Expression of MART-1 in human uveal melanoma cell lines_Chinese Journal of Ocular Fundus Diseases

Authors：

 Wang Yingli , Zhou Yumei , Jin Yangyang , Tao Jun , Chen Ran

Department of Ophthalmology, Emergency General Hospital, Beijing 100028, China;

Corresponding author：

Wang Yingli, Email: wangylnancy@163.com

Keywords：

MART-1 antigen; Choroid neoplasms; Melanoma; Methylation

DOI：

10.3760/cma.j.issn.1005-1015.2019.03.014

Video：

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Objective To observe the expression and transcription of MART-1 in human uveal melanoma cell lines 92-1, 92-2, Ocm3, Me1285, as well as the possible effect of methylation on its expression.Methods The cell lines 92-1, 92-2, Ocm3 and Mel285 were cultured routinely and tested for MART-1 expression at protein and mRNA level by FACS analysis, Western blot and RT-PCR respectively. Methylation status of the MART-1 promoter region in all the cell lines were checked by Southern blots of DNA digested with methylation sensitive restriction enzymes.Results As observed in FACS analysis and Western blot, 92-1, 92-2 and Ocm3 were MART-1 positive cell lines while Me1285 was negative cell line. Consistent with protein analysis, 92-1 and Ocm3 cell lines showed MART-1 specific PCR products and there was no product in Me1285 cell line in RT-PCR. The MART-1 positive cell lines, 92-1, 92-2, and Ocm3 show methylation at the MspI/HpaⅡ site, and the NruⅠ sites of all positive cell lines are not methylated. The MART-1 negative cell line Mel285 shows hypermethylation at the NruⅠsite and the MspⅠ/HpaⅡ site is not methylated.Conclusions MART-1 could be expressed in human uveal melanoma cell lines 92-1, 92-2 and Ocm3. The change of methylation status of MART-1 promoter may correlate with the transcription of MART-1.

Citation： Wang Yingli, Zhou Yumei, Jin Yangyang, Tao Jun, Chen Ran. Expression of MART-1 in human uveal melanoma cell lines. Chinese Journal of Ocular Fundus Diseases, 2019, 35(3): 279-283. doi: 10.3760/cma.j.issn.1005-1015.2019.03.014 Copy

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14.	Danielli R, Ridolfi R, Chiarion-Sileni V, et al. Ipilimumab in pretreated patients with metastatic uveal melanoma: safety and clinical efficacy[J]. Cancer Immunol Immunother, 2012, 61(1): 41-48. DOI: 10.1007/s00262-011-1089-0.
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22.	Venza M, Visalli M, Biondo C, et al. Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma[J]. Biochim Biophys Acta, 2015, 1849(3): 247-256. DOI: 10.1016/j.bbagrm.2014.12.004.

