A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells.
Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4DeltaN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4DeltaN1 increased the p53 protein level, but decreased the p21(Cip1) transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4DeltaN1-binding protein. MAGE-A4DeltaN1 was recruited through association with Miz-1 to the p21(Cip1) promoter and down-regulated transcription of p21(Cip1). In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4DeltaN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4DeltaN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4.
Sakurai
Toshiharu
T
Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan.
Itoh
Katsuhiko
K
Higashitsuji
Hiroaki
H
Nagao
Toshikazu
T
Nonoguchi
Kohsuke
K
Chiba
Tsutomu
T
Fujita
Jun
J
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D017209:Apoptosis; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015696:Gene Products, tat / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D020287:In Situ Nick-End Labeling; D007425:Intracellular Membranes / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins; D010957:Plasmids / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D051020:bcl-X Protein
D000590:Amino Acid Chloromethyl Ketones; D000951:Antigens, Neoplasm; C494811:BCL2L1 protein, human; C494812:Bcl2l1 protein, mouse; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D051741:Kruppel-Like Transcription Factors; C095204:MAGEA4 protein, human; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C107961:ZBTB17 protein, human; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004317:Doxorubicin; D020169:Caspases