T-leukemia Cells Research Articles

Selected Adaptor Proteins NTAL, LAT, PAG, LIME Function in Proximal Signaling, Corticosteroid Driven Apoptosis and Expression of IKAROS Isoforms in the T-Leukemic Cells.

Abstract 5034Transmembrane adaptor proteins NTAL, LAT, PAG and LIME have an important role in proximal signaling of T-lymphocytes. They lack enzymatic or kinase function, but when tyrosine-phosphorylated they bind other signaling molecules and mediate signaling from T-cell receptor (TCR) to nucleus. In our previous studies, we showed that in childhood T-cell acute lymphoblastic leukemia (T-ALL) the level of NTAL significantly correlates with early response to treatment. Patients with high NTAL levels responded favorably (prednisone good-responders) whereas patients with low NTAL were prednisone poor responders (p=0.05). We confirmed this clinical data also in in-vitro experiment - derivative Jurkat cell line (human T-ALL) transfected with NTAL expression construct (Jurkat/NTAL+) was more sensitive to corticosteroid treatment compared to wild-type Jurkat cell line (Jurkat/wt, NTAL negative).In the present study, we performed a more detailed analysis of a relationship between adaptor proteins, TCR signaling and apoptosis in T-ALL. Monoclonal IgM antibody C305 binding the Jurkat TCR with high affinity was used to stimulate TCR signalization in Jurkat/wt and Jurkat/NTAL+ cell lines. To assess the influence of TCR signaling on corticosteroid-driven apoptosis, we treated both the Jurkat cell lines with the C305 antibody and/or with methylprednisolone. At 24 hours we detected higher percentage of cells with the stimulation marker CD69 in Jurkat/NTAL+ cells compared to the Jurkat/wt (median 70% vs. 60%, p<0.05). More cells treated with C305 and methylprednisolone together expressed higher CD69 than cells treated by C305 alone (median 75% vs. 65%, p<0.004). At 24 and 48 hours we determined the level of apoptosis. At each time point the number of living cells in the untreated control was set to 100%. Jurkat/NTAL+ cell line was more prone to apoptosis than the wild-type cell line in all settings. At 24 hours, the percentage of surviving cells Jurkat/NTAL+ vs. Jurkat/wt was 44% vs. 50% when treated with methylprednisolone alone (p<0.1), 24% vs. 42% (C305 alone, p<0.1) and 15% vs. 26% (methylprednisolone + C305, p<0.1). Similar differences were detected after 48hours of treatment. Using flow cytometry, we further determined phosphorylation status of key downstream kinases ERK, p38 and JNK after TCR stimulation by the C305 antibody. We detected hyperphosphorylation of ERK higher in Jurkat/NTAL+ compared to Jurkat/wt (1.57, 1.53 and 1.46 fold at 5, 15 and 30 minutes after stimulation, respectively). Levels of phosphorylated P38 and JNK did not differ from the unstimulated controls or between the two cell lines. Our data show that the differences in apoptosis are not driven by an upregulation of FAS or FAS ligand as any significant increase in the levels of FAS/FASL was not detected by flow cytometry throughout the above described experiments.It was shown previously that aberrant TCR signaling affects the incidence of dominant-negative isoforms of IKAROS. Thus, to further elucidate the role of the signaling in T-ALL we analyzed presence and expression profile of IKAROS splicing variants (using on-chip electrophoresis) in a cohort of 29 pediatric T-ALL patients. While we did not observe any link between NTAL or LAT levels and expression of different IKAROS isoforms, we found a close correlation between IKAROS isoforms IK1 and IK4a and another adaptors, PAG (major inhibitor of Src kinases in lymphocytes) and LIME (adaptor involved in signalization via CD4 and CD8). The two patients expressing IK1 and IK4A as dominant transcripts had >2 logs lower PAG (p=0.02) and LIME (p<0.1) expression levels compared to the rest of the cohort (n=27) showing IK2 as a dominant transcript. Based on this data, we propose that NTAL acts as a tumor suppressor enhancing proximal signaling of lymphocytes. Via phosphorylation of ERK, the NTAL sensitizes T-leukemic cells to corticosteroid induced apoptosis that is not mediated by FAS or FASL. The role of IKAROS isoforms and its connection with adaptor proteins is under investigation.The work was supported by grants MSM0021620813 and GAUK35607. DisclosuresNo relevant conflicts of interest to declare.

