Interferon-gamma induces a dual and alternative early biological response in neuroblastoma cells: differentiation and apoptosis

Interferon-gamma (IFN-gamma) is a well known differentiation inducer for many neuroblastoma (MB) cell lines in vitro and its activity is currently being evaluated in vivo. In sensitive cells, this process involves progressive loss of cell proliferation, loosening of cell clusters, a tighter adhesion of cells to their substrate, often accompanied by emission of dendritic and neuritic processes. Moreover, changes in the expression of peculiar cytoskeletal proteins are often observed. Depending on the model employed, IFN-gamma has also been reported to affect positively or negatively the events leading to apoptosis. We investigated whether IFN-gamma, besides differentiation, also triggers apoptosis in NB cells and, if so, what is the relationship between the two processes. To this purpose, LAN-5 cells (an IFN-gamma-sensitive human NB cell line), were cultured for 5 days without or with IFN-gamma (1000 IU/ml). At defined times, apoptosis was evaluated by several parameters, including [3H]-thymidine labeling of fragmented DNA, agarose gel electrophoresis of DNA, electron microscopy, cytofluorimetric and fluorescent microscopic analysis of nuclear staining with propidium iodide (PI); this latter also allowed the simultaneous detection of differentiation markers with specific antibodies in a double labelling assay. After 24-36 h. of IFN-gamma treatment, a small proportion of LAN-5 cells underwent internucleosomal DNA fragmentation, chromatin condensation and bright PI nuclear staining. These events increased quantitatively and qualitatively after 48 hours and, by day 5, almost 50% of the cells were frankly apoptotic, vs. 8% in control cultures. Interestingly, differentiation markers (phosphorylated 200 kDa neurofilaments and microtubule-associated proteins) increased and organized themselves intracellularly only in cells with differentiated morphology and uncondensed PI nuclear staining, while condensed PI staining (apoptotic) cells showed disorganized cytoskeleton and a weak signal for the above markers. The mutual exclusion of differentiative and apoptotic features and the simultaneous evolution of these processes in different cell subpopulations suggest that these two cell fates are independent of each other and represent a different response to IFN-gamma. Whether this difference is due to bi- or polyclonality of LAN-5 cells or is linked to different (for instance cell cycle-related) metabolic conditions of the cells remains to be clarified. Nonetheless, our findings further support the potential for the employement of IFN-gamma in the clinical management of NB