Lymphoma Jurkat Cells Research Articles

Impact of high (1950 MHz) and extremely low (50 Hz) frequency electromagnetic fields on DNA damage caused by occupationally relevant exposures in human derived cell lines

Epidemiological studies indicate that electromagnetic fields (EMF) are associated with cancer in humans. Exposure to mobile phone specific high frequency fields (HF-EMF) may lead to increased glioma risks, while low frequency radiation (LF-EMF) is associated with childhood leukemia. We studied the impact of HF-EMF (1950 MHz, UMTS signal) on DNA stability in an astrocytoma cell line (1321N1), and the effect of LF-EMF (50 Hz) in human derived lymphoma (Jurkat) cells. To find out if these fields affect chemically induced DNA damage, co-exposure experiments were performed. The cells were exposed to HF-EMF or LF-EMF and treated simultaneously and sequentially with mutagens. The compounds cause DNA damage via different molecular mechanisms, i.e. pyrimidine dimers which are characteristic for UV light (4-nitroquinoline 1-oxide, 4NQO), bulky base adducts (benzo[a]pyrene diolepoxide, BPDE), DNA-DNA and DNA-protein cross links and oxidative damage (NiCl2, CrO3). DNA damage was measured in single cell gel electrophoresis (comet) assays. We found a moderate reduction of basal and 4NQO-induced DNA damage in the astrocytoma line, but no significant alterations of chemically induced DNA migration by the HF and LF fields under all other experimental series. The biological consequences of the moderate reduction remain unclear, but our findings indicate that acute mobile phone and power line specific EMF exposures do not enhance genotoxic effects caused by occupationally relevant chemical exposures.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma RD cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In RD cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.

Seleno-vs. thioether triazine derivatives in search for new anticancer agents overcoming multidrug resistance in lymphoma

Lymphomas are still difficult to treat even with modern therapies as, among others, multidrug resistance (MDR) is often counteracting a successful cancer therapy. P-gp/ABC-transporters are well-known for their crucial role in the main tumour MDR mechanism, eliminating drugs and cytotoxic substances from the cancer cell by efflux, and their modulators are promising for innovative therapy, but none has been approved in the pharmaceutical market yet. Herein, we have designed, synthesised and analysed 30 novel seleno- and thioether 1,3,5-triazine derivatives conducting comprehensive studies to evaluate their potential application in human JURKAT lymphoma cells. Among the new compounds, four (11, 12, 13 and 23) were much more effective than the reference inhibitor verapamil, being potent ABCB1 inhibitors already at 2 μM, while 5 and 15 showed very potent ABCB1 inhibitory activity only at 20 μM. Results of P-gp ATPase assays, supported with docking studies, indicated the competitive substrate mode of modulating action for 15, while ABCB1, ABCC1 and ABCG2 genes expression induction by 15 with q-PCR was confirmed. All compounds were evaluated for their cytotoxic and antiproliferative properties in both sensitive (PAR) and resistant (MDR) mouse T-lymphoma cell lines, and compound 15, also considering its promising ABCB1 inhibition properties, was revealed to be the best compound in terms of its cytotoxic effect (IC50: 16.73 μM) as well as concerning the antiproliferative effect (IC50: 5.35 μM) in MDR cells.Regarding the mechanistic studies looking at the cell cycle, the thioether 15 and selenium derivatives 26 and 29 were significantly effective in the regulation of cell cycle-related genes alone or in co-treatment with doxorubicin counteracting Cyclin D1 and E1 expression and increasing p53 and p21 levels, shedding first light on their mechanism of action. In summary, we explored the chemical space of seleno- and thioether 1,3,5-triazine derivatives with interesting activity against lymphoma. Especially compound 15 is worthy of being studied deeper to evaluate its precise mode of action further as well it can be improved regarding its potency and drug-likeness.

Preparative Production and Purification of Recombinant Human Cyclophilin A.

In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.

Landfill soil leachates from Nigeria and India induced DNA damage and alterations in genes associated with apoptosis in Jurkat cell.