TRESK channel as a potential target to treat T-cell mediated immune dysfunction

In this review, we propose that TRESK background K + channel could serve as a potential therapeutic target for T-cell mediated immune dysfunction. TRESK has many immune function-related properties. TRESK is abundantly expressed in the thymus, the spleen, and human leukemic T-lymphocytes. TRESK is highly activated by Ca 2+, calcineurin, acetylcholine, and histamine which induce hypertrophy, whereas TRESK is inhibited by immunosuppressants, such as cyclosporin A and FK506. Cyclosporine A and FK506 target the binding site of nuclear factor of activated T-cells (NFAT) to inhibit calcineurin. Interestingly, TRESK possesses an NFAT-like docking site that is present at its intracellular loop. Calcineurin has been found to interact with TRESK via specific NFAT-like docking site. When the T-cell is activated, calcineurin can bind to the NFAT-docking site of TRESK. The activation of both TRESK and NFAT via Ca 2+–calcineurin–NFAT/TRESK pathway could modulate the transcription of new genes in addition to regulating several aspects of T-cell function.

Cytotoxic effect of 2’, 3’-epoxy isocapnolactone and 8- hydroxyisocapnolactone-2’3’-diol isolated from Micromelum minutum (G.Forst.) Wight and Arn. in human T-lymphocyte leukemia CEM-SS cells

2’,3’-Epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol are two bioactive compounds isolated from the leaves of Micromelum minutum. In this study, the induction of apoptosis by 2’,3’-epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol on CEM-SS cells were investigated. The cytotoxicity of 2’,3’-epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol was screened on CEM-SS by using MTT assay. The apoptotic effect of both compounds on CEM-SS was evaluated by using fluorescence and electron microcopies morphological observation and DNA laddering analysis. The inhibition effect of 2’,3’-epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol at 50% of cell population (IC50) was found to be 4.6 µg/ml (13.5 µM) and 3 µg/ml (7.8 µM) on CEM-SS cells, respectively. From DNA fragmentation, Acridine orange and Propidium iodide staining and electron microscope analyses, the compounds were confirmed to have ability in promoting apoptosis. However, the % of apoptosis induced is low and the event is time-dependent. At high concentration of 10 µg/mL,2’,3’-epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol induced necrosis. Furthermore, 8-hydroxyisocapnolactone-2’,3’-diol also exhibited better cytotoxicity compared to 2’,3’-epoxyisocap-nolactone. The induction time for apoptosis by 8-hydroxyisocapnolactone-2’,3’-diol in CEM-SS is earlier than 2’,3’-epoxyisocapnolactone, which is 4 h and 12 h after treatment. Both 2’,3’-epoxyisocapnolactone and 8-hydroxyisocapnolactone-2’,3’-diol are potential as anticancer agent. Key words: Cytotoxic, 2’,3’-epoxy isocapnolactone, 8-hydroxyisocapnolactone-2’3’-diol, Micromelum minutum, CEM-SS.

