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.

Figure 1

Cell lines induced to undergo apoptosis with treatment of staurosporine for 19 h, representative images of >20 separate experiments. Treated cell cultures were live stained with mAb2C4:AF568 and annexin V:AF488 prior to fixation and staining with anti-cleaved caspase 3 antibody. (a–e) A172 cells treated with 1 μM staurosporine: (a) merged image, arrows indicate examples of nuclei from unaffected cells; (b) mAb2C4:AF568; (c) annexin V:AF488, the arrowhead indicates an example with no uptake of mAb2C4:AF568; (d) caspase 3; and (e) DAPI (4,6-diamidino-2-phenylindole). (f–j) GBM-L2 cells treated with 1 μM staurosporine: (f) merged image, arrows indicate examples of nuclei from unaffected cells; (g) mAb2C4:AF568; (h) annexin V:AF488; (i) caspase 3; and (j) DAPI. The arrowheads in panels (g and i) indicate apoptotic cells for which there is little or no annexin V:AF488 signal. (k–o) COS-7 cells treated with 1 μM staurosporine: (k) merged image; (l) mAb2C4:AF568; (m) annexin V:AF488; (n) caspase 3; and (o) DAPI. (p–t) Primary mouse neural precursor cells treated with 0.1 μM staurosporine: (p) merged image; (q) mAb2C4:AF568; (r) annexin V:AF488; (s) caspase 3; and (t) DAPI. (u–x) MG63 cells were treated with 1 μM staurosporine: (u), a merged image; (v) mAb2C4:AF568; (w) annexin V:AF488; and (x) cleaved caspase 3. MG63 cells were treated with 1 μM staurosporine showing merged images with annexin V:AF488 plus ((y), HFEs) and minus ((z), autofluorescence) mAb2C4:AF568. Venn diagrams of relative cell counts for the cell line MG63 and GBM-L2, and overlap for annexin V, mAb2C4:AF568 and caspase 3. The calibration bar shown in panel (z) represents (a–e) 25 μm; (f–j) 80 μm; (k–o) 25 μm; (p–t) 60 μm; (u–x) 25 μm; (y and z) 20 μm.

Figure 2

MG63 cells were treated with cytotoxins for 19 h to induce PCD. Live staining with mAb2C4:AF568 and annexin V:AF488, was followed by fixation and staining with additional primary antibodies (caspase 8, LC3B or α-tubulin) followed by specific species or isotype secondary antibody:AlexaFluors. (a–d) MG63 cells were treated with 1 μM staurosporine: (a), a merged image, with arrows indicating examples of nuclei from unaffected cells; (b) mAb2C4:AF568; (c) annexin V:AF488; and (d) cleaved caspase 8. (e and f) merged images of MG63 cells were treated with 50 μM etoposide+30 μM necrostatin-1 (DAPI (4,6-diamidino-2-phenylindole), blue; mAb2C4:AF568, red; annexin V:AF488, green). (g and h) mAb2C4:AF568 (red channel) and merged image of MG63 cells treated with 50 μM chloroquine to induce autophagy (DAPI, blue; mAb2C4:AF568, red; LC3B, green). (i and j) MG63 cells treated with tumor necrosis factor α and zVAD-fms underwent necroptosis (mAb2C4:AF568, red; annexin V, green). (k) MG63 cells were untreated as controls (DAPI, blue; α-tubulin, green). (l–p) MG63 cells were treated with 1% DMSO+1 μM paclitaxel: (l, DAPI, blue; α-tubulin, green; mAb2C4:AF568, red) induced into the PACSR. (q–t) separate experiment with cells treated with 1% DMSO+1 μM paclitaxel: (DAPI, blue; mAb2C4:AF568, red; α-tubulin, pseudo-green) The calibration bar shown in (t) represents (a–e, k and l) 25 μm and (f–j, m-t) 10 μm.

Figure 3

Flow cytometry (FACS analysis) of Jurkat cells treated with (a) 0.5 μM staurosporine or (b) 50 μM etoposide. In (c), treatments for 24 h were (1) 10 μM rapamycin; (2) 50 μM etoposide; (3) 50 μM etoposide plus pan caspase inhibitor z-VAD-fmk; (4) 15 ng/ml TRAIL; (5) 15 ng/ml TRAIL plus z-VAD-fmk; (6) untreated. (a–c) ■, Annexin V-positive; ■, Annexin V-positive and mAb2C4:AF568-positive; ■, mAb2C4:AF568-positive and ■; double-negative, viable cells.

Figure 4

A model in which CTR is externalized during preapoptotic cell stress. In this model, mAb2C4:AF568 accumulates in the absence of α-tubulin within the lysosomes and with shrinkage of the cytoplasm results in a high fluorescent signal.