A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death

Abstract

We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis.

Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma

“…The principal aim of this study was to synthesize conjugates of mAb2C4 antibody including the immunotoxins mAb2C4:dianthin-30, mAb2C4:gelonin and the antibody:drug conjugate (ADC) mAb2C4:MMAE, for comparison of their efficacies to promote cell death in high grade glioma cell lines and U87MG. The validation of mAb2C4, which binds to an extracellular linear epitope (epitope 4) of human CTR, has been presented elsewhere [24].…”

“…These studies involved the use of a mouse monoclonal anti-human CTR antibody [24]) and further data published elsewhere [25].…”

“…In Fig. 4 The opportunity for this study began with our identification of the expression of CTR in glioblastoma and the validation of the antibody mAb2C4 which specifically binds an extracellular epitope of hCTR [24].…”

“…Seck et al [47] originally proposed that CTR is embedded in the endosomal membrane and that this structure is bound to the cytoskeleton through the intermediate filament filamin. We have extended this idea [24] in which carboxyl terminal arginines together with a PDZ docking site are identified in all species and are predicted to be important sites of binding to tubulin. We proposed that the mechanism of uptake of mAb2C4 conjugates was driven by receptor-mediated endocytosis with endosomes trafficked along the microtubules driven by molecular motors as reviewed [48].…”

“…It is expressed by activated lymphocytes [19,20] and during foetal development [21][22][23]. Recently, we have proposed that CTR is externalised during the pre-apoptotic cell stress response [24].…”