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

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 th at 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 m ethylation in the TIMP-1 5' regulatory area. In the H-2K(b) 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 inhib itor of histone deacetylase trichostatin A resulted in up-regulation of TIM P-1 expression. These results indicate that methylation and histone deacety lation play an important role in transcription repression of TIMP-1 in BL6 melanoma cells. Some data showed that nitric oxide (NO) could affect methyl ation and expression of various gene. Therefore we analyzed NO production i n B16 melanoma cell lines with different expression of TIMP-1. We have foun d that B16F10 and BL6-8 melanoma cells do not express TIMP-1 and do not pro duce nitric oxide (NO) even after stimulation with IFN-gamma and LPS. Howev er, BL6-8 cells transfected with H-2K(b) or H-2K(d), but not H-2D(d) or H-2 L(d) gene expressed TIMP-1 and produced NO constitutevely. NO production in these cells was further stimulated by IFN-gamma and LPS. Northern blot ana lysis 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 melano ma cells. Thus, TIMP-1 expression and NO production in BL6 melanoma cells t ransfected with MHC class I gene coincides but it remains unclear whether N O is responsible for the change in TIMP-1 methylation and expression.