Piotr Smolewski

Detection of caspases activation by fluorochrome‐labeled inhibitors: Multiparameter analysis by laser scanning cytometry

BACKGROUND The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. METHODS Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. RESULTS Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (> 90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. CONCLUSIONS The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation.

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patients prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold‐standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta‐associated protein (ZAP‐70) expression was associated with unmutated IgVH genes and ZAP‐70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP‐70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP‐70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti‐ZAP‐70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti‐ZAP‐70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP‐70 levels were expressed as percentage positive (POS) relative to isotype control or normal B‐cells or normal T‐cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T‐cells. The reported level of ZAP‐70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T‐cell ZAP‐70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP‐70 expression. We assessed the variation of ZAP‐70 expression levels in fresh cells according to storage time, which demonstrated that ZAP‐70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.

The inhibitor of apoptosis protein family and its antagonists in acute leukemias

The Inhibitor of Apoptosis Protein family (IAP) functions as inhibitors of apoptotic pathways, both death receptor- and mitochondrial mediated. We detail the current body of knowledge for the IAP family with regard to their structure and function, their expression in normal and leukemic cells, and their prognostic importance in acute leukemia. Although there is some evidence that IAPs play an important role in the chemoresistance of leukemia cell lines, little is known about their influence on this phenomenon in acute leukemia cells of human origin. IAPs are also explored as a specific target for new antitumor strategies, including antisense oligonucleotides of XIAP (X-chromosome-linked IAP) or survivin and small molecules of polyphenylurea-based XIAP inhibitors.Several proteins negatively regulate the function of the IAP family. One of those antagonists is Smac/DIABLO. Short peptides of Smac were found to enhanced apoptosis, induced by chemo- or immunotherapy, in the leukemic cells in vitro. Moreover, small-molecule agents, resembling Smac/DIABLO in function, were shown to potentiate cytotoxicity of chemotherapy in different malignancies.IAPs, exhibiting downstream influence on both external and intrinsic pathways as well as on some caspase-independent mechanisms of apoptosis, are potentially attractive target for anti-tumor therapy, although their role in the pathology and prognosis of acute leukemia has to be further elucidated.

Expression and prognostic significance of the inhibitor of apoptosis protein (IAP) family and its antagonists in chronic lymphocytic leukaemia.

Impaired apoptosis is still considered to be an important event in the development and progression of chronic lymphocytic leukaemia (CLL). However, mechanisms of this defect have not been fully elucidated. In this study, expression of inhibitor of apoptosis proteins, IAPs (cIAP1, cIAP2, XIAP and survivin), and their antagonists (Smac/DIABLO and HtrA2/Omi) was comprehensively analysed in 100 untreated CLL patients, using flow cytometry and Western blot techniques. Expression of anti-apoptotic cIAP1 and cIAP2 in leukaemic cells was significantly higher than in non-tumour lymphocytes (p=0.000001 and p=0.014, respectively), whereas the IAP-antagonist, Smac/DIABLO, was decreased in CLL (p=0.010). Higher expression of all analysed IAPs (cIAP1, p=0.002; cIAP2, p=0.026; XIAP, p=0.002; survivin, p=0.00006) and lower levels of Smac/DIABLO (p=0.006) were found in patients with progressive disease, compared to those with stable CLL. High baseline expression of cIAP1 and survivin correlated with worse response to treatment. Co-expression of these proteins was associated with shorter overall survival of CLL patients (p=0.005). In conclusion, CLL cells show the apoptosis-resistant profile of IAPs/IAP-antagonist expression. Upregulation of IAPs is associated with a progressive course of the disease. Co-expression of cIAP1 and survivin seems to be an unfavourable prognostic factor in CLL patients. Further studies with longer follow up are warranted to confirm and expand these findings.

Rituximab plus cladribine with or without cyclophosphamide in patients with relapsed or refractory chronic lymphocytic leukemia.