Landfill soil leachates, containing myriad of xenobiotics, increase genotoxic and cytotoxic stress-induced cell death. However, the underlying mechanism involved in the elimination of the damaged cells is yet to be fully elucidated. This study investigated the apoptotic processes induced in lymphoma (Jurkat) cells by landfill soil leachates from Olusosun (OSL, Nigeria) and Nagpur (NPL, India). Jurkat was incubated with sub-lethal concentrations of OSL and NPL for 24 h and analyzed for DNA fragmentation and apoptosis using agarose gel electrophoresis and Hoechst 33258-PI staining, respectively. Complementary DNA expression profiling of some pro-apoptotic and anti-apoptotic genes regulating apoptosis was also analyzed using real-time PCR (RT-PCR) method. Agarose gel electrophoresis revealed DNA fragmentations in OSL and NPL-treated cells. Hoecsht-33258 - Propidium Iodide (PI) based apoptotic analysis confirmed apoptotic cell death in exposed Jurkat. RT-PCR analysis revealed different fold changes in the pro- and anti-apoptotic genes in OSL and NPL-treated Jurkat. There was significant increase in fold change of the up-regulated genes; apoptosis inducing factor mitochondrion-associated 2 (AIFM2), Fas-associated death domain (FADD), Caspase-2, Caspase-6, BH3 interacting domain death agonist (BID), tumor suppressor (p53), and BCL2 associated agonist of cell death (BAD) and down-regulation of apoptosis inhibitor 5 (API5). Results suggest that OSL and NPL elicited genotoxic stress-related apoptosis in Jurkat. The dysregulation in the expression of genes involved in apoptotic processes in wildlife and human exposed to landfill emissions may increase aetiology of various pathological diseases including cancer.

Exploring the Mechanism of Paclitaxel Inhibiting T-cell Lymphoma based on High-throughput Sequencing and Public Databases

To analyze gene expression profile of T cell lymphoma Jurkat cell line treated with paclitaxel by computational biology based on next generation sequencing and to explore the possible molecular mechanism of paclitaxel resistance to T cell lymphoma at gene level. IC50 of paclitaxel on Jurkat cell line was determined by CCK-8 assay. Gene expression profile of Jurkat cells treated with paclitaxel was acquired by next generation sequencing technology. Gene microarray data related to human T cell lymphoma were screened from Gene Expression Omnibus (GEO) database (including 720 cases of T cell lymphoma and 153 cases of normal tissues). Combined with the sequencing data, differential expression genes (DEGs) were intersected and screened. DAVID database was used for enrichment analysis of GO function and KEGG pathway to determine and visualize functional entries of DEGs, and protein-protein interactions network of DEGs was drawn. The levels of gene expression were detected and verified by RT-qPCR. CCK-8 results showed that the proliferation of Jurkat cells was inhibited by paclitaxel depended on the concentration apparently. Treated by paclitaxel for 48 h, P<0.05 and |log2(FC)|≥1 were used as filter criteria on the results of RNA Sequencing (RNA-Seq) and GeoChip, 351 DEGs were found from Jurkat cells, including 323 up-regulated genes and 28 down-regulated genes. The GO functional annotation and KEGG pathway enrichment analysis showed that the role of paclitaxel was mainly concentrated in protein heterodimerization activity, nucleosome assembly and transcriptional dysregulation in cancer, etc. The results of RT-qPCR were consistent with those of the sequencing analysis, which verified the reliability of this sequencing. Paclitaxel can affect the proliferation and apoptosis of T-cell lymphoma by up-regulating JUN gene, orphan nuclear receptor NR4A family genes and histone family genes.

40 Years of Research on Polybrominated Diphenyl Ethers (PBDEs)—A Historical Overview and Newest Data of a Promising Anticancer Drug

Polybrominated diphenyl ethers (PBDEs) are a group of molecules with an ambiguous background in literature. PBDEs were first isolated from marine sponges of Dysidea species in 1981 and have been under continuous research to the present day. This article summarizes the two research aspects, (i) the marine compound chemistry research dealing with naturally produced PBDEs and (ii) the environmental toxicology research dealing with synthetically-produced brominated flame-retardant PBDEs. The different bioactivity patterns are set in relation to the structural similarities and dissimilarities between both groups. In addition, this article gives a first structure–activity relationship analysis comparing both groups of PBDEs. Moreover, we provide novel data of a promising anticancer therapeutic PBDE (i.e., 4,5,6-tribromo-2-(2′,4′-dibromophenoxy)phenol; termed P01F08). It has been known since 1995 that P01F08 exhibits anticancer activity, but the detailed mechanism remains poorly understood. Only recently, Mayer and colleagues identified a therapeutic window for P01F08, specifically targeting primary malignant cells in a low µM range. To elucidate the mechanistic pathway of cell death induction, we verified and compared its cytotoxicity and apoptosis induction capacity in Ramos and Jurkat lymphoma cells. Moreover, using Jurkat cells overexpressing antiapoptotic Bcl-2, we were able to show that P01F08 induces apoptosis mainly through the intrinsic mitochondrial pathway.

