Drp1 mediates caspase-independent type III cell death in normal and leukemic cells.

Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.

Caspase-independent type III programmed cell death in chronic lymphocytic leukemia: the key role of the F-actin cytoskeleton.

BACKGROUND: Programmed cell death has been traditionally related with caspase activation. However, it is now accepted that caspase-independent forms of programmed cell death also regulate cell death. In chronic lymphocytic leukemia, CD47 ligation induces one of these alternative forms of cell death: type III programmed cell death. This poorly understood process is characterized by cytoplasmic hallmarks, such as mitochondrial damage. To gain insights into the molecular pathways regulating type III programmed cell death in chronic lymphocytic leukemia, we performed extensive biochemical and cell biology assessments. DESIGN AND METHODS: After CD47 triggering, purified B-cells from 20 patients with chronic lymphocytic leukemia were studied by flow cytometry, immunofluorescence and three-dimensional imaging, immunoblotting, electron microscopy, and fibrillar/globular actin measurements. Finally, we subjected CD47-treated chronic lymphocytic leukemia cells to a phagocytosis assay. RESULTS: We first confirmed that induction of type III programmed cell death is an efficient means of triggering cell death in chronic lymphocytic leukemia. Further, we demonstrated that the signaling events induced by CD47 ligation provoked a reduction in cell size. This alteration is related to F-actin disruption, as the two other cytoskeleton networks, microtubules and intermediate filaments, remain undisturbed in type III programmed cell death. Strikingly, we revealed that the pharmacological modulation of F-actin dynamics regulated this type of death. Finally, our data delineated a new programmed cell death pathway in chronic lymphocytic leukemia initiated by CD47 triggering, and followed by serine protease activation, F-actin rearrangement, mitochondrial damage, phosphatidylserine exposure, and cell clearance. CONCLUSIONS: Our work reveals a key molecular tool in the modulation of cell death in chronic lymphocytic leukemia: F-actin. By assessing the regulation of F-actin and type III programmed cell death, this analysis provides new options for destroying chronic lymphocytic leukemia cells, such as a combination of therapies based on apoptosis regulators (e.g., caspases, Bcl-2, Bax) along with alternative therapies based on type III death effectors (e.g., F-actin).

IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma.

PURPOSE: To determine the value of IL-10 measurement in aqueous humor (AH) for screening in primary intraocular lymphoma (PIOL). METHODS: One hundred consecutive diagnostic or therapeutic vitrectomies were performed in patients with uveitis. During surgery, 100 microL of both AH and pure vitreous was taken. IL-10 levels were determined with a standard quantitative sandwich enzyme immunoassay technique. Patients were distributed in two groups: 51 patients with proven PIOL, 108 patients with uveitis divided into 74 with uveitis of proven etiology and 34 with idiopathic uveitis. Groups were compared by ANOVA and the Tukey-Kramer test or nonparametric Wilcoxon test. Distributions were compared by using the chi(2) test. Segmentation was derived from the ROC curves by choosing a tradeoff between sensitivity and specificity. RESULTS: In patients with PIOL, IL-10 mean values were 2205.5 pg/mL (median: 1467 pg/mL) in the vitreous and 543.4 pg/mL (median: 424 pg/mL) in AH. In patients with uveitis (idiopathic and diagnostic uveitis), mean values were 26.6 pg/mL (median: 8 pg/mL) in the vitreous, and 21.9 pg/mL (median: 8 pg/mL) in AH. IL-10 mean values were significantly different between patients with PIOL and patients with uveitis (P < 10(-3)). The areas under the curves were 0.989 and 0.962 for vitreous and AH, respectively. A cutoff of 50 pg/mL in the AH was associated with a sensitivity of 0.89 and a specificity of 0.93. In the vitreous, a cutoff value of 400 pg/mL yielded a specificity of 0.99 and a sensitivity of 0.8. CONCLUSIONS: Diagnosis of PIOL is often made months or years after the initial onset of ocular symptoms. Cytology remains the gold standard for diagnosis. However, measurement of IL-10 in the AH is a good screening test to reduce diagnostic delays.

Cytokine profile in human eyes: contribution of a new cytokine combination for differential diagnosis between intraocular lymphoma or uveitis.

