Effect of 3-methyladenine on autophagy in transgenic rice cell suspension cultures

초록

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in transgenic rice cell cultures (Oryza sativa L.) using RAmy3D promoter. Due to the inducing property of the promoter, it was necessary to change the growth medium into sugar-free medium to produce hCTLA4Ig. However, depletion of carbon source cause autophagy, which is known as type II programmed cell death. There are many reports regarding the autophagy in mammalian cells and tobacco BY-2 cell. Sucrose-free medium for the production of hCTLA4Ig in rice cell cultures can cause autophagy. Such autophagy formed double membrane-bound structure called autophagosomes and the intracellular organelles are degraded through the lysosomal machinery. In the previous study, we found that autophagosomes were produced from day 2 and the cell viability also decreased sharply. In this study, we investigated the effect of 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, on the autophagy of rice cells in culture. The addition of 5 mM 3-MA to the culture media was reported to block cellular protein degradation in tobacco BY-2 cell cultures. Therefore, we thought that 3-MA might be useful for the enhanced production of hCTLA4Ig and cell viability in transgenic rice cultures. Effect of 3-MA on the production of recombinant protein are going to be discussed.