Histone deacetylase inhibitors up-regulate the expression of cell surface MHC class-I molecules in B16/BL6 cells.

Abstract

In a previous report, we showed that anthracyclines up-regulate the cell surface expression of MHC class-I molecules in B16/BL6 cells1). By using the same assay system, we found an other type of MHC class-I upregulator in the culture broth of a newly isolated soil bacterium, Bacillus megaterium 6N38. Here we report the isolation and characterization of this up-regulator, and its possible molecular mechanism. Starting from 1 liter of culture supernatant of Bacillus megaterium 6N38, the MHC class-I up-regulator was purified. After the pH of the supernatant had been adjusted to 13 with NaOH, solvent extraction was performed with 1 liter of dichloromethane. The water fraction was adjusted to pH 2 with HCl, and extracted 3 times with 500ml of dicholoromethane each time. The organic fraction was condensed in vacuo to give 2.81g of dark brown oil. The extract was applied onto a silica gel column (bed volume=147ml) equilibrated with dicholoromethane, and eluted with 1 liter of the same solvent. The eluate was collected and evaporated to give 1.97g of a yellowish oil. The oil was applied to the second silica gel column (bed volume=100ml) equilibrated with cyclohexane, and then eluted stepwisely with 250ml of cyclohexane, 500ml of cyclohexane: dichloromethane=8:2, and 700ml of cyclohexane: dichloromethane=6:4. The last fraction was evaporated to give 880mg of pale yellowish oil. The sample was finally purified by reverse-phase HPLC using a TOSO HPLC system (SC-8010, UV-8010, COMP) equipped with a semi-preparative column (Nacalai Tesque Cosmosil 5C18-AR, i. d.=22mm, length=25cm). The elution pattern was monitored by measuring the absorbance at 210nm. The elution condtions were as follows: solvent A, 0.1M sodium phosphate (pH=2); solvent B, equal mixture of 0.05M sodium phosphate (pH=2) and acetonitrile; linear gradient from B=0% to 100% over 100 minutes; flow rate, 5ml/minute. The active peak was collected, extracted by dichloromethane, and evaporated in vacuo to afford 20.1mg of a colorless oil (compound 1). The molecular ion peak of 1 detected by FAB-MS was 101 ([M-H]-ion), indicating the molecular weight of 1 to be 102. The 1H and 13C NM R spectra of 1 could be explained and assigned consistently, assuming com-