Objectives: The aim of our study was to determine the feasibility, effectiveness and toxicity of combined regimens consisting of rituximab and cladribine (2‐CdA) (RC) and RC plus cyclophosphamide (RCC) in the treatment of patients with recurrent or refractory chronic lymphocytic leukemia (CLL).

Rituximab combined with cladribine or with cladribine and cyclophosphamide in heavily pretreated patients with indolent lymphoproliferative disorders and mantle cell lymphoma

In vitro studies have shown synergistic or additive interactions between rituximab and purine nucleoside analogues. The results of recent clinical trials seem to confirm these preclinical observations.

Circulating endothelial cells in patients with acute myeloid leukemia.

Abstract:  Objectives: The circulating endothelial cells (CEC) are proposed to be a non‐invasive marker of angiogenesis. The level of CEC in peripheral blood (PB) of acute myeloid leukemia (AML) patients has not been investigated prior to this study. We evaluated the count of resting (rCEC), activated (aCEC) and endothelial progenitor cells (CEPC) in the PB of AML and healthy subjects. In addition we correlated the levels of CEC with disease status, known prognostic factors and response to treatment. Methods: CEC were quantified by utilizing four‐color flow cytometry procedures in 48 AML patients at the time of diagnosis and 29 healthy controls. Additionally, measurements were again taken after the first course of induction treatment in 12 of the patients. Results: The numbers of aCEC, rCEC and CEPC were significantly higher in the AML patients than in the controls (P < 0.0001, P < 0.0001 and P < 0.001, respectively). The CEC count was significantly higher in the AML patients with white blood cell count (WBC) >15 G/L, elevated lactic dehydrogenase (LDH) levels and a higher (over median) absolute blasts count (ABC) in PB than in the group with WBC <15 G/L (P < 0.03), a normal LDH level (P < 0.03) and a lower (

Proapoptotic activity of alemtuzumab alone and in combination with rituximab or purine nucleoside analogues in chronic lymphocytic leukemia cells

Proapoptotic activity of anti-CD52 monoclonal antibody, alemtuzumab (ALT) as well as ALT-affected apoptosis-regulatory mechanisms were assessed in tumor cells from 36 patients with chronic lymphocytic leukemia (CLL). Cells were treated in vitro for 24 - 48 h with ALT alone or in combination with rituximab (RTX), or purine nucleoside analogues (PNA), fludarabine and cladribine. Moreover, eight ALT-treated patients were examined in vivo. In 22/36 patients with the pre-treatment overexpression of Bax, Bak and Bid proteins, ALT induced a distinct (more than 50% from the baseline) increase in the incidence of apoptosis after 24 h of in vitro treatment. ALT-attributed CLL cell apoptosis was also detected after 24 h from in vivo ALT administration, with significantly downregulated Bcl-2 (P = 0.012) and Mcl-1 (P = 0.031). ALT combined with PNA or RTX exerted significantly higher proapoptotic effect in vitro than single agents, downregulating FLIP and Bcl-2 (ALT + PNA) or significantly increasing Bax expression (ALT + RTX; P = 0.007). In conclusion, the evidence of apoptotic CLL cells death in response to ALT, with deregulation of intrinsic apoptotic pathway, is presented. ALT and PNA or RTX trigger complementary changes in expression of proteins regulating cell propensity to undergo apoptosis, what provides molecular rationale for combining ALT with those agents.

