Search

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.

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.

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.

Tumor necrosis factor alpha (TNFalpha) is constitutively produced by B-chronic lymphocytic leukemia (B-CLL) cells and may act as an autocrine factor for their growth and survival. However, very few data are available on the possible cytotoxic effect of TNFalpha produced by B-CLL cells. This study investigated whether B-CLL cells exert in vitro cytotoxicity by TNFalpha and if so, whether this cytotoxicity can be modulated by cytokines. In 8 of 12 patients (66.6%), B-CLL cells in vitro constitutively produced TNFalpha and exerted a TNFalpha-mediated cytotoxicity, evaluated in an 18-h 51Cr release assay, against the TNFalpha-sensitive Jurkat, U937 and K562 cell lines but not against the TNFalpha-resistant Raji cell line. Involvement of TNFalpha in B-CLL cell cytotoxicity is demonstrated by the fact that anti-TNFalpha antibodies strongly inhibited it and supernatants of cytotoxic cultures contained TNFalpha and mediated a completely TNFalpha-dependent cytotoxicity. When the cytotoxic B-CLL cells were stimulated with interleukin (IL)-2 plus IL-12, there was increased TNFalpha mRNA expression, TNFalpha production and TNFalpha-mediated cytotoxicity. All eight patients with cytotoxic leukemic cells had progressive disease and six of these also expressed high levels of ZAP-70 protein. In the other four patients (33.3%), B-CLL cells did not produce TNFalpha in vitro and were not cytotoxic, either spontaneously or after IL-2 plus IL-12 stimulation. Of these four patients, three had stable disease and one had progressive disease. The patient with progressive disease and one of the three with stable disease expressed low levels of ZAP-70 protein. We conclude that a group of B-CLL patients with progressive disease have leukemic B cells able to exert in vitro a TNFalpha-mediated cytotoxicity, which is modulated by cytokines.

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.

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.