Hydrodynamic particle focusing enhanced by femtosecond laser deep grooving at low Reynolds numbers

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re < 1. Moreover, we achieved continuous focusing of live cells with different sizes ranging from 10 to 15 µm, i.e. human T-cell lymphoma Jurkat cells, rat adrenal pheochromocytoma PC12 cells and dog kidney MDCK cells. The glass grooves fabricated by fs laser are expected to be integrated with on-chip detection components, such as contact imaging and fluorescence lifetime-resolved imaging, for various biological and biomedical applications, where particle focusing at a relatively low flow rate is desirable.

Cell death induction by Ranunculus ternatus extract is independent of mitochondria and dependent on Caspase-7.

Ranunculus ternatus is a traditional Chinese medicine with an anticancer effect, but its underlying mechanism is unknown. In this study, we demonstrated by MTT assay that ethyl acetate extract (RTE) from R. ternatus exerts cytotoxic effects on human T cell lymphoma Jurkat cells. Then, to test the apoptosis induction ability of RTE to induce apoptosis, we analyzed phosphatidylserine exposure, DNA fragmentation, and caspase cleavage. RTE induced phosphatidylserine exposure and caspase-7 cleavage, but not caspase-3 cleavage. Sub-G1 cells were accumulated but DNA fragmentation was not observed. A pan-caspase inhibitor Z-Asp-CH2-DCB suppressed RTE-induced caspase cleavage and the above-described events. RTE also induced cell death in caspase-3 null human breast cancer MCF-7 cells, indicating that RTE-induced apoptotic-like cell death depends on the activation of one or more caspases, but not caspase-3. Moreover, RTE-induced cell death was not suppressed in Bcl-2 overexpressing Jurkat cells, suggesting that mitochondria were not involved in RTE-induced cell death. In conclusion, RTE-induced cell death was independent of mitochondria and dependent on caspase-7.

Sinensetin induces apoptosis and autophagy in the treatment of human T-cell lymphoma.

The present study was carried out to explore the effect of sinensetin in human T-cell lymphoma Jurkat cells and to reveal the underlying molecular mechanisms. We found that sinensetin significantly impeded Jurkat cell proliferation in a dose-dependent and time-dependent manner. Additionally, sinensetin treatment triggered apoptosis and autophagy in Jurkat cells. The apoptosis induction was related to a loss of mitochondrial membrane potential and to increased caspase-3/-8/-9 and poly(ADP-ribose) polymerase (PARP) cleavage. Sinensetin also induced autophagy, as evidenced by the formation of acidic vacuoles, the upregulation of LC3-II and beclin-1, and the downregulation of p62. In addition, the inhibition of autophagy by 3-methyladenine significantly enhanced the apoptosis rate and improved the sensitivity of the Jurkat cells to sinensetin. Moreover, sinensetin induced cell death, apoptosis, and autophagy through the activation of the reactive oxygen species/ c-Jun N-terminal kinase signaling pathway and the inhibition of the Akt/mTOR signaling pathways. In summary, our results revealed that sinensetin induced apoptosis and autophagy in human T-cell lymphoma Jurkat cells by activating reactive oxygen species/ c-Jun N-terminal kinase and blocking the Akt/mTOR signaling pathways. Thus, sinensetin might be a potential candidate in the development of antitumor drugs targeting T-cell leukemia.

Abstract A098: SLC12A2 as a novel “brake” on immunogenic apoptotic cell clearance