Irradiation Alters Selection for Oncogenic Mutations in Hematopoietic Progenitors

Exposure to ionizing radiation and other DNA-damaging carcinogens is strongly associated with induction of malignancies. Prevailing paradigms attribute this association to the induction of oncogenic mutations, as the incidence of oncogenic events is thought to limit initiation and progression of cancers. On the other hand, random mutagenic and genotoxic effects of irradiation are likely to alter progenitor cell populations and the microenvironment, thus altering the selective effects of oncogenic mutations. Using competitive bone marrow transplantation experiments in mice, we show that ionizing irradiation leads to a persistent decline in the numbers and fitness of hematopoietic stem cells, in part resulting from persistent induction of reactive oxygen species. Previous irradiation dramatically alters the selective effects of some oncogenic mutations, substantially inhibiting clonal expansion and leukemogenesis driven by Bcr-Abl or activated N-Ras oncogenes but enhancing the selection for and leukemogenesis driven by the activated Notch1 mutant ICN. Irradiation-dependent selection for ICN expression occurs in a hematopoietic stem cell-enriched pool, which should facilitate the accumulation of additional oncogenic events at a committed T-progenitor stage critical for formation of T-lymphocytic leukemia stem cells. Enhancement of ICN-driven selection and leukemogenesis by previous irradiation is in part non-cell autonomous, as partial restoration of normal hematopoiesis can reverse these effects of irradiation. These studies show that irradiation substantially alters the adaptive landscape in hematopoietic progenitors and suggest that the causal link between irradiation and carcinogenesis might involve increased selection for particular oncogenic mutations.

Intracellular delivery of bovine lactoferricin’s antimicrobial core (RRWQWR) kills T-leukemia cells

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide with potent cytotoxic activity against cancer cells. The antimicrobial activity of LfcinB resides in its RRWQWR amino acid sequence (referred to here as LfcinB6); however, the anticancer activity of LfcinB6 is not known. Here, we show that free LfcinB6 did not kill T-leukemia or breast cancer cells but LfcinB6 was strongly cytotoxic when delivered to the cytosolic compartment by fusogenic liposomes. LfcinB6 bound weakly to isolated mitochondria but, unlike LfcinB, did not permeabilize mitochondria or cause cytochrome c to be released. Cathepsin B and caspase activity were important for cytotoxicity caused by intracellular LfcinB6 whereas reactive oxygen species were not involved. The mechanism of LfcinB6-induced cytotoxicity is therefore different from that of LfcinB. We suggest that LfcinB6, in combination with a fusogenic liposome delivery system that selectively targets malignant cells, has potential as a novel anticancer agent.

Transcriptional regulation of the human ALDH1A1 promoter by the oncogenic homeoprotein TLX1/HOX11

The homeoprotein TLX1, which is essential to spleen organogenesis and oncogenic when aberrantly expressed in immature T cells, functions as a bifunctional transcriptional regulator, being capable of activation or repression depending on cell type and/or promoter context. However, the detailed mechanisms by which it regulates the transcription of target genes such as ALDH1A1 remains to be elucidated. We therefore functionally assessed the ability of TLX1 to regulate ALDH1A1 expression in two hematopoietic cell lines, PER-117 T-leukemic cells and human erythroleukemic (HEL) cells, by use of luciferase reporter and mobility shift assays. We showed that TLX1 physically interacts with the general transcription factor TFIIB via its homeodomain, and identified two activities in respect to TLX1-mediated regulation of the CCAAT box-containing ALDH1A1 promoter. The first involved CCAAT-dependent transcriptional repression via perturbation of GATA factor-containing protein complexes assembled at a non-canonical TATA (GATA) box. A structurally intact homeodomain was essential for repression by TLX1 although direct DNA binding was not required. The second activity, which involved CCAAT-independent transcriptional activation did not require an intact homeodomain, indicating that the activation and repression functions of TLX1 are distinct. These findings confirm ALDH1A1 gene regulation by TLX1 and support an indirect model for TLX1 function, in which protein-protein interactions, rather than DNA binding at specific sites, are crucial for its transcriptional activity.