Brentuximab Vedotin with Chemotherapy for Stage III or IV Hodgkin’s Lymphoma

BACKGROUND Brentuximab vedotin is an anti‐CD30 antibody–drug conjugate that has been approved for relapsed and refractory Hodgkins lymphoma. METHODS We conducted an open‐label, multicenter, randomized phase 3 trial involving patients with previously untreated stage III or IV classic Hodgkins lymphoma, in which 664 were assigned to receive brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A+AVD) and 670 were assigned to receive doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). The primary end point was modified progression‐free survival (the time to progression, death, or noncomplete response and use of subsequent anticancer therapy) as adjudicated by an independent review committee. The key secondary end point was overall survival. RESULTS At a median follow‐up of 24.9 months, 2‐year modified progression‐free survival rates in the A+AVD and ABVD groups were 82.1% (95% confidence interval [CI], 78.7 to 85.0) and 77.2% (95% CI, 73.7 to 80.4), respectively, a difference of 4.9 percentage points (hazard ratio for an event of progression, death, or modified progression, 0.77; 95% CI, 0.60 to 0.98; P=0.03). There were 28 deaths with A+AVD and 39 with ABVD (hazard ratio for interim overall survival, 0.72 [95% CI, 0.44 to 1.17]; P=0.19). All secondary efficacy end points trended in favor of A+AVD. Neutropenia occurred in 58% of the patients receiving A+AVD and in 45% of those receiving ABVD; in the A+AVD group, the rate of febrile neutropenia was lower among the 83 patients who received primary prophylaxis with granulocyte colony‐stimulating factor than among those who did not (11% vs. 21%). Peripheral neuropathy occurred in 67% of patients in the A+AVD group and in 43% of patients in the ABVD group; 67% of patients in the A+AVD group who had peripheral neuropathy had resolution or improvement at the last follow‐up visit. Pulmonary toxicity of grade 3 or higher was reported in less than 1% of patients receiving A+AVD and in 3% of those receiving ABVD. Among the deaths that occurred during treatment, 7 of 9 in the A+AVD group were associated with neutropenia and 11 of 13 in the ABVD group were associated with pulmonary‐related toxicity. CONCLUSIONS A+AVD had superior efficacy to ABVD in the treatment of patients with advanced‐stage Hodgkins lymphoma, with a 4.9 percentage‐point lower combined risk of progression, death, or noncomplete response and use of subsequent anticancer therapy at 2 years. (Funded by Millennium Pharmaceuticals and Seattle Genetics; ECHELON‐1 ClinicalTrials.gov number, NCT01712490; EudraCT number, 2011‐005450‐60.)

In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells

Abstract:  Objective: The anti‐tumour in vitro activity of proteasome inhibitor bortezomib (PS‐341, VELCADE) in combination with purine nucleoside analogues, cladribine (2‐CdA) and fludarabine (FA) was tested in lymphocytes derived from 26 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL). Methods: Cell viability was assessed by propidium iodide staining, and apoptosis by annexin‐V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis‐regulating proteins Bax, Bak, Bid, Bcl‐w, Bcl‐2, XIAP and Mcl‐1 was evaluated in B‐CLL lymphocytes. Results: Bortezomib alone induced significant, dose‐dependent cytotoxicity starting from the low concentration 2.5 nm, inducing apoptosis of B‐CLL cells. Combination of this agent with 2‐CdA or FA resulted in an increase of cytotoxicity when compared with that mediated by single drugs. The observed increase was especially evident when 5 nm of bortezomib were combined with suboptimal doses of 2‐CdA or FA. The combination index (CI) was 0.87 for bortezomib + 2‐CdA and 0.82 for bortezomib + FA, indicating an evident additive effect of these combinations. Moreover, B‐CLL cells were more sensitive to proteasome inhibitor used alone or combined with 2‐CdA or FA comparing to CD3+ lymphocytes. Corresponding to enhanced apoptosis, the expression levels of several apoptosis‐regulating proteins were altered. The most pronounced changes were down‐regulation of XIAP and up‐regulation of Bid proteins by the combination of bortezomib with either 2‐CdA or FA. Conclusions: This study suggest that the in vitro cytotoxic effect through proteasome inhibition by bortezomib can be increased substantially with low doses of the purine nucleoside analogues, 2‐CdA and FA, and that this effect on B‐CLL cell is selectively higher than on normal, CD3‐positive lymphocytes.