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MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma., (©2010 AACR.)

Pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-ethyl-pyrimidine), a folic acid antagonist, may exert, in addition to antiprotozoan effects, immunomodulating activities, including induction of peripheral blood lymphocyte apoptosis. However, the molecular mechanisms underlying this proapoptotic activity remain to be elucidated. Here we show that pyrimethamine, used at a pharmacologically relevant concentration, induced per se apoptosis of activated lymphocytes via the activation of the caspase-8- and caspase-10-dependent cascade and subsequent mitochondrial depolarization. Importantly, this seems to occur independently from CD95/Fas engagement. The proapoptotic activity of pyrimethamine was further confirmed in a patient with autoimmune lymphoproliferative syndrome, an immune disorder associated with a defect of Fas-induced apoptosis. In this patient, pyrimethamine treatment resulted in a "normalization" of lymphocyte apoptosis with a significant amelioration of laboratory parameters. Altogether, these results suggest a mechanism for pyrimethamine-mediated apoptosis that seems to bypass CD95/Fas engagement but fully overlaps CD95/Fas-induced subcellular pathway. On these bases, a reappraisal of the use of pyrimethamine in immune lymphoproliferative disorders characterized by defects in CD95/Fas-mediated apoptosis should be taken into account.

The mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon-gamma (IFN-gamma)) or type 2 (producing IL-4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper-IgE/hypereosinophilia, one with Churg-Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine-producing cells was assessed by flow cytometry. T cells with a type-2 cytokine profile, i.e. producing IL-4 alone, were significantly more resistant to activation-induced apoptosis than those producing IFN-gamma alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8+, as well as in the patients with Churg-Strauss and with hypereosinophilic syndrome. CD4+ and CD8+ IL-4-producing cells were equally resistant to apoptosis. Lower susceptibility to apoptosis of type-2 T cells was also observed in subjects with normal cytokine balances. Bcl-2 expression was high in type-2 cells and in viable type-1 cells, whereas it was low in apoptotic type-1 cells. Resistance to activation-induced apoptosis may explain the expansion of cells producing type-2 cytokines in certain immune disorders.

The measurement of apoptosis in peripheral blood might represent a useful tool in acquired immunodeficiency syndrome (AIDS) and cancer research. Among the many assays that are currently used to identify apoptotic leukocytes, flow cytometric methods are the most valuable in terms of rapidity, simplicity, and level of analytical detail. Some flow cytometric assays may also offer the additional advantage of detecting the earliest phases of apoptosis, which is paramount importance for measuring apoptotic cells in vivo before they are destroyed by phagocytes.