CD26 expression on T cell lines increases SDF-1-α-mediated invasion

Background:CD26 is a multifunctional membrane-bound glycoprotein that regulates tumour growth in addition to its other activities. Because disease aggressiveness is correlated with CD26 expression in several T-cell malignancies, we decided to investigate the invasiveness of cells expressing different levels of CD26.Methods:To assess CD26 involvement in cell invasion, we performed in vitro invasion assays with human T cell lines expressing different levels of CD26. These included the parental CD26-positive T-lymphoblast cell line HSB-2 and clones infected with a retrovirus expressing siRNA vectors that either targeted CD26 or encoded a missense siRNA, and the parental CD26-negative T-leukaemia cell line Jurkat and clones expressing CD26. CD26 expression in these cell lines was evaluated by flow cytometry and western immunoblotting. CXCR4 expression, phosphorylation of signalling kinases, and MMP-9 secretion were also evaluated by western immunoblotting, whereas MMP-9 activity and the effect of kinase and CD45 inhibitors on activity were measured by zymography of conditioned media.Results:The presence of CD26 enhanced stromal-cell-derived factor-1-α (SDF-1-α)-mediated invasion of T cell lines. This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-α and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion. In addition, CD26-associated enhancement of SDF-1-α-induced invasion was decreased when CD45 was inhibited.Conclusions:Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-α-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways. The data also suggest that CD26 enhancement of invasion may be mediated by CD45, however, more studies are required to confirm this involvement.

Alteration of enhancer of polycomb 1 at 10p11.2 is one of the genetic events leading to development of adult T‐cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a malignant tumor caused by latent human T-lymphotropic virus 1 (HTLV-1) infection. We previously identified a common breakpoint cluster region at 10p11.2 in acute-type ATLL by spectral karyotyping. Single nucleotide polymorphism array comparative genomic hybridization analysis of the breakpoint region in three ATLL-related cell lines and four patient samples revealed that the chromosomal breakpoints are localized within the enhancer of polycomb 1 (EPC1) gene locus in an ATLL-derived cell line (SO4) and in one patient with acute-type ATLL. EPC1 is a human homologue of the E(Pc) enhancer of polycomb gene of Drosophila. Inappropriate expression of the polycomb group gene family has been linked to the loss of normal gene silencing pathways, which can contribute to the loss of cell identity and malignant transformation in many kinds of cancers. In the case of the SO4 cell line, which carried a der(10)t(2;10)(p23;p11.2) translocation, EPC1 was fused with the additional sex combs-like 2 (ASXL2) gene at 2p23.3 (EPC1/ASXL2). In the case with an acute-type ATLL, who carried a der(10)del(10)(p11.2)del(10)(q22q24) translocation, a putative truncated EPC1 gene (EPC1tr) was identified. Overexpression of EPC1/ASXL2 enhanced cell growth in T-leukemia cells, and a GAL4-EPC1/ASXL2 fusion protein showed high transcriptional activity. Although a GAL4-EPC1tr fusion protein did not activate transcription, overexpression of EPC1tr accelerated cell growth in leukemia cells, suggesting that the EPC1 structural abnormalities in the SO4 cell line and in the patient with acute-type ATLL may contribute to leukemogenesis.

Combining Radiotherapy with APO010 in Cancer Treatment

Various proapoptotic agents are currently being explored to improve the outcome of radiotherapy. We have evaluated whether APO010-a novel recombinant ligand of the Fas/CD95 death receptor-enhanced the cytotoxic effect of radiation on lymphoid and solid tumor cell types. A Bcl-2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line (HCT116), and a mesothelioma cell line were used as model systems in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by tumor growth delay using bioluminescence and palpation in vivo. Whereas the three cell lines resisted apoptosis induction by irradiation and APO010 alone, combined treatment greatly enhanced their apoptotic response. In clonogenic survival assays, APO010 reduced the outgrowth of Jurkat-Bcl-2 and HCT116 cells and sensitized the mesothelioma cell line to radiation. In vivo, systemic treatment with APO010 alone caused tumor growth delay in Jurkat-Bcl-2 and HCT116 cells. However, APO010 did not improve the efficacy of radiotherapy in any of the model systems at the selected single dose, which had moderate and reversible systemic toxicity. Although APO010 and radiation had a clear combined cytotoxic effect on tumor cells in vitro, a combined therapeutic effect was not achieved on the same cells subcutaneously grafted in mice, at APO010 doses approximating the maximally tolerable level. These findings suggest that it will be difficult to identify a therapeutic window for this combined modality approach in a clinical setting.

