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Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.

The nucleophosmin (NPM1) gene encodes for a multifunctional nucleocytoplasmic shuttling protein that is localized mainly in the nucleolus. NPM1 mutations occur in 50% to 60% of adult acute myeloid leukemia with normal karyotype (AML-NK) and generate NPM mutants that localize aberrantly in the leukemic-cell cytoplasm, hence the term NPM-cytoplasmic positive (NPMc+ AML). Cytoplasmic NPM accumulation is caused by the concerted action of 2 alterations at mutant C-terminus, that is, changes of tryptophan(s) 288 and 290 (or only 290) and creation of an additional nuclear export signal (NES) motif. NPMc+ AML shows increased frequency in adults and females, wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD, CD34 negativity, and a distinct gene-expression profile. Analysis of mutated NPM has important clinical and pathologic applications. Immunohistochemical detection of cytoplasmic NPM predicts NPM1 mutations and helps rationalize cytogenetic/molecular studies in AML. NPM1 mutations in absence of FLT3-ITD identify a prognostically favorable subgroup in the heterogeneous AML-NK category. Due to their frequency and stability, NPM1 mutations may become a new tool for monitoring minimal residual disease in AML-NK. Future studies should focus on clarifying how NPM mutants promote leukemia, integrating NPMc+ AML in the upcoming World Health Organization leukemia classification, and eventually developing specific antileukemic drugs.

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We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AMLassociated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus. IntroductionIn acute myeloid leukemia (AML), a clinically and molecularly heterogeneous disease, 1 recurrent cytogenetic abnormalities help define subgroups with different prognosis, and identify patients who might benefit from targeted therapies. 1 However, almost half adult AMLs display normal karyotype at conventional cytogenetics, 2 and the clinical and molecular features of this large subgroup of patients are still poorly understood. [3][4][5][6][7] We recently observed that about 60% of adult AML with normal karyotype display aberrant cytoplasmic expression of nucleophosmin (NPM). 8 A multifunctional protein [9][10][11][12][13][14][15] that characteristically shuttles between the nucleus and the cytoplasm, 16 NPM is found mainly in the nucleolus, [17][18][19] where it is one of the most abundant of the approximately 700 proteins so far identified by proteomic techniques. 20 Cytoplasmic NPM identifies a distinct subgroup of AML, named NPMc ϩ AML, that accounts for about 35% of all adult AML and is characterized by wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD mutations, absence of CD34, and relatively good response to induction therapy. 8 NPMc ϩ AML also has a distinct gene expression profile 21 and carries mutations in exon-12 of the NPM gene 8 that serve as predictor of favorable prognosis in AML with normal karyotype, [22][23][24] and as a marker for monitoring of minimal residual disease. 25 In spite of the close association between the aberrant cytoplasmic expression of NPM and exon-12 NPM mutations, 8 the mechanism underlying cytoplasmic accumulation of NPM in leukemic cells and its interference with wild-type NPM protein remaine...

Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplasmic shuttling protein that shows a restricted nucleolar localization. Mutations of NPM1 gene leading to aberrant cytoplasmic dislocation of nucleophosmin (NPMc þ ) occurs in about one third of acute myeloid leukaemia (AML) patients that exhibit distinctive biological and clinical features. We discuss the latest advances in the molecular basis of nucleophosmin traffic under physiological conditions, describe the molecular abnormalities underlying altered transport of nucleophosmin in NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukaemogenesis. We then outline how a highly specific immunohistochemical assay can be exploited to diagnose NPM1-mutated AML and myeloid sarcoma in paraffin-embedded samples by looking at aberrant nucleophosmin accumulation in cytoplasm of leukaemic cells. This procedure is also suitable for detection of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surrogate for molecular analysis can serve as first-line screening in AML and should facilitate implementation of the 2008 World Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc þ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleophosmin transport in AML with mutated NPM1.

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.