 antibody reacted with purified NB-p260 as well as with a predominant protein fragment of approximately 90 kDa that appeared during the process of NB-p260 autodegradation. The anti-HSP90 antibody was also capable of binding to the surface of LA-N-1 cells and inhibiting the binding of human anti-NB IgM in a dose-dependent manner. CONCLUSIONS: Collectively, our data suggest that NB-p260, the apoptosis-mediating receptor for natural human anti-NB IgM, represents a novel surface protein of human NB cells containing polypeptide sequences related to the subunit c of the mitochondrial ATP synthase and the heat shock protein HSP90.