Human T-leukemia and T-lymphoma express glutamate receptor AMPA GluR3, and the neurotransmitter glutamate elevates the cancer-related matrix-metalloproteinases inducer CD147/EMMPRIN, MMP-9 secretion and engraftment of T-leukemia in vivo

Glutamate is the major excitatory neurotransmitter of the nervous system. We previously found that glutamate activates normal human T-cells, inducing their adhesion and chemotaxis, via its glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3) expressed in these cells. Here, we discovered that human T-leukemia (Jurkat) and cutaneous sezary T-lymphoma (HuT-78) cells also express high levels of GluR3. Furthermore, glutamate (10 nM) elevates CD147/EMMPRIN, a cancer-associated matrix metalloproteinases (MMPs) inducer, promoting spread of many tumors. Glutamate-induced CD147 elevation in both cancerous and normal human T-cells was mimicked by AMPA (glutamate/AMPA-receptor agonist) and blocked by CNQX (glutamate/AMPA-receptor antagonist). Importantly, glutamate also increased gelatinase MMP-9 secretion by T-lymphoma. Finally, ex vivo pre-treatment of T-leukemia with glutamate enhanced their subsequent in vivo engraftment into chick embryo liver and chorioallantoic membrane. Together, these findings reveal that glutamate elevates cancer associated proteins and activity in T-cell cancers and by doing so may facilitate their growth and spread, especially to and within the nervous system. If so, glutamate receptors in T-cell malignancies should be blocked.

Gamma-Radiation-Induced Phosphorylation of p53 on Serine 15 Is Dose-Dependent in MOLT-4 Leukaemia Cells

Molecular indicators of the absorbed dose of ionizing radiation are powerful tools in biodosimetry. The studies reported here were undertaken with the motivation to find such a marker among the mo lecules involved in ataxia-telangiectasia mutated kinase-dependent signalling induced by ionizing radiation (ATM-kinase, checkpoint kinase-2, protein p53, and oncoprotein Mdm2). In our previous work on T-lymphocyte leukaemia MOLT-4 cells we described the mentioned molecules of ATM-dependent pathway and none of them showed a pronounced dosedependent response. Here we employed Western blotting and ELISA assay to investigate the response of post-translationally modified p53 (particularly phosphorylated on serine 15) after gamma-irradiation. We have found the amount of phosphorylated p53 to be homogenously increased after irradiation by the doses of 0.5 to 7.5 Gy. The dose-dependent response was pronounced especially after the doses up to 3.0 Gy. The presented data indicate that p53 phosphorylated on serine 15 might be used as a potential biodosimetric marker.

Abstract A23: Expression profile of genes altered by ester-protected hydroxybenzyl phosphates (EHBP) in T-leukemia cells

