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T. S. Cunneen, G. Anastassiou, R. M. Conway, M. Madigan; Expression of Matrix Metalloproteinase (MMP) and Tissue Inhibitors of Metalloproteinase (TIMP) mRNA in Conjunctival Melanoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4801.
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© ARVO (1962-2015); The Authors (2016-present)
Conjunctival melanoma is a rare disease with a poor prognosis whose distinct pathogenesis has been minimally studied. The observed survival in patients with conjunctival melanoma has not improved in recent times. Many adjuvant treatments are now being examined. MMPs and TIMPS are proteolytic enzymes important in tumor angiogenesis, invasion and metastasis. The expression of MMPs and TIMPs in conjunctival melanoma, and their role in tumour progression has not been investigated. In this study we examined the mRNA profile of several classes of MMPs and TIMPs in two recently established conjunctival melanoma cell lines.
Two conjunctival melanoma cell lines (CRMM-1 and CRMM-2) established from recurrent bulbar conjunctival melanoma were used. Melanocyte origin was confirmed using HMB45+HMB50 (Ab3, Neomarkers) immunocytochemistry. Total RNA was isolated from cells cultured in serum-free medium, and qualitative reverse-transcriptase PCR (RT-PCR) performed to assess the mRNA expression of MMPs 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20 and TIMPs 1, 2, 3, and the MMP-inducer, EMMPRIN. Gelatin zymography was performed to investigate gelatinolytic activity (MMP-2 and -9) in serum-free supernatants from control and 50µM phorbol 12-myristate 13-acetate (PMA) stimulated cells.
Both cell lines expressed mRNA for the same classes of MMPs and TIMPs. Collagenases MMP-1 and -8 were detected while collagenases MMP-13 and MMP-18 were not. Membrane type MMPs 1-4 were detected. The stromelysin/matrilysin MMP-11 was detected but stromelysins MMP-3 and -10 were not found. The neutrophil associated metalloelastase MMP8 was also expressed. MMP-12, -19 and -20 were not detected. mRNA for TIMP-1, -2 and -3 was detected as was mRNA for EMMPRIN. Low levels of latent MMP-2 (72kD) and MMP-9 (90kD) gelatinase activity were detected by zymography, and increased slightly with PMA stimulation up to 72 hours.
mRNA for several classes of MMPs and TIMPs, and EMMPRIN (an MMP-inducer) were expressed in conjunctival cell lines. The low levels of secreted MMP-2 and -9 activity suggest that conjunctival melanoma growth is dependent on other classes of MMPs. Characterisation of these enzymes is important in our understanding of the distinct pathogenesis of these rare but serious tumours.
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