Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.

High prevalence of antibodies reacting to mimotopes of Simian virus 40 large T antigen, the oncoprotein, in serum samples of patients affected by non-Hodgkin lymphoma.

A new immunological investigation was carried out to study the association between non-Hodgkin lymphoma and Simian virus 40 (SV40). To this end, a new indirect ELISA was employed with two mimotopes from SV40 large T antigen (Tag), the viral oncoprotein, to analyse for specific reactions to antibodies in sera from non-Hodgkin lymphoma patients and controls, represented by healthy subjects (HS) and breast carcinoma (BC) patients. This study allowed us to assay a new sera collection from non-Hodgkin lymphoma patients (NHL, n = 254). To verify the association between NHL and SV40 Tag, two totally independent cohorts were analysed: NHL1 n = 150 and NHL2 n = 104. The epidemiological survey included sera from HS1, n = 150; HS2, n = 104 and BC, n = 78. This new indirect ELISA revealed that antibodies against SV40 Tag mimotopes are detectable in NHL1 and NHL2 sera with a prevalence of 37 and 36%, respectively. The prevalence of SV40-antibodies detected in both NHL1 and NHL2 cohorts differs statistically from controls, at 19% for HS1 (p < 0.01), HS2 (p < 0.05) and BC patients (p < 0.05). This study, carried out with an immunological assay with specific Tag oncoprotein mimotopes of Simian virus 40, reports the presence of IgG antibodies against the large Tumour antigen in non-Hodgkin lymphomas for the first time. Our immunological data with two independent NHL cohorts show a statistically significant association between Simian virus 40 Tag and non-Hodgkin lymphoma. These results suggest that SV40-positive non-Hodgkin lymphomas could be treated differently from those tested SV40-negative.

The antineoplastic agent α-bisabolol promotes cell death by inducing pores in mitochondria and lysosomes.

The sesquiterpene α-bisabolol (α-BSB) has been shown to be an effective cytotoxic agent for a variety of human cancer cells in culture and animal models. However, much of its intracellular action remains elusive. We evaluated the cytotoxic action of α-BSB against CML-T1, Jurkat and HeLa cell lines, as preclinical models for myeloid, lymphoid and epithelial neoplasias. The approach included single cell analysis (flow cytometry, immunocytology) combined with cytotoxicity and proliferation assays to characterize organelle damage, autophagy, cytostatic effect, and apoptosis. The study focuses on the relevant steps in the cytotoxic cascade triggered by α-BSB: (1) the lipid rafts through which α-BSB enters the cells, (2) the opening of pores in the mitochondria and lysosomes, (3) the activation of both caspase-dependent and caspase-independent cell death pathways, (4) the induction of autophagy and (5) apoptosis. The effectiveness of α-BSB as an agent against tumor cells is grounded on its capability to act on different layers of cell regulation to elicit different concurrent death signals, thereby neutralizing a variety of aberrant survival mechanisms leading to treatment resistance in neoplastic cell.

Immunologic evidence of a strong association between non-Hodgkin lymphoma and simian virus 40.

BACKGROUND: Non-Hodgkin lymphoma (NHL), the most common cancer of the lymphatic system, is of unknown etiology. The identification of etiologic factors in the onset of NHL is a key event that could facilitate the prevention and cure of this malignancy. Simian virus 40 (SV40) has been considered an oncogenic agent in the onset/progression of NHL. METHODS: In this study, an indirect enzyme-linked immunosorbent assay with 2 synthetic peptides that mimic SV40 antigens of viral capsid proteins 1 to 3 was employed to detect specific antibodies against SV40. Serum samples were taken from 2 distinct cohorts of NHL-affected patients (NHL1 [n = 89] and NHL2 [n = 61]) along with controls represented by oncologic patients affected by breast cancer (BC; n = 78) and undifferentiated nasopharyngeal carcinoma (UNPC; n = 64) and 3 different cohorts of healthy subjects (HSs; HS1 [n = 130], HS2 [n = 83], and HS3 [n = 87]). RESULTS: Immunologic data indicated that in serum samples from NHL patients, antibodies against SV40 mimotopes were detectable with a prevalence of 40% in NHL1 patients and with a prevalence of 43% in NHL2 patients. In HSs of the same median age as NHL patients, the prevalence was 16% for the HS1 group (57 years) and 14% for the HS2 group (65 years). The difference was statistically significant (P < .0001 and P < .001). Interestingly, the difference between NHL1/NHL2 patients and BC patients (40%/43% vs 15%, P < .001) and between NHL1/NHL2 patients and UNPC patients (40%/43% vs 25%, P < .05) was significant. CONCLUSIONS: Our data indicate a strong association between NHL and SV40 and thus a need for innovative therapeutic approaches for this hematologic malignancy.

Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells.

BACKGROUND: We previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells. METHODS: We tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML) for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours. RESULTS: A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 µM α-bisabolol IC50) included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 µM IC50). Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential. CONCLUSION: Our study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

Macrophages may promote cancer growth via a GM-CSF/HB-EGF paracrine loop that is enhanced by CXCL12.

BACKGROUND: Increased numbers of tumour-associated macrophages correlate with shortened survival in some cancers. The molecular bases of this correlation are not thoroughly understood. Events triggered by CXCL12 may play a part, as CXCL12 drives the migration of both CXCR4-positive cancer cells and macrophages and may promote a molecular crosstalk between them. RESULTS: Samples of HER1-positive colon cancer metastases in liver, a tissue with high expression of CXCL12, were analysed by immunohistochemistry. In all of the patient biopsies, CD68-positive tumour-associated macrophages presented a mixed CXCL10 (M1)/CD163 (M2) pattern, expressed CXCR4, GM-CSF and HB-EGF, and some stained positive for CXCL12. Cancer cells stained positive for CXCR4, CXCL12, HER1, HER4 and GM-CSF. Regulatory interactions among these proteins were validated via experiments in vitro involving crosstalk between human mononuclear phagocytes and the cell lines DLD-1 (human colon adenocarcinoma) and HeLa (human cervical carcinoma), which express the above-mentioned ligand/receptor repertoire. CXCL12 induced mononuclear phagocytes to release HB-EGF, which activated HER1 and triggered anti-apoptotic and proliferative signals in cancer cells. The cancer cells then proliferated and released GM-CSF, which in turn activated mononuclear phagocytes and induced them to release more HB-EGF. Blockade of GM-CSF with neutralising antibodies or siRNA suppressed this loop. CONCLUSIONS: CXCL12-driven stimulation of cancer cells and macrophages may elicit and reinforce a GM-CSF/HB-EGF paracrine loop, whereby macrophages contribute to cancer survival and expansion. The involvement of mixed M1/M2 GM-CSF-stimulated macrophages in a tumour-promoting loop may challenge the paradigm of tumour-favouring macrophages as polarized M2 mononuclear phagocytes.

The Evolving Knowledge on T and NK Cells in Classic Hodgkin Lymphoma: Insights into Novel Subsets Populating the Immune Microenvironment.

Classic Hodgkin lymphoma (cHL) is a unique lymphoid neoplasm characterized by extensive immune infiltrates surrounding rare malignant Hodgkin Reed-Sternberg (HRS) cells. Different subsets of T and NK cells have long been recognized in the cHL microenvironment, yet their distinct contribution to disease pathogenesis has remained enigmatic. Very recently, novel platforms for high dimensional analysis of immune cells, such as single-cell RNA sequencing and mass cytometry, have revealed unanticipated insights into the composition of T- and NK-cell compartments in cHL. Advances in imaging techniques have better defined specific T-helper subpopulations physically interacting with neoplastic cells. In addition, the identification of novel cytotoxic subsets with an exhausted phenotype, typically enriched in cHL milieu, is shedding light on previously unrecognized immune evasion mechanisms. This review examines the immunological features and the functional properties of T and NK subsets recently identified in the cHL microenvironment, highlighting their pathological interplay with HRS cells. We also discuss how this knowledge can be exploited to predict response to immunotherapy and to design novel strategies to improve PD-1 blockade efficacy.

