Characterization of Monoclonal Antibodies Against GRIM-19, a Novel IFN-βand Retinoic Acid-Activated Regulator of Cell Death

Abstract

A combination of interferon-beta (IFN-beta) and all-trans retinoic acid (IFN/RA) induces tumor cell apoptosis via some unknown mechanisms. Apoptosis is a gene-directed process that limits the proliferation of undesired cells. Several genes are required to regulate cell death in the higher-order animals. Earlier, we employed a gene expression knockout technique to isolate cell death-related genes. A novel gene, the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), was found to be essential for tumor cell death induced by IFN/RA. Here, we describe the development and characterization of three monoclonal antibodies (mAbs) against GRIM-19. GRIM-19 is present in the nucleus and cytoplasm. Its expression is induced by the IFN/RA combination. We also show that GRIM-19 inhibits the cell-transforming property of viral oncogenic protein viral IFN regulatory factor-1 (vIRF-1) via a physical interaction. mAbs developed in this study should be useful for studying the other physiologic roles of GRIM-19 and serve as a potent tool for studying tumor responses to IFN/RA therapy.

Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

“…It has been shown to interact exclusively with the transcriptional activating domain of Stat3 and inhibited transcriptional activation. 27,28,38 Recent studies have also shown that GRIM-19 expression is reduced or eliminated in a number of primary tumors. 29,35 Survivin is a downstream target of Stat3.…”

“…A mouse monoclonal antibody against GRIM-19 was described earlier. 27 The criteria for grading immunohistochemical data for Survivin and GRIM-19 were similar to those described earlier. 28 Semiquantitative reverse transcription-PCR (RT-PCR) mRNA levels were determined using semiquantitative RT-PCR.…”

“…GRIM-19 antibody was applied as before. 27 One percent of bovine serum albumin was used as control antibody. The absorbance of each band in western blots was quantified using densitometry, and the results are shown as relative expression of each protein from different samples.…”

“…The U6 promoter and the sequence of Survivin-shRNA or Scramble-shRNA were then PCR amplified with primers: P1, 59-GC(AGATCT)TGCTTCGCGATGTACGGGCC-39; P2, 59-CG(TCGCGA)GGGCTATGAACTAATGACCC-39, then cloned into T vector to construct plasmids. The human GRIM-19 open reading frame was PCR amplified using the pCXN2mycA GRIM-19 plasmid 27 as a template with primers P3, 59-AGGTACCATGGCG-GCGTCAAAGGTGA-39 and P4, 59-ACGAATTCCTACGAATTCC-TACGTGTACCACATGAAGCC-39, then cloned into KpnI and EcoRI sites of plasmid pcDNA3.1(1) to construct a plasmid pGRIM-19, in which the GRIM-19 gene was placed under the control of a cytomegalovirus enhancer. T vector was digested with BglII and NurI enzymes and cloned into BglII and NurI sites of pGRIM-19 to generate pGRIM-19-si-Survivin, which could co-express both shRNA-Survivin and protein GRIM-19.…”

Sinorhizobium meliloti nifA mutant induces different gene expression profile from wild type in Alfalfa nodules

“…However, expression patterns of up-regulated G-protein-coupled receptors (G7) and down-regulated G-protein α-subunit (G16) indicate a complex regulation pattern of G proteins in nodules. The retinoid-interferon-induced mortality-19 gene (GRIM-19) and cytokine IK are novel cell death regulators characterized only in animals [43][44][45]. Further studies on their homologous genes in plants may provide new insights into apoptotic plant cell death.…”