Citation

  • Authors: Shivange G. et al.
  • Year: 2021
  • Journal: Cell Rep 37 109953
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: 4T1
      Description: Malignant neoplasms of the mouse mammary gland cells, also known as 4T1-A
    2. Name: A549
      Description: Human lung carcinoma cells, type II pneumocytes
      Known as: A-549
    3. Name: Cavo-3
    4. Name: HCC1806
      Description: Human breast carcinoma cell line
    5. Name: MC-38
    6. Name: MDA-MB-231
      Description: Human breast adenocarcinoma cells
      Known as: MDAMB231
    7. Name: MDA-MB-436
    8. Name: OVCAR-3
      Description: Human ovary adenocarcinoma cells

Method

Transfection of various DR5 constructs into the different tumor cell lines was achieved by jetOPTIMUS DNA transfection reagent for recombinant DR5 cloned in pCDN3.1 vector. In brief, 60%–70% of confluent cells were grown in a 10cm culture dish. Mixing 10ug of plasmid DNA and 10ul of transfection reagent into 1ml of jetOPTIMUS buffer made transfection solution. After incubating for 10min at room temperature, the transfection mix was added on the cells. The cells were further allowed to grow for 24 hours and then selected using 2 mg/ml of G418. In detail, 10 μg DNA was diluted into 1000 μL jetOPTIMUS buffer and vortexed. This was followed by the addition of 10 μL jetOPTIMUS into the DNA solution (ratio 1:1 corresponding to μg DNA: μL reagent) and vortexed and spun down briefly. The mixture was incubated for 10 minutes at room temperature. Next, the transfection mix was added dropwise onto the cells in a serum-containing medium and distributed evenly. Plates were incubated at 37°C for 24hrs. The next day transfection medium was replaced with by cell growth medium and cells were allowed to grow for another day before starting G418 (2 mg/ml) selection. Media was changed every day, and a reliable GFP signal was evident 72 hours of transfection.

Abstract

Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.

Pubmed