Rita Sabatini

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Group B Streptococcus Induces Apoptosis in Macrophages

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS β-hemolytic activity, suggesting that GBS β-hemolysin could be involved in apoptosis. β-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

γ-Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and Notch down-regulation.

γ‐Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z‐Leu‐Leu‐Nle‐CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time‐course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up‐regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl‐1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I‐induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down‐regulation by small‐interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL.

Apoptosis of human primary B lymphocytes is inhibited by N-acetyl-L-cysteine

Thiols are important molecules to control apoptosis. This study examined the effect of N‐acetyl‐L‐cysteine (NAC) on in vitro spontaneous apoptosis of human tonsillar B lymphocytes (TBL). Results show that NAC inhibits TBL apoptosis and maintains their survival in vitro. The antiapoptotic action of NAC is progressively reduced when its addition to culture is delayed, is reversible, and is not blocked by cycloheximide. The antiapoptotic activity of NAC is associated with its ability to inhibit caspase‐3 and ‐7 proteolytic processing, DNA‐fragmentation factor 45 cleavage, and DNA fragmentation. Furthermore, NAC inhibits BID cleavage and cytochrome c release from mitochondria and increases the expression of Bcl‐2 and BclXL survival proteins. However, it has no effect on caspase‐9 cleavage and increases that of caspase‐8 and poly(adenosine 5′‐diphosphate‐ribose)polymerase. We conclude that NAC‐induced inhibition of TBL apoptosis is associated with inhibition of caspase‐3 and ‐7 processing and is accompanied by changes in several regulatory components of the apoptotic process. These results pose the question of whether microenvironment thiols may in part contribute to in vivo B cell survival.

Activation of cytokine genes during primary and anamnestic immune response to inactivated c. albicans.

Recent evidence suggests that after repeated stimulations with inactivated C. albicans (CA) cells, CD2F1 mice respond with a cytokine pattern typical of T‐helper 1 (Th1) subset development. The purpose of this study was to analyse the sequence of immunological events which, soon after priming mice with CA, lead to the development of primary and anamnestic response. A comprehensive kinetics analysis of cytokine mRNA expression was performed by Northern blot assay, in peritoneal exudate cells (PEC), at different phases of immune response to CA: after priming (one i.p. injection of 2×107 CA cells/mouse), during development of the primary immune response (five progressive CA i.p. injections over a 2‐week period) and in the anamnestic response (CA booster 30days after the primary response). In vitro assays were performed 2 and 24hr after every CA stimulation. The response to CA priming was characterized by an early and high expression of interleukin‐2 (IL‐2) and IL‐1β mRNAs. At 24hr, IL‐2 mRNA was still at a high level, while IL‐1β had greatly decreased. A weak expression of IL‐10 was only induced at 2hr, whereas IL‐12 p40 subunit, interferon‐γ (IFN‐γ), IL‐4 and IL‐5 mRNAs were undetectable. In this phase no in vitro proliferative response of PEC to CA was observed, whereas a significant natural killer (NK) activity was induced. From the second CA injection, the IFN‐γ mRNA was already induced at 2hr. Its expression level increased progressively with the number of CA injections persisting up to 24hr after the fifth stimulation. A progressive increase of IL‐2 mRNA expression was also induced whereas IL‐1β and IL‐10 mRNAs were always transiently expressed at 2hr at levels similar to those observed after the priming. IL‐12 p40 subunit, IL‐4 and IL‐5 mRNAs were never detectable. The expression of this selected cytokine pattern typical of Th1 response was correlated with the development of CA‐specific T lymphocytes as confirmed by the in vitro proliferative response of CA‐5d‐induced PEC to CA. NK activity also increased progressively with the number of CA injections and after the fifth stimulation lymphokine‐activated killer (LAK) activity was also induced. The anamnestic response to CA was characterized by a very quick induction of high levels of IL‐2, IFN‐γ and IL‐1β mRNAs. IL‐2 and IFN‐γ mRNAs remained high up to 24hr while IL‐1β mRNA decreased strongly. A weak, transient expression of IL‐10 mRNA was induced at 2hr whereas the IL‐12 p40 subunit, IL‐4 and IL‐5 mRNAs were not detectable. The presence of CA‐specific memory lymphocytes was confirmed by the in vitro specific proliferative response of PEC to CA. CA booster caused also a very rapid and high level of NK/LAK activation. In conclusion, these results indicate that CA is able to progressively trigger differentiation of the Th1 subset which develops in the absence of IL‐12, and that Th memory cells retain the same selected Th1 cytokine profile developed in the primary immune response.

Constitutive phosphorylation of the active Notch1 intracellular domain in chronic lymphocytic leukemia cells with NOTCH1 mutation

Alterations in Notch signaling are involved in chronic lymphocytic leukemia (CLL) pathogenesis, a hematological disease characterized by the accumulation of CD19+/CD5+ B cells resistant to apoptosis. We previously reported that constitutive Notch1/2 activation contributes to apoptosis resistance of CLL cells.1 Furthermore, a NOTCH1 PEST domain mutation, resulting in a truncated protein more stable and active than wild-type (WT) protein, has recently emerged as a recurrent genetic lesion in CLL patients with adverse prognosis and poor outcome.2, 3 Despite the progress achieved on the role of NOTCH1 mutations in CLL outcome, little is known regarding their role in CLL cell biology. There is evidence that NOTCH1 mutation stabilizes Notch1 signaling in CLL cells,4 but the molecular mechanisms underlying this effect remain to be defined.

In vitro effects of Meropenem and Imipenem/Cilastatin on some functions of human natural effector cells

Meropenem, a new carbapenem antibiotic, was assessed to evaluate its effects on some functional parameters of human polymorphonuclear (PMN) and natural killer (NK) cells in comparison with imipenem/cilastatin. Both drugs significantly inhibited PMN phagocytosis and chemotaxis at concentrations of 2,000 and 4,000 μg/ml. They affected PMN microbicidal activity, evaluated against Candida albicans, only at 4,000 μg/ml. A study of the effects of both drugs on peripheral NK populations and the human NK line (NK-92) showed that even at 4,000 μg/ml there was no effect on antitumor activity. These data indicate that meropenem can reduce some PMN antimicrobial functions only at very high concentrations like imipenem/cilastatin, whereas no concentration influenced NK activity.

Erratum: γ-Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and Notch down-regulation (International Journal of Cancer (2013) 132: 8 (1940-1953) DOI: 10.1002/ijc.27863)

Rosati E, Sabatini R, De Falco F, Del Papa B, Falzetti F, Di Ianni M, Cavalli L, Fettucciari K, Bartoli A, Screpanti I, Marconi P. gSecretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and Notch down-regulation. Int J Cancer. 2013 Apr 15;132(8):1940–1953. doi: 10.1002/ijc.27863. Epub 2012 Oct 17. In this article, Figure 1b contained an error in the plot relative to DMSO treatment at 1 hr. The plot image of DMSO was mistakenly the same plot image relative to GSI treatment at 1 hr, but the gating numbers were correct. The corrected Figure 1b, showing the correct DMSO plot image, is now provided. The authors regret this error, which however, does not alter the conclusions of the study.