Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells

Abstract

Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM.

Keywords: TYR-related protein 1; differential expression; immunocytochemistry; immunofluorescence; uveal melanoma.

Figure 1.

TYRP1 mRNA expression in human UM cells. (A) TYRP1 mRNA expression in the four UM cell lines and the RPE cells was determined by reverse transcription-PCR. (B) The relative quantitative analysis of TYRP1 mRNA expression in the 4 UM cell lines and the RPE cells was performed using quantitative PCR. The data represents three independent experiments and GAPDH was used as the control. TYRP1, tyrosinase-related protein 1; UM, uveal melanoma; RPE, retinal pigment epithelium; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

TYRP1 protein expression in human UM cells. (A) Western blot analysis of TYRP1 protein expression was performed using the TYRP1 antibody in RPE cells and four UM cell lines. (B) The protein bands were analyzed by a gradation scan, and the relative value was obtained from TYRP1/actin. IOD, integrated optical density. TYRP1, tyrosinase-related protein 1; UM, uveal melanoma; RPE, retinal pigment epithelium.

Figure 3.

Immunohistochemical staining of TYRP1 in four UM cell lines and RPE cells. Nuclei were counterstained with Harris (blue), and TYRP1 was visualized with diaminobenzidine (brown). Phosphate-buffered saline was used instead of TYRP1 in the blank control group. Original magnification, x10. TYRP1, tyrosinase-related protein 1; UM, uveal melanoma; RPE, retinal pigment epithelium.

Figure 4.

Immunofluorescence detection of TYRP1 in four uveal melanoma cell lines and RPE cells. Nuclei were stained with DAPI (blue), and TYRP1 was visualized with goat anti-rabbit immunoglobulin G secondary antibody (red). Original magnification, x20. TYRP1, tyrosinase-related protein 1; RPE, retinal pigment epithelium; DAPI, 4′,6-diamidino-2-phenylindole.