Abstract A23 Introduction Acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality in children. Novel compounds and different therapeutic approaches are required for more effective treatments. Nitric oxide-releasing aspirin (NO-ASA), comprised of an ASA molecule covalently bound to an NO liberating moiety through an aromatic spacer was reported to inhibit Jurkat T cell growth and other cancer cell lines of colon, pancreatic and breast origin, including experimentally-induced tumors in various animal models. Recently, we and others showed that the mechanism of action of NO-ASA did not involve released NO or aspirin but that the spacer molecule joining these two entities was the biologically active component. The role of the NO-releasing moiety was simply that of a leaving group in the production of a reactive quinone methide (QM) intermediate. The ASA component apparently had a very limited or no biological contribution. On this basis, we synthesized two compounds as models in which the NO-releasing group was replaced with a substituted phosphate (Agent 1: No NO, NO-ASA); and one in which the aspirin component was replaced with an acetyl group and the -ONO2 leaving group was replaced by a substituted phosphate group (Agent 2: No NO, No-aspirin, NO-ASA). In this study we investigated whether these three compounds may alter the gene expression of biological signaling pathways that are potential targets in carcinogenesis, and to elucidate the role of the NO-releasing moiety and the aspirin component of NO-ASA in the differential expression of these genes. Methods Agents: NO-ASA (2-(acetyloxy)-4-[(nitrooxy)methyl] phenyl ester, Agent 2: (2-(acetyloxy)-4-[(diethylphosphate)methyl] phenyl ester, Agent 3: (4-acetyloxybenzyl diethylphosphate) were synthesized, purified and NMR verified by us. Cell line: Jurkat T-acute lymphoblastic leukemia; the IC50 for cell growth inhibition for the three compounds was determined by an MTT assay. Following treatment of the Jurkat cells with NO-ASA, agent 2 or agent 3, at their respective IC50s, mRNA was isolated and labeled according to standard protocols and kits. The oligo arrays set from SuperArray were used. The blots were hybridized and the ratios of the expression levels quantified and calculated. Potential candidate genes that were differentially expressed were validated by real time RT-PCR. Results All three drugs were potent growth inhibitors of human leukemia Jurkat T cells. The genes that were prominently induced by all three drugs included FMO4, HSPA1A, HSPA6, HSPH1, FOS, CYP24 A1, DDIT3; this induction occurred in a concentration dependent manner. These may provide mechanistic insights into the growth inhibition observed with these agents. Other moderately altered genes included HYOU1, SOD1, SOD2. Interestingly, all 3 agents had the same effect in terms of fold-changes in gene expression for many of these candidate genes. This strongly suggests that the NO-releasing group and the aspirin component of NO-ASA have a negligible role in modulating these gene expressions. We propose that the growth inhibitory properties of all three agents are associated with the spacer component of the molecules which generates a quinone methide intermediate. The significance of the genes induced or repressed and the relationship of their gene expression with the cell growth inhibition will be discussed. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A23.

Paraoxon induced cell death and alterations of M1–M5 lymphocytic cholinergic muscarinic subtypes in human leukemic T-lymphocytes

Paraoxon induced cell death and alterations of M1–M5 lymphocytic cholinergic muscarinic subtypes in human leukemic T-lymphocytes

Carcinogenic chromium(VI)‐induced protein oxidation and lipid peroxidation: implications in DNA–protein crosslinking

Hexavalent chromium [Cr(VI)] compounds are Group-I human carcinogens. Cr(VI)-induced DNA-protein crosslinks (DPCs) have been implicated in the mutagenic and carcinogenic effects of Cr(VI). Although multiple mechanisms have been suggested for Cr(VI)-induced DNA-protein crosslinking, the mechanism of formation of DNA-protein crosslinks is not well understood. In this study, we explored the hypothesis that Cr(VI)-induced DPCs could be formed via generation of protein carbonyls and malonaldehyde (MDA) through protein oxidation and lipid peroxidation, respectively. Treatment of human leukemic T-lymphocyte MOLT4 cells with potassium chromate induced the formation of protein carbonyls and DPCs within 2 h, but increased the level of MDA only after 4 h, in a dose-dependent manner. Chromate treatment of MOLT4 cell homogenates also resulted in increased formation of MDA and protein carbonyls in a dose-dependent manner. EPR spectrometry in combination with spin trapping techniques revealed that reaction of Cr(VI) with biological reductants such as NADPH, glutathione reductase or H(2)O(2) generates Cr(V) and (*)OH radicals. Pretreatment of cells with antioxidants such as alpha-tocopherol or Tiron inhibited chromate-induced increase in formation of protein carbonyls, MDA and DPCs, but pretreatment of cells with riboflavin or 3-aminotriazole, a catalase inhibitor, had the opposite effect. Our results, for the first time, demonstrate that Cr(VI) exposure increases the cellular level of protein carbonyls and that Cr(VI)-induced DPCs may be formed, at least in part, via generation of protein carbonyls.

Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Is defect in phosphorylation of Nbs1 responsible for high radiosensitivity of T-lymphocyte leukemia cells MOLT-4?

Mutations in NBS1 gene are related to higher occurrence of malignancies. In this work we studied response of T-lymphocyte leukemia cells MOLT-4 to ionizing radiation. We detected IRIF (ionizing radiation forming foci) containing histone γH2A.X, protein 53BP1, and Nbs1, which were formed around double-strand breaks of DNA. We found dose-dependent increase in foci number (colocalization of γH2A.X and 53BP1) and γH2A.X amount (integral optical density) 1 h after irradiation. After the dose of 1.5 Gy the number of foci decreases with time, but 72 h after irradiation 9% of live cells still contained big foci around unrepaired DNA damage. Western blot method revealed massive phosphorylation of H2A.X during apoptosis induction, 6–24 h after irradiation by the doses 1.5 and 3 Gy. Cells with apoptotic morphology showed strong phosphorylation of H2A.X, but it was not accompanied by 53BP1. 1 h after irradiation by the lethal doses 5 and 10 Gy we detected by Western blot a decrease in repair proteins Mre11, Rad50, and Nbs1. While phosphorylation of H2A.X 1 h after irradiation was detected by both confocal microscopy and Western blot, phosphorylation of Nbs1 on serine 343 was not detectable in MOLT-4 cells. Despite functional ATM and p53 the phosphorylation of Nbs1 on serine 343 was impaired in these cells, and might be responsible for high radiosensitivity of MOLT-4 cells.

In vivooptical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integrin

Increasing literature suggests that cell adhesion molecule alpha4beta1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of alpha4beta1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies. Due to the lack of high-affinity targeting ligands, molecular imaging of alpha4beta1 integrin is much less explored than that of alphavbeta3 and alphavbeta5 integrins. We have recently reported using the one bead-one compound combinatorial library method to identify a peptidomimetic, LLP2A, that preferentially binds to activated alpha4beta1 integrin. Here, we described the use of LLP2A-Cy5.5 conjugate as an in vivo optical imaging probe in a human lymphoma xenograft model. This univalent LLP2A-Cy5.5 conjugate retained the binding activity and specificity to alpha4beta1 integrin as shown by cell binding assays using alpha4beta1-positive Molt-4 T-leukemia cells. The subcutaneous Molt-4 tumor was clearly visualized from 1 to 24 h after tail vein injection of the conjugate. Direct imaging and confocal microscopic examination of excised tumors and organs confirmed the accumulation of LLP2A in tumors and revealed very little or no uptake in normal organs except for lymph nodes. Kidney uptake was high when the whole organ was scanned but it was negative when examined microscopically, suggesting that LLP2A bound to the renal tubules loosely. Tumor uptake of LLP2A-Cy5.5 conjugate was blocked by excess unlabeled LLP2A. This study showed that the combinatorial chemical library-derived peptidomimetic LLP2A can be easily developed into an optical imaging probe for noninvasively monitoring of activated alpha4beta1 integrin in vivo.

