Abnormal Translocation of Tyrosinase and Tyrosinase-Related Protein 1 in Cutaneous Melanocytes of Hermansky–Pudlak Syndrome and in Melanoma Cells Transfected with Anti-Sense HPS1 cDNA  Rangaprasad Sarangarajan, Ashish Budev, Yang Zhao, Raymond E. Boissy  Journal of Investigative Dermatology  Volume 117, Issue 3, Pages 641-647 (September 2001) DOI: 10.1046/j.0022-202x.2001.01435.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 HPS melanocytes exhibit DOPA-positive membranous complexes.Ultrastructure of melanocytes in the basal layer of the epidermis from an HPS patient homozygous for the 16 bp duplication in HPS1 (a), an HPS patient heterozygous for an A1195del mutation (b), and a control individual (c). Punch biopsies were processed for DOPA histochemistry and electron microscopy. Melanocytes of the HPS patients exhibited complexes with DOPA-positive cisterna (arrows) and 50 nm vesicles (arrowheads) throughout the periphery of the HPS melanocytes that were absent from the control. K, keratinocyte. Scale bar: (a, c) 2.0 µm; (b) 0.7 µm. Journal of Investigative Dermatology 2001 117, 641-647DOI: (10.1046/j.0022-202x.2001.01435.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of the HPS1 gene product in pigmented melanoma cells by Western blot analysis. SK-MEL-188 (M-188) cells were transfected with human HPS1 cDNA inserted into an EcoRI/EcoRI site of the pcDNA3.1 expression vector in the sense (S-188) and antisense (A-188) orientations. Following selection with G-418, the cells were analyzed for expression of HPS1p by Western blot analysis using HPS-C and p-80 antisera specific for HPS1p. As expected, both the M-188 and S-188 lanes contain a band of approximately 79 kDa using either the HPS-C or HPS-p80 antiserum. In contrast, this band was significantly reduced in the lane containing A-188 cell extract. Journal of Investigative Dermatology 2001 117, 641-647DOI: (10.1046/j.0022-202x.2001.01435.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 In vitro and in situ determination of tyrosine hydroxylase activities of tyrosinase in SK-MEL-188 (M-188) cells transfected with human HPS1 cDNA in the sense (S-188) or antisense (A-188) orientations reveal altered activity. In vitro (cell lysate) and in situ (intact cell) tyrosine hydroxylase activities of tyrosinase in SK-MEL-188 cells transfected with the human HPS1 cDNA in the sense (S-188) and antisense (A-188) orientations were determined as described in Materials and Methods. The in vitro determinations in M-188, S-188, and A-188 were not significantly different (A). Tyrosine hydroxylase activities in A-188 cells were significantly lower (p <0.05), however, compared to M-188 and S-188 as determined by the in situ method (B). Tyrosine hydroxylase activities were expressed in dpm per mg protein (in vitro method) and dpm per cell (in situ method). Journal of Investigative Dermatology 2001 117, 641-647DOI: (10.1046/j.0022-202x.2001.01435.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Melanin content and cellular localization of tyrosinase and TRP-1 was assessed in pigmented melanoma cells (M-188) untransfected and transfected with sense (S-188) or antisense (A-188) human HPS1 cDNA. Approximately equal numbers of cells were centrifuged to form a pellet (a, top panel) and cell suspension (b, bottom panel) photographed to demonstrate large (M-188, S-188) and small (A-188) amounts of melanin pigmentation. Soluble melanin content, expressed in µg melanin per mg protein (c), was also determined. There was a significant reduction in the amount of pigmentation in A-188 cells (a, b) and of soluble melanin in A-188 (c) compared to M-188 and S-188 cells (p <0.05). Cellular localization of tyrosinase and TRP-1 in S-188 and A-188 cells was determined by immunofluorescent techniques. In contrast to M-188 (M column, frames d, g, j) and S-188 (S column, frames e, h, k), both tyrosinase (frames d, e) and TRP-1 (frames g, h) in A-188 cells (A column, frames f, i, l) were localized to distinct large granular structures throughout the cytoplasm and dendrites (frames f, i). Images in frames j, k, and l are the merged images of frames d, e, f with frames g, h, I, respectively, to document colocalization of tyrosinase and TRP-1. Journal of Investigative Dermatology 2001 117, 641-647DOI: (10.1046/j.0022-202x.2001.01435.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Electron microscopy of M-188, S-188, and A-188 cells processed by DOPA histochemistry for the localization of functional tyrosinase. SK-MEL-188 (M-188) cells transfected with sense (S-188) or antisense (A-188) human HPS1 cDNA were processed for electron microscope analysis. DOPA reaction products were present in the trans-Golgi network (block arrows) and melanosomes (block arrowheads) of M-188 (a), S-188 (b), and A-188 cells (c, d). The A-188 cells (c, d), however, also exhibited the presence of DOPA reaction products in large membranous complexes (block arrows in c and d), in contrast to M-188 and S-188 cells. Scale bar: (a, b, c) 0.6 µm; (d) 0.45 µm. Journal of Investigative Dermatology 2001 117, 641-647DOI: (10.1046/j.0022-202x.2001.01435.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions