An atypical caspase-independent death pathway for an immunogenic cancer cell line

Abstract

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.

Apoptotic, necrotic, or fused tumor cells: An equivalent source of antigen for dendritic cell loading

“…Plasma membrane disruption was determined by staining tumor cells with trypan blue or propidium iodide (Sigma). PROb cells treated with Fas-ligand constituted positive control for apoptosis [26].…”

“…Cell extraction and western blot analysis of equal amounts of proteins (30 µg) were performed as described [26]. Protein loading was verified by Ponceau staining.…”

Dendritic Cells Trigger Tumor Cell Death by a Nitric Oxide-Dependent Mechanism

“…Chromatin condensation and fragmentation were monitored after staining with Hoechst 33258 fluorochrome (Sigma-Aldrich) as previously reported (19).…”

Th-1 Lymphocytes Induce Dendritic Cell Tumor Killing Activity by an IFN-γ–Dependent Mechanism

“…LPS (1 µg/ml) - activated DCs were used as a positive control. Tumor cell killing was then evaluated as previously described (4, 5, 35–37). Briefly, the cells were rinsed with PBS and remaining adherent cells were fixed with 95% ethanol and stained with the Crystal Violet dye (100 µl in each well of a 96-well plates) for less than 10 seconds.…”

“…Data were presented as the percentage of relative absorbance calculated from the formula: A test /A control , where A test is the absorbance of tumor cells cultured with DCs in different conditions and A control is the absorbance of tumor cells cultured alone. DCs are very poorly stained with the dye and minimally contribute to the detected absorbance (4, 5, 35–37). …”