MUNO Daisaku JUNTENDO UNIVERSITY, DEPARTMENT OF BIOCHEMISTRY, RESEARCH ASSISTANT, 医学部, 助手 (00146771)
TANIDA Isei JUNTENDO UNIVERSITY, DEPARTOEMTNOF BIOCHEMISTRY, ASSISTANT PROFESSOR, 医学部, 講師 (30296868)
|Budget Amount *help
¥14,300,000 (Direct Cost : ¥14,300,000)
Fiscal Year 2003 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 2002 : ¥9,300,000 (Direct Cost : ¥9,300,000)
Autophagy is a universal mechanism, by which bulky cell proteins are degraded via lysosomal/vacuolar system. It has been demonstrated that more than 15 autophagy-related genes (ATG genes) are essential to this process and that numbers of the ATG gene products including Atg7, Atg3, Atg 10, Atgl2, Atg5, and Atg8 operate in two ubiquitin-like conjugation pathways. In this paper, we report that in some cultured melanoma cell lines such as B16-F0, B16-F1, M3, and G581, Atg7 and Atg12-Atg5 conjugate are abundantly expressed at extremely high levels. In order to clarify functional relevance of the elevated levels of these ATG gene products to possible enhancement of autophagy in melanomas, we further investigated the two Atg8 homologues, LC3 and GABARAP whose phospholipid-conjugated forms (LC3-II and GABARAP-II) are recruited to autophagosomal membranes and subsequently degraded after fusion of autophagosome with lysosome during starvation-induced autophagy. In B16-F1 melanoma, both LC3-II and GABARAP-II accumulated markedly when lysosomal proteolysis was inhibited with E64d and pepstatin. However, the accumulation occurred under not only nutrient-deprived condition but also nutrient-rich condition. Thus, LC3-II-or GABARAP-II-loaded membranes are continuously turned over via lysosomes in ordinary culture medium containing abundant amino acids and fetal calf serum. In cell fractionation analysis using discontinuous OptiPrep gradients, distribution of the accumulated LC3-II and GABARAP-II was found to well coincide with those of lysosomal makers (cathepsin L and LGP120) and melanosomal markers (tyrosinase and TRP-1). These biochemical data were further confirmed by immunofluorescence analyses. Taken together, these data indicate that LC3-II and GABARAP-II on melanosomal membranes facilitate melanosomes to fuse with lysosomes to be degraded in cultured B16-F1 melanoma.