1. 魏文斌. 进一步提高我国脉络膜黑色素瘤的诊断治疗水平[J]. 中华眼底病杂志, 2006, 22(3): 147-149. DOI: 10.3760/j.issn:1005-1015.2006.03.001.Wei WB. More efforts should be made to improve the level of diagnosis and treatment of choroidal melanoma[J]. Chin J Ocul Fundus Dis, 2006, 22(3): 147-149. DOI: 10.3760/j.issn:1005-1015.2006.03.001.
2. Ordóñez NG. Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update[J]. Hum Pathol, 2014, 45(2): 191-205. DOI: 10.1016/j.humpath.2013.02.007.
3. Chodon T, Comin-Anduix B, Chmielowski B, et al. Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma[J]. Clin Cancer Res, 2014, 20(9): 2457-2465. DOI: 10.1158/1078-0432.CCR-13-3017.
4. Spagnoli GC, Zajac P, Marti WR, et al. Cytotoxic T-cell induction in metastatic melanoma patients undergoing recombinant vaccinia virus-based immuno-gene therapy[J]. Recent Results Cancer Res, 2002, 160: 195-201. DOI: 10.1007/978-3-642-59410-6_23.
5. Jungbluth AA. Serological reagents for the immunohistochemical analysis of melanoma metastases in sentinel lymph nodes[J]. Semin Diagn Pathol, 2008, 25(2): 120-125. DOI: 10.1053/j.semdp.2008.05.002.
6. Zubovits J, Buzney E, Yu L, et al. HMB-45, S-100, NK1/C3, and MART-1 in metastatic melanoma[J]. Hum Pathol, 2004, 35(2): 217-223. DOI: 10.1016/j.humpath.2003.09.019.
7. Dunn IS, Haggerty TJ, Kono M, et al. Enhancement of human melanoma antigen expression by IFN-beta[J]. J Immunol, 2007, 179(4): 2134-2142. DOI: 10.4049/jimmunol.179.4.2134.
8. Teullins HE, willemsen KJ, Glykofridis I, et al. The antibody response against MART-1 differs in patients with melanoma-associated leucoderma and vitiligo[J]. Pigment Cell Melanoma Res, 2014, 27(6): 1086-1096. DOI: 10.1111/pcmr.12294.
9. Etzkorn JR, Sobanko JF, Elenitsas R, et al. Low recurrence rates for in situ and invasive melanenomas using Mohs micrographic surgery with melanoma antigen recognized by T cells 1 (MART-1) immunostaining: tissue processing methodology to optimize pathologic staging and margin assessment[J]. J Am Acad Dermatol, 2015, 72(5): 840-850. DOI: 10.1016/j.jaad.2015.01.007.
10. Kurnick JT, Ramirez-Montagut T, Boyle LA, et al. A novel antocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage MART-1 antigen through down-modulation of its promoter[J]. J Immunol, 2001, 167(3): 1204-1211. DOI: 10.4049/jimmunol.167.3.1204.
11. Butterfield LH, Stoll TC, Lau R, et al. Cloning and analysis of MART-1/Melan-A human melanoma antigen promoter regions[J]. Gene, 1997, 191(2): 129-134. DOI: 10.1016/s0378-1119(96)00789-5.
12. Bosch JJ. Immunotherapy of uveal melanoma[J]. Dev Ophthalmol, 2012, 49: 137-149. DOI: 10.1159/000328268.
13. Saleh FH, Crotty KA, Hersey P, et al. Primary melanoma tumour regression associated with an immune response to the tumour-associated antigen melan-A/MART-1[J]. Int J Cancer, 2001, 94(4): 551-557. DOI: 10.1002/ijc.1491.
14. Danielli R, Ridolfi R, Chiarion-Sileni V, et al. Ipilimumab in pretreated patients with metastatic uveal melanoma: safety and clinical efficacy[J]. Cancer Immunol Immunother, 2012, 61(1): 41-48. DOI: 10.1007/s00262-011-1089-0.
15. Tietze JK, Angelova D, Heppt MV, et al. The proportion of circulating CD45RO⁺CD8⁺ memory T cells is correlated with clinical response in melanoma patients treated with ipilimumab[J]. Eur J Cancer, 2017, 75: 268-279. DOI: 10.1016/j.ejca.2016.12.031.
16. Triozzi PL, Aldrich W, Crabb JW, et al. Spontaneous cellular and humoral tumor antigen responses in patients with uveal melanoma[J]. Melanoma Res, 2015, 25(6): 510-518. DOI: 10.1097/cmr.0000000000000207.
17. van Dinten LC, Pul N, van Nieuwpoort AF, et al. Uveal and cutaneous melanoma: shared expression characteristics of melanoma-associated antigens[J]. Invest Ophthalmol Vis Sci, 2005, 46(1): 24-30. DOI: 10.1167/iovs.04-0961.
18. Jager MJ, Magner JA, Ksander BR, et al. Uveal melanoma cell lines: where do they come from? (An American Ophthalmological Society Thesis)[J]. Trans Am Ophthalmol Soc, 2016, 114: 1-16.
19. Martina A, Mieke V, Helen K. Culturing uveal melanoma cells[J]. Ocul Oncol Pathol, 2015, 1(3): 126-132. DOI: 10.1159/000370150.
20. Berdasco M, Gomez A, Rubio MJ, et al. DNA methylomes reveal biological networks involved in human eye development, functions and associated disorders[J]. Sci Rep, 2017, 7(1): 11762-11776. DOI: 10.1038/s41598-017-12084-1.
21. Brantley MA Jr, Harbour JW. Deregulation of the Rb and p53 pathways in uveal melanoma[J]. Am J Pathol, 2000, 157(6): 1795-1801. DOI: 10.1016/s0002-9440(10)64817-1.
22. Venza M, Visalli M, Biondo C, et al. Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma[J]. Biochim Biophys Acta, 2015, 1849(3): 247-256. DOI: 10.1016/j.bbagrm.2014.12.004.

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