A decisive role of mitochondria in defining rate and intensity of apoptosis induction by different alkaloids

Sanguinarine, chelerythrine and chelidonine possess prominent apoptotic effects towards cancer cells. In this study, we found that sanguinarine and chelerythrine induce apoptosis in human CEM T-leukemia cells, and that is accompanied by an early increase in cytosolic cytochrome c that precedes caspases-8, -9 and -3 processing. During apoptosis induction by sanguinarine and chelerythrine, reactive oxygen species (ROS) was rapidly generated and Δ Ψ mt dissipated, while Bax, Bcl-2 and Bcl-X (L/S) proteins’ content in the mitochondrial fraction did not change significantly. Caspase-3 activation and DNA fragmentation were considerably inhibited by N-acetyl-cysteine (NAC). Chelidonine induced only a slight release of cytochrome c (12 h), parallel to caspase-3 activation. Effect of sanguinarine or chelerythrine towards mitochondria was confirmed by marked changes in morphology of this organelle (3 h), while chelidonine did not affect mitochondria intactness. Sanguinarine or chelerythrine also caused an intensive DNA damage in cells in 1 h, however a massive increase in number of such impaired cells occurred in 6 h, while chelidonine induced intensive DNA damage in 15–20% cells only in 24 h. Thus, our results demonstrated that rapid cytochrome c release in CEM T-leukemia cells exposed to sanguinarine or chelerythrine was not accompanied by changes in Bax, Bcl-2 and Bcl-X (L/S) proteins in the mitochondrial fraction, and preceded activation of the initiator caspase-8.

Apoptosis-associated antigens recognized by autoantibodies in patients with the autoimmune liver disease primary biliary cirrhosis

There is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. Since defects in the elimination of apoptotic cells lead to secondary necrosis and subsequent release of intracellular components, this might explain the generation of autoantibodies against intracellular antigens. Accordingly, we wanted to investigate, whether antibodies from patients with the autoimmune liver disease primary biliary cirrhosis (PBC) recognize self-proteins generated and released during apoptosis. Using Western blot analyses we could detect intracellular antigens with serum IgG from PBC patients but not with serum IgG from healthy donors in lysates of Jurkat T-leukemia, HepG2 hepatoma, and HT-29 colon-carcinoma cells. Interestingly, PBC serum IgG also recognized caspase substrates in cells undergoing apoptosis induced by staurosporine or TRAIL (TNF-related apoptosis inducing ligand). In addition to intracellular antigens, serum IgG from PBC patients detected caspase-dependent antigens in the supernatants of apoptotic (secondary necrotic) cells and antigens on the surface of apoptotic Jurkat cells. Among the caspase substrates recognized by PBC serum IgG we could identify the components PDC-E2 and -E1beta of the known autoantigen PDC (pyruvate dehydrogenase complex). Thus, caspase-mediated processing of intracellular proteins might generate de novo autoantigens that upon release contribute to the generation of autoantibodies and autoimmune diseases as PBC.

Differential involvement of reactive oxygen species in apoptosis caused by the inhibition of protein phosphatase 2A in Jurkat and CCRF-CEM human T-leukemia cells

A better understanding of dysregulated signaling pathways in cancer cells may suggest novel strategies to prevent tumor development and/or progression. Here we show that Jurkat and CCRF-CEM human T-leukemia cell lines were more sensitive than normal human T cells to the cytotoxic effect of inhibiting protein phosphatase 2A (PP2A). Inhibition of PP2A by okadaic acid (OA) caused T-leukemia cells to die by apoptosis, as indicated by DNA fragmentation, caspase-3 activation, loss of mitochondrial membrane potential (Δ Ψ m), and changes in nuclear morphology that were consistent with apoptosis. PP2A might therefore be a useful intracellular target for the treatment of T cell-derived leukemias. We also observed that reactive oxygen species (ROS) were generated in response to PP2A inhibition in T-leukemia cells. However, loss of Δ Ψ m that resulted from PP2A inhibition was not prevented by exogenous antioxidants (glutathione and N-acetyl-cysteine), indicating that OA-induced changes in mitochondrial membrane permeability were not a consequence of ROS production. Moreover, exogenous antioxidants protected CCRF-CEM T-leukemia cells from apoptosis caused by PP2A inhibition but failed to prevent OA-induced apoptosis in Jurkat T-leukemia cells, indicating a differential role for ROS in apoptosis caused by PP2A inhibition in two different human T-leukemia cell lines.