Classical Hodgkin lymphoma cells may promote an IL-17-enriched microenvironment.

In classical Hodgkin lymphoma (cHL), the significance of the interplay between Hodgkin and Reed-Sternberg cells (HRS) and reactive T cells remains poorly defined. By immunohistochemistry on bioptic cHL specimens, we found that HRS and surrounding T lymphocytes stained positive for IL-17 in 40% of cases. IL-17 was detectable in a similar proportion of patients' sera and correlated with disease burden. Supernatants of KM-H2 and HDLM-2 cHL cell lines guided preferential chemotaxis of CCR6+ T lymphocytes. Coculture of cHL cell lines with PBMC promoted the enrichment of Th17 lymphocytes and Foxp3+/IL-17+ cells, whereas T regulatory cells slightly decreased. Soluble CD30 downmodulated membrane CD30 expression on T cells and contributed to their polarization shift by stimulating IL-17 production and reducing IFN-γ synthesis. Thus, HRS and a number of reactive CD4+ T cells, attracted by tumor-secreted chemokines, produce an IL-17 tumor-shaped inflammatory milieu in a cHL subset.

Monocyte-to-macrophage switch reversibly impaired by Ibrutinib.

Ibrutinib is increasingly adopted for treating lymphoid malignancies. While growing amounts of data pile up about Ibrutinib mechanism of action on neoplastic B cells, little is known about its impact on other immune cells. Here we investigated the effect of Ibrutinib on monocyte/macrophage functions. (1) Ibrutinib treatment of purified human monocytes affected both chemoattractant-triggered inside-out as well as integrin-mediated outside-in signaling events, thus provoking defective adhesion and spreading on purified integrin ligands, respectively. (2) In in vitro cell-culture experiments, Ibrutinib promoted a differentiation shift of monocytes to fibrocyte-like cells, characterized by the acquisition of a typical elongated cell morphology. Importantly, this clear-cut shape transition also occurred upon culturing monocytes with sera derived from Ibrutinib-treated patients, thus clearly suggesting that the drug concentrations achievable in vivo can generate the phenotypic shift. (3) Ibrutinib-induced fibrocyte-like cells showed adhesion deficiency, altered phagocytic properties, and, with respect to macrophages, they acquired the capability of generating larger amounts of reactive oxygen species, possibly displaying different metabolic activities. Taken together, our results indicate that Ibrutinib has profound effects on the monocyte/macrophage immunobiology. They may finally shed some light about the biological ground of several Ibrutinib-related toxicities.

West Nile Virus Encephalitis in Haematological Setting: Report of Two Cases and a Brief Review of the Literature.

West Nile virus is a zoonotic agent causing life-threatening encephalitis in a proportion of infected patients. Older age, immunosuppression, and mutations in specific host genes (e.g., CCR5 delta-32 mutation) predispose to neuroinvasive infection. We report on two cases of severe West Nile encephalitis in recently-treated, different-aged, chronic lymphocytic leukemia patients. Both patients developed high-grade fever associated with severe neurological impairment. The younger one harboured germ-line CCR5 delta-32 mutation, which might have played a role in the pathogenesis of its neuroinvasive manifestations.

BID and the α-bisabolol-triggered cell death program: converging on mitochondria and lysosomes.

α-Bisabolol (BSB) is a plant-derived sesquiterpene alcohol able to trigger regulated cell death in transformed cells, while deprived of the general toxicity in several mouse models. Here, we investigated the involvement of lysosomal and mitochondrial compartments in the cytotoxic effects of BSB, with a specific focus on the BH3-only activator protein BID. We found that BSB particularly accumulated in cancer cell lines, displaying a higher amount of lipid rafts as compared to normal blood cells. By means of western blotting and microscopy techniques, we documented rapid BSB-induced BID translocation to lysosomes and mitochondria, both of them becoming dysfunctional. Lysosomal membranes were permeabilized, thus blocking the cytoprotective autophagic flux and provoking cathepsin B leakage into the cytosol. Multiple flow cytometry-based experiments demonstrated the loss of mitochondrial membrane potential due to pore formation across the lipid bilayer. These parallel events converged on neoplastic cell death, an outcome significantly prevented by BID knockdown. Therefore, BSB promoted BID redistribution to the cell death executioner organelles, which in turn activated anti-autophagic and proapoptotic mechanisms. This is an example of how xenohormesis can be exploited to modulate basic cellular programs in cancer.

Bone marrow stromal cells and the upregulation of interleukin-8 production in human T-cell acute lymphoblastic leukemia through the CXCL12/CXCR4 axis and the NF-kappaB and JNK/AP-1 pathways.

BACKGROUND: Cytokines released in the bone marrow and thymic microenvironments play a key role in the growth of T-cell acute lymphoblastic leukemia. Among such cytokines, interleukin-8 is highly expressed in T-cell acute lymphoblastic leukemia cells refractory to chemotherapy. In this study we explored whether bone marrow stromal cells can regulate IL-8 expression in T-cell acute lymphoblastic leukemia and investigated the role of the stromal CXCL12 chemokine in this event. We also investigated the roles of the nuclear factor-kappaB and Jun-N-terminal kinase (JNK)/activating protein (AP)-1 signaling pathways, which contribute to regulate interleukin-8 production in some cells. DESIGN AND METHODS: We analyzed the expression of interleukin-8 in primary cells from ten adult patients with T-cell acute lymphoblastic leukemia when these cells were cultured with bone marrow stromal cells or stimulated with exogenous CXCL12. Interleukin-8 mRNA was analyzed by a colorimetric assay. Cytokine production was assayed by cytometric antibody array and flow cytometry. Nuclear factor-kappaB and JNK/AP-1 activation was investigated by using specific inhibitors of these pathways, immunoblotting, electrophoretic mobility-shift assay and cell transfection assays. RESULTS: Bone marrow stromal cells upregulated interleukin-8 mRNA in T-cell acute lymphoblastic leukemia cells through the activity of CXCR4, the CXCL12 receptor, as assessed by the use of neutralizing antibodies. Exogenous CXCL12 induced a significant increase in the production of IL-8 mRNA and protein in all T-cell acute lymphoblastic leukemia cases. We showed that CXCL12 activates the nuclear factor-kappaB and JNK/AP-1 pathways, and that these events are required for increased expression of interleukin-8. Furthermore, the nuclear factor-kappaB and AP-1 elements of the interleukin-8 promoter are necessary for both constitutive and CXCL12-induced interleukin-8 expression. CONCLUSIONS: Interleukin-8 is physiologically regulated by the CXCL12/CXCR4 axis and the nuclear factor-kappaB and JNK/AP-1 pathways are required for interleukin-8 expression in T-cell acute lymphoblastic leukemia. We propose that, by upregulating interleukin-8, the bone marrow microenvironment and the CXCL12/CXCR4 axis may play a role in the pathogenesis of T-cell acute lymphoblastic leukemia.

A single amino acid substitution in CXCL12 confers functional selectivity at the beta-arrestin level.

CXCL12/CXCR4 axis relies on both heterotrimeric Gi protein and ß-arrestin coupling to trigger downstream responses. G protein activation allows for calcium flux, chemotaxis and early extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation, whereas ß-arrestin recruitment leads to late signaling, receptor desensitization and internalization. Together they may regulate the balance between transactivation and transinhibition of epithelial growth factor receptor 1 (HER1). Since we have previously noted significant differences between CXCL12 and its structural variant [N33A]CXCL12 in CXCR4 signaling, we sought to better characterize them by performing cAMP inhibition and ß-arrestin recruitment assays, as well as functional tests that separately investigate G protein and ß-arrestin-induced responses. [N33A]CXCL12 showed reduced potency both in Gαi coupling and ß-arrestin recruitment as compared to the wild type chemokine, acting as an unbiased ligand. While these findings translated into reduced potency within Gαi-dependent functions, ß-arrestin-dependent modules were affected in a more peculiar way. Unlike CXCL12, the mutant analogue did not restore HB-EGF-stimulated HER1 from CXCR4-induced transinhibition, and did not trigger the late wave of ERK1/2 phosphorylation. Instead, CXCR4 internalization was not impaired upon [N33A]CXCL12 stimulation. These differences highlight the novel opportunity to dissect CXCL12 signaling within the ß-arrestin layer, in which the mutant chemokine clearly favors the internalization module over the other pathways. Such functional selectivity has an impact on HER1 activation status and may play a relevant part in the crosstalk between tyrosine kinase and seven transmembrane receptors.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Efficient lysis of B-chronic lymphocytic leukemia cells by the plant-derived sesquiterpene alcohol α-bisabolol, a dual proapoptotic and antiautophagic agent.

The sesquiterpene α-bisabolol (α-BSB) is a cytotoxic agent against acute leukemia and chronic myeloid leukemia cells. Here the profile of α-BSB citotoxicity was evaluated ex vivo in primary mononuclear blood cells isolated from 45 untreated B-chronic lymphocytic leukemia (B-CLL) patients. We studied the effects of α-BSB by flow cytometric and western blotting techniques with the following findings: (1) α-BSB was an effective proapoptotic agent against B-CLL cells (IC50 42 ± 15 µM). It was also active, but to a lesser extent, on normal residual B cells and monocytes (IC50 68 ± 34 and 74 ± 28 µM, respectively; p < 0.01), while T-cells, though not achieving IC50, were nevertheless decreased. (2) Lipid raft content positively correlated with α-BSB cell sensitivity, while neither the phenotype of B-CLL cells nor the disease clinical stage did affect the sensitivity to α-BSB. (3) Flow cytometry analysis evidenced the induction of pores in mitochondrial and lysosomal membrane after 3- to 5-hour exposure of B-CLL cells to α-BSB, leading to apoptosis; in contrast, western blotting analysis showed inhibition of the autophagic flux. Therefore, according to cellular selectivity, α-BSB is a cytotoxic agent preferentially active against leukemic cells, while its lower activity on normal B cells, monocytes and T cells may account for an additive anti-inflammatory effect targeting the leukemia-associated pro-inflammatory microenvironment. Consistent with the observed effects on intracellular processes, α-BSB should be regarded as a dual agent, both activating mitochondrial-based apoptosis and inhibiting autophagy by disrupting lysosomes.

Interleukin 7 requirement for survival of T-cell acute lymphoblastic leukemia and human thymocytes on bone marrow stroma.

We explored the role of interleukin-7 (IL-7) in the bone marrow (BM) stroma-mediated survival of primary T-cell acute lymphoblastic leukemia (T-ALL) cells and normal thymocytes. We presented evidence that IL-7 has a major role in the enhanced survival mediated by BM stroma both in T-ALL cells and thymocytes.

Flow cytometry analysis of receptor internalization/shedding.

BACKGROUND: Differentiation, proliferation, and chemotaxis responses stem from biological programs that recognize checkpoints at the level of membrane receptor internalization and shedding. Therefore, receptor trafficking represents a crucial regulator of cell functions. METHODS: Here, we present a survey of analyses of receptor internalization vs. shedding based on simple flow cytometry-based techniques. A relevant basic observation is that a fluorochrome-bearing antibody bound to a specific receptor that is translocated from the membrane to the cytoplasm continues to emit light, i.e., the cell remains equally positive for that marker even if the receptor is strongly downregulated or no longer detectable on the membrane. In contrast, fluorescence is lost following receptor shedding. RESULTS: The combined uses of standardized hyperosmolar sucrose, acidic treatment and flow cytometry staining at different times allows for fully informative studies of the internalization or shedding pattern of a given receptor. The procedure can be simplified into a straightforward, simple-to-use, and flexible flow cytometry method based on two sequential steps with in-between receptor stimulation. This method obviates the need for time-consuming fluorescence techniques and even confocal microscopy. CONCLUSION: We validate this procedure via comparisons of three receptors, i.e., CXCR4, CD30, and CD25, with membrane trafficking patterns that are involved in biological functions that are relevant to immunity and cancer. © 2016 International Clinical Cytometry Society.