ON THE ANALYSIS OF THE PATHOPHYSIOLOGY OF CHEDIAK-HIGASHI-SYNDROME - DEFECTS EXPRESSED BY CULTURED MELANOCYTES

BACKGROUND: The Chediak-Higashi syndrome (CHS) is a disorder that affe cts the synthesis and/or maintenance of storage/secretory granules in various types of cells. Lysosomes of leukocytes and fibroblasts, dense bodies of platelets, azurophilic granules of neutrophils and melanoso mes of melanocytes are generally larger in size and irregular in morph ology, indicating that a common pathway in storage organellogenesis is affected in patients with CHS. EXPERIMENTAL DESIGN: A pure line of me lanocytes has been established using a 2 cm(2) shave biopsy from a chi ld with CHS, This 4-week-old male patient had oculocutaneous albinism and expressed neutropenia, impaired platelet function, and no natural killer cell activity. The cultured CHS melanocytes were analyzed for c ell biological and biochemical aberrancies. RESULTS: Cultured melanocy tes demonstrated some large and/or complexed melanosomes that resemble d those observed in melanocytes from ultrastructural sections of the b iopsy, Cytoplasmic localization of tyrosinase, tyrosinase-related prot ein-1 and granulophysin (a 40 kilodalton membrane protein originally i dentified as a component in dense bodies of platelets) demonstrated a prominent perinuclear accumulation. The basal synthesis of melanin and the activity levels of tyrosine hydroxylase, dihydroxyphenylalanine ( DOPA) oxidase, or DOPAchrome tautomerase were comparable to control Ca ucasian melanocytes in culture. However, melanin synthesis as well as the catalytic activities of tyrosinase were not dramatically upregulat ed in CHS melanocytes by the addition of isobutyl methylxanthine and c holera toxin in the growth medium when parameters were assayed in cell lysates. In contrast, when assays were performed using live cells, ty rosine hydroxylase demonstrated dramatic upregulation. Medium conditio ned by CHS melanocytes demonstrated phenylthiourea-inhibitable tyrosin ase activity. Melanocyte lysates and conditioned medium analyzed by so dium dodecyl sulfate-polyacrylamide gel electrophoresis and DOPA stain ing showed an extra, approximately 100 kilodalton soluble protein band with DOPA positivity and tyrosinase immunoreactivity. In addition to tyrosinase, one of three lysosomal enzymes assayed (B-glucuronidase) w as aberrantly secreted into the medium. CONCLUSIONS: These results dem onstrate that melanocytes cultured from CHS express a defect in the st ructure and/or function of the melanosome and abnormal trafficking of some cellular proteins.