Biological and Biochemical Studies on Melanogenesis and M elanoma Cells

For the previous ten years, we have been studying intracellular transport of melanosoma1 proteins and their biological and biochemical functions in melanoma cells. Melanosoma1 proteins,tyrosinase, tyrosinase-related protein 1 (TYRP1) and TYRP2/DOPAchrome tautomerase (DCT), gp100/Pme117 are transported from the trans-Golgi network (TGN) to earlystage melanosomes via endosoma1 compartments. We suggest that Rab7 is involved in the vesicular transport of tyrosinase and TYRP1 and in the melanogenesis through the regulation of gp100/Pme117 maturation. TYRP1 and TYRP 2/DCT were shown to play an essential role in suppressing TYR-mediated cytotoxicity in melanocytic cells, possibly through interaction with TYR in melanosomes. We also studied apoptotic cell death of melanoma cells and death mediators. Among p53 family members, p51A (p63) induced apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and 73β. Interferon(IFN) exerts anti-tumor activities possibly by regulating IFN-stimulated genes. Caspase-2 activation was commonly associated with induction of apoptosis in IFN一β一sensitive melanoma cells. The diacylglycero1 kinase (DGK) , expressed in several human melanoma cell lines but not in melanocytes, was a novel positive regulator of NF-KB, which suppresses TNF一α一induced melanoma cell apoptosis

Identification of rab7 as a Melanosome-Associated Protein Involved in the Intracellular Transport of Tyrosinase-Related Protein 1

The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8–6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3′-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome