Enhancement of interferon-gamma-induced major histocompatibility complex class II gene expression by expressing an antisense RNA of poly(ADP-ribose) synthetase.

Abstract

We studied the effect of poly(ADP-ribose) synthetase on the interferon-gamma (IFN-gamma)-inducible expression of major histocompatibility complex (MHC) class II molecules by expressing an antisense RNA of poly(ADP-ribose) synthetase. We constructed two expression plasmids capable of expressing an antisense RNA for poly(ADP-ribose) synthetase, carrying 0.7-kilobase long fragment of 5'-coding region (pAS-5') and full-length cDNA (pAS-FL) of poly(ADP-ribose) synthetase in an antisense orientation under control of metallothionein I promoter. We transfected the plasmid into human leukemia THP-1 cells and isolated transformants. Metal-inducible reduction in poly(ADP-ribose) synthetase activity was observed in two pAS-5'-transfected clones out of 72 neo-resistant clones examined, and metal-independent reduction in the activity was exhibited in pAS-FL-transfected clones. The antisense RNA was induced in a metal-dependent manner in the clones transfected with pAS-5', as judged by hybridizing with a sense riboprobe of the synthetase gene. The mRNA of the synthetase decreased 1 day after an addition of metal ions, and the synthetase activity of the transformants decreased by more than 90% 3 days after an addition of metal ions. Thus, we incubated the transformant clones in the presence of metal ions for 3 days and then treated them with IFN-gamma. The IFN-gamma-inducible expression of MHC class II molecules was amplified in the transformant clones, as judged by RNA blot analysis and flow cytometry. These results indicate that the decrease in poly(ADP-ribose) synthetase makes THP-1 cells favorable to induce MHC class II molecules by IFN-gamma.