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Nitric oxide (NO), methylation and TIMP-1 expression in BL6 melanoma cells transfected with MHC class I genes

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Abstract

We have previously found that transfection of BL6-8 melanoma cells with the H-2K, but not H-2D/L genes resulted in loss of their metastatic ability that was associated with decrease in their invasiveness and up-regulation of TIMP-1 expression. In the present study using the methylation-specific PCR (MSP) we found that lack of TIMP-1 expression in BL6-8 is associated with methylation in the TIMP-1 5′ regulatory area. In the H-2Kb transfected CL8-1 melanoma cells up-regulation of TIMP-1 was in parallel with loss of TIMP-1 gene methylation. Treatment of BL6-8 with 5-azacytidine or with an inhibitor of histone deacetylase trichostatin A resulted in up-regulation of TIMP-1 expression. These results indicate that methylation and histone deacetylation play an important role in transcription repression of TIMP-1 in BL6 melanoma cells. Some data showed that nitric oxide (NO) could affect methylation and expression of various gene. Therefore we analyzed NO production in B16 melanoma cell lines with different expression of TIMP-1. We have found that B16F10 and BL6-8 melanoma cells do not express TIMP-1 and do not produce nitric oxide (NO) even after stimulation with IFN-γ and LPS. However, BL6-8 cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld gene expressed TIMP-1 and produced NO constitutevely. NO production in these cells was further stimulated by IFN-γ and LPS. Northern blot analysis showed that expression of iNOS was paralleled with TIMP-1 expression in the tested melanoma cells. However, NO produced by SNAP or inhibition of NO production by NMA did not affect TIMP-1 expression in the tested melanoma cells. Thus, TIMP-1 expression and NO production in BL6 melanoma cells transfected with MHC class I gene coincides but it remains unclear whether NO is responsible for the change in TIMP-1 methylation and expression.

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Authors and Affiliations

  1. University of Pittsburgh Cancer Institute and Department of Pathology, University of Pittsburgh, Pennsylvania, USA

    Xiaojun Huang, Mengfeng Li & Elieser Gorelik

  2. Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Amila Orucevic

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  1. Xiaojun Huang
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  2. Amila Orucevic
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  3. Mengfeng Li
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  4. Elieser Gorelik
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Huang, X., Orucevic, A., Li, M. et al. Nitric oxide (NO), methylation and TIMP-1 expression in BL6 melanoma cells transfected with MHC class I genes. Clin Exp Metastasis 18, 329–335 (2000). https://doi.org/10.1023/A:1010867618014

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