A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs / 药物分析学报

Suppression of cellular O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) can repress prolifera-tion and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells.The fluorescent reporter mainly consists of a reporter (green fluorescent protein (GFP)),an internal reference (red fluorescent protein),and an operator (neuronal differentiation 1),which serves as a "sweet switch" to control GFP expression in response to cellular O-GlcNAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-GlcNAcylation in HeLa and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for con-ducting gene transcription studies.

IL-12 p40-Expressing Immune Cells Revealed by Cytokine Reporter Mouse System / 체질인류학회지

Interleukin-12 (IL-12), consisting of p35 and p40, plays important roles in linking innate and adaptive immunity. While p35 is constitutively expressed, IL-12 p40 gene expression is induced upon activation by Toll-like receptor ligands. Recently, with gene targeting technology, the cytokine IL-12 p40 reporter mouse has been developed to express the p40 gene linked via a viral IRES element with yellow fluorescence protein (YFP) fluorescent reporter. We investigated whether this novel system would be useful to reveal IL-12 p40-producing immune cells. We first investigated whether macrophages and dendritic cells from these mice faithfully reported p40 induction. Next, we tested if microglial cells, macrophages in the brain, could induce IL-12 p40. Finally we tested whether B cells could produce IL-12 p40 because there were very few reports for IL-12 production by B cells. Our results confirmed that macrophages and dendritic cells are main producer of IL-12 p40. Then, we found that microglial cells could produce IL-12 p40 upon stimulation with various TLR ligands. Finally we found that a subset of B cells could produce IL-12 p40 in TLR9-dependent manner. Taken all together, our system will be a valuable tool to identify the type of immune cells that produce IL-12 p40.

The Immunostimulatory Effect of B16 Freezing/thawing Anti-tumor Vaccine / 체질인류학회지

Since cancer has become the second most common cause of death, next to heart disease and approximately 20% of human population dies from cancer, it is much desired to develop therapeutic anti-tumor vaccine with safety and efficacy. Here we investigated the immunostimulatory effects of B16 freezing/thawing (F/T) anti-tumor vaccine (hereafter F/T vaccine), one of whole cell anti-tumor vaccines. To this end, we took advantage of the IL12 p40 reporter system which is designed for monitoring the induction of IL12 expression via the detection of co-expressed yellow fluorescent protein. First, we examined whether F/T vaccine can induce IL12 expression using bone marrow-derived dendritic cells (BMDCs) from IL12 p40 reporter mice. Second, we examined whether F/T vaccine can change the expression level of MHC molecules and co-stimulatory molecules during the activation of dendritic cells. Third, to dissect what component of F/T vaccines accounts for the immunostimulatory activities, we examined the effect of F/T vaccine on BMDC activation after treating it with DNase or proteinase. Lastly, we used MyD88 knockout mice to investigate whether F/T vaccine activates BMDCs in a TLRdependent manner. We found that treatment of BMDCs with F/T vaccine induced IL12 expression as well as the increase of MHC II expression and co-stimulatory molecules such as CD86. Interestingly, we also found that F/T vaccine increased CD1d expression on BMDCs, which may influence the activation of natural killer T cells known to be involved in anti-tumor immune responses. In addition, we found that treatment of F/T vaccine with proteinase but not DNase abolished its immunostimulatory effect, indicating that proteins in F/T vaccine mainly have its adjuvant activity. Furthermore, the activation of BMDCs with F/T vaccine was dependent on MyD88 adaptor molecule. Taken together, our findings in this study demonstrated that the F/T vaccine might be one of the valuable reagents to provide a new insight for underlying mechanism of whole-cell anti-tumor vaccines and an important clue for the development of better therapeutic anti-cancer vaccines.