Primary intraocular lymphoma (PIOL), also called primary vitreoretinal lymphomas, often masquerades as uveitis. This misdiagnosis can result in subsequent brain involvement and oculocerebral lymphoma (OCL). In this study, we sought to characterize the helper T-cell type 1 (Th1)/Th2 cytokine profile in vitreous samples from patients with PIOL, OCL, uveitis and controls with non-inflammatory disease. Vitreous and aqueous humor samples from 87 patients with PIOL (n = 30), OCL (n = 12), uveitis (n = 34), and retinal detachment (RD) without hemorrhage (n = 11) were analyzed and their concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were determined by flow cytometric bead arrays (CBA). The IL-10 levels determined by CBA were compared with those by ELISA. IL-10 concentrations measured by CBA and ELISA were highly correlated. IL-2, IL-4, and TNFα were not detected in any sample. The only cytokine detected at a significant level in samples from RD vitreous was IL-6. The IL-10/IL-6 ratio, as previously reported, was slightly higher in PIOL than in uveitis samples, but not for all patients. Cytokine profiles from PIOL and OCL samples did not differ. The combination of the IL-10/IL-6 and IL-10/IFNγ ratios was highly informative for discriminating PIOL/OCL from uveitis samples and for therapeutic follow up of PIOL. This strategy might be very helpful as an initial screening to rule out PIOL in patients thought to have uveitis.

A study of bone marrow neoangiogenesis in chronic lymphocytic leukemia patients.

OBJECTIVE: To use a purely geometric data-unravelling method to identify, without a priori, direct links of neoangiogenesis with known clinicobiologic variables in 23 untreated chronic lymphocytic leukemia (CLL) patients at the time of marrow sampling. STUDY DESIGN: We measured neovascular surface density (CD34 endothelial marker) in the 10 most labeled fields (hVA), cellularity (CA) and the percentage of avascular fields (AVF) in bone marrow biopsy specimens from 34 patients with CLL. Vascular endothelial growth factor (VEGF) was assayed in thawed serum samples from 8 of the 23 untreated patients (12 Rai stage 0, 6 Rai stage I-II, 5 Binet stage B-C). RESULTS: Neoangiogenesis hVA was lower in the 12 stage O patients (p < 0.039) and in the 6 patients with nodular interstitial mixed infiltration (p = 0.058). The link between hVA and serum VEGF (sVEGF) was negative in 8 patients (R = -0.49, -0.70 for a given age). Age was linked to moderate interstitial infiltration (p < 0.02), AVF (R = 0.37) and lower sVEGF levels (R = -0.676). The blood platelet count showed numerous correlations, including links with AVF (R = 0.40), low hVA (R = -0.38), female sex (p = 0.068), absence of splenomegaly (p < 0.001), mixed-type infiltration (p < 0.01) and sVEGF (R = 0.38, 0.61 for a given age). CONCLUSION: The links revealed by the iconography of correlations were described statistically. Surprisingly, negative links of hVA with the sVEGF level, 2 known prognostic factors, on the one hand, and with the blood platelet count, on the other hand, were found. The direct link between disease progression and the amount of avascular hematopoietic tissue (NS), not previously described, will require further study. Age should be taken into account.

Novel flow-cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia.

We describe a new flow-cytometric analysis using quadruple labelling with anti-CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b-/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity-determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0.2. The sensitivity of the BCD5+R was 1 x 10-4vs 5 x 10-2 for CDR3 PCR and 1 x 10-5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0.2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0.2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.

Biological diagnosis of primary intraocular lymphoma.

Primary intraocular lymphoma (PIOL) is a rare presentation of lymphoma that is particularly difficult to recognize. In our institution, 36 cases of PIOL were diagnosed between March 1997 and July 2002. The recognition of lymphoma cells by cytology with or without immunophenotyping on slides generated a strong suspicion of the diagnosis in 34 of 36 cases. The diagnosis was confirmed by measurement of interleukin-10 (IL-10) in the vitreous humour or aqueous humour; high levels were observed in 35 of 36 cases, all were of B-cell origin. As expected, the only case with T-cell lymphoma had a very low level of IL-10. Furthermore, IL-10 levels excluded this diagnosis in two cases that were incorrectly suspected of PIOL after cytological examination. Finally, detection of clonality by polymerase chain reaction techniques, performed in 29 cases, represented a helpful tool in diagnosing PIOL as this approach definitively confirmed the diagnosis of B- or T-cell lymphoma in 17 cases.

Sildenafil and vardenafil, types 5 and 6 phosphodiesterase inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells.

Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3'5' cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-alpha (SDF-1alpha). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.