Abstract Phagocytes face a key challenge during efferocytosis – they must engulf an apoptotic cell, often similar in size, while maintaining their own cell integrity, shape, and volume. Yet little is known about phagocyte volume homeostasis during apoptotic cell clearance. To this end, we performed an unbiased transcriptomics screen designed to reveal alterations of phagocyte gene expression after engulfment of apoptotic Jurkat lymphoma cells and identified several transcriptional programs distinctly modified in engulfing phagocytes. We identified a program involving cell volume regulation and potassium/chloride flux, the latter of which is thought to underlie immunogenic responses. Disruption of one member of this cell volume program, SLC12A2, resulted in “overeating” of apoptotic JurkaT-cells by individual phagocytes and a significant increase in cell size. Importantly, we demonstrate that SLC12A2-deficient phagocytes actively engulfing apoptotic JurkaT-cells led to a downregulation of the hallmark antiinflammatory efferocytosis program and robust upregulation of a proinflammatory program. In particular, we observed robust upregulation of immunogenic programs, including a type I interferon signature, in SLC12A2-deficient engulfing phagocytes that were not observed in phagocytes over-expressing the phosphatidylserine receptor TIM4, Mechanistically, we found that the chloride-sensing kinase WNK1 and downstream kinases OSR1 and SPAK function to regulate chloride flux into engulfing phagocytes via SLC12A2, and that this pathway regulates the apoptotic JurkaT-cell “overeating” observed in SLC12A2-deficient phagocytes. Collectively, these data elucidate a novel mechanism by which phagocytes protect themselves from potentially dangerous apoptotic cell-derived inflammatory ligands and suggest that SLC12A2 serves to actively suppress immunogenic responses by phagocytes and limits the rate of corpse uptake during efferocytosis. Citation Format: Justin Shaun Arnold Perry, Sho Morioka, Christopher Medina, Michael Raymond, Kodi Ravichandran. SLC12A2 as a novel “brake” on immunogenic apoptotic cell clearance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A098.

Inhibition of Caspase-2 Activity in Human Jurkat T-cell Lymphoma Cells by Splice Switching Oligonucleotide to its pre-mRNA

Caspase-2 is a key enzyme thinvolved in induction of apoptosis. The caspase-2 level is regulated by alternative splicing (AS) of its mRNA. The aim of this work was to determine the ability of an oligonucleotide complementary to Casp-2 pre-mRNA to induce AS. This oligonucleotide blocked the binding of splicing-regulating proteins to their sites at the end of exon 9 of Casp-2 pre-mRNA, leading to induction of AS of Casp-2 mRNA. The decrease in expression of full-size active splice-variant (Casp-2L) and the increase the expression of a shortened variant (Casp-2S) was demonstrated in human T-cell lymphoma Jurkat cell line. The expression level of total Casp-2 remained unchanged. Disproportion of splice variants of Casp-2 led to inhibition of enzymatic activity of caspase-2.

Dihydroartemisinin triggers c-Myc proteolysis and inhibits protein kinase B/glycogen synthase kinase 3β pathway in T-cell lymphoma cells.

Recent studies have revealed a positive therapeutic effect of dihydroartemisinin (DHA) on tumor cells. However, the underlying mechanism of this has not yet been elucidated. The present study examined the potential therapeutic role and mechanism of DHA in T-cell lymphoma cells. It was revealed that DHA inhibited the proliferation of Jurkat and HuT-78 T-cell lymphoma cells in a concentration- and time-dependent manner. Furthermore, DHA reduced c-Myc protein expression at the transcriptional level, and induced the phosphorylation of c-Myc and the degradation of c-Myc oncoprotein levels. DHA treatment resulted in decreased phosphorylation of protein kinase B (Akt) and glycogen synthase 3β (GSK3β) in T-cell lymphoma cells. In addition, DHA treatment induced cell apoptosis, which was accompanied by an increased ratio of Bax/Bcl-2. Taken together, the results of the present study suggested that DHA may exert its antitumor role by accelerating c-Myc proteolysis and inhibiting the Akt/GSK3β pathway in T-cell lymphoma cells.

Arsenic trioxide induces the apoptosis and decreases NF-κB expression in lymphoma cell lines.

Lymphoma is a type of cancer that develops from certain immune system cells. Arsenic trioxide (ATO) has attracted wide attention owing to its antitumor activities. However, the role of ATO in tumorigenesis and progression remains to be investigated. In the present study, the antitumor function of ATO was investigated in in lymphoma Raji and Jurkat cell lines and the effect of ATO on nuclear factor (NF)-κB expression levels. A Cell Counting kit-8 assay was used to assess cellular proliferation and the degree of cell apoptosis was measured by flow cytometric analysis; these assays demonstrated that ATO inhibited proliferation and promoted the apoptosis of Raji and Jurkat cells in a dose- and time-dependent manner. Western blot analysis revealed that ATO treatment affected the expression of apoptosis-associated proteins by downregulating the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and upregulating the pro-apoptotic protein Bcl-2-associatedX and the degree of caspase-3 cleavage. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis showed that the mRNA and protein expression levels of NF-κB were downregulated significantly following treatment with 2 µM ATO for 24, 48 and 72 h in the two cell lines. Additionally, immunofluorescence staining indicated that NF-κB expression diminished following ATO treatment in a time-dependent manner. These data indicated that ATO inhibited the proliferation of lymphoma cells by inducing cell apoptosis, which may be associated with the inhibition of the NF-κB signaling pathway. The findings of the present study may lay the foundation for developing a personalized medicine strategy using ATO via targeting of the NF-κB signaling pathway in lymphoma.

Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells.

γδ T cells represent less than 5% of circulating T cells; they exert a potent cytotoxic function against tumor or infected cells and secrete cytokines like conventional αβ T cells. As αβ T cells γδ T cells reside in the typical T cell compartments (the lymph nodes and spleen), but are more widely distributed in tissues throughout the body. For these reasons, some investigators are exploring the possibility of immunotherapies aimed to expand and activate Vδ2 T cells, or using them as Chimeric Antigen Receptor carriers. However, the role of immunosuppressive microenvironment on Vδ2 T cells during infections and cancers has not been completely elucidated. In particular, the effects of myeloid-derived suppressor cells (MDSC), largely expanded in such pathologies, were not explored. In the present work, we demonstrated that MDSC may inhibit IFN-γ production and degranulation of phosphoantigen-activated Vδ2 T cells. Moreover, the Vδ2 T cells cytotoxic activity against the Burkitt lymphoma cell line Daudi and Jurkat cell line were impaired by MDSC. The Arginase I seems to be involved in the impairment of Vδ2 T cell function induced by both tumor cells and MDSC. These data open a key issue in the context of Vδ2-targeted immunoteraphy, suggesting the need of combined strategies aimed to boost Vδ2 T cells circumventing tumor- and MDSC-induced Vδ2 T cells suppression.

Relationship between the agonist activity of synthetic ligands of TRAIL-R2 and their cell surface binding modes.

Tumor Necrosis Factor Receptor Apoptosis Inducing Ligand (TRAIL) appears as an interesting candidate for targeted cancer therapy as it induces apoptosis in cancer cells without toxicity to normal cells. TRAIL elicits apoptosis through agonist death receptor TRAIL-R1 and TRAIL-R2 engagement. Nevertheless, recombinant soluble TRAIL and monoclonal antibodies against these receptors demonstrated insufficient efficacy in clinical trials. This may be explained by the cell-type dependency of the apoptotic response, itself influenced by the effect on ligand binding mode of factors such as the level of receptor oligomerization or glycosylation. To investigate the relation between binding mode and signaling, we used previously described synthetic divalent and monovalent peptides specific for TRAIL-R2. We measured their pro-apoptotic activity on three cancer cell lines sensitive to rhTRAIL induced-apoptosis and monitored their cell-surface binding kinetics. The two divalent peptides bound with strong affinity to TRAIL-R2 expressed on B lymphoma BJAB cells and induced a high degree of apoptosis. By contrast, the same peptides bound weakly to TRAIL-R2 expressed at the surface of the human colon cancer HCT116 or T lymphoma Jurkat cell lines and did not induce their apoptosis. Cross-linking experiments suggest that these differences could be afforded by variations in the TRAIL-R2 oligomerization state at cell surface before ligand addition. Moreover divalent peptides showed a different efficiency in BJAB apoptosis induction, and kinetic distribution analysis of the BJAB binding curves suggested subtle differences in binding mechanisms. Thus our data support a relation between the cell-surface binding mode of the peptides and their pro-apoptotic activity. In this case the precise characterization of ligand binding to the surface of living cells would be predictive of the therapeutic potential of TRAIL-R2 synthetic ligands prior to clinical trials.

Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells.

Mitochondria are central to apoptosis. However, apoptosis progression involving mitochondria is not fully understood. A factor involved in mitochondria-mediated apoptosis is 7A6 antigen. 7A6 localizes to mitochondria from the cytosol during apoptosis, which seems to involve 'effector' caspases. In this study, we investigated the precise role of effector caspases in 7A6 localization to mitochondria during apoptosis. Human T-cell lymphoma Jurkat cells were treated with an antibody against FAS. 7A6 localization was analyzed by confocal laser scanning microscopy and flow cytometry. Caspases activation was determined by western blot analysis. 7A6 localization to mitochondria during anti-FAS-induced apoptosis was significantly reduced by the caspase-6 inhibitor, N-acetyl-Val-Glu-Ile-Asp-aldehyde, but not by the caspase-3 inhibitor, N-acetyl-Asp-Asn-Leu-Asp-aldehyde, nor caspase-7/3 inhibitor, N-acetyl-Asp-Gln-Thr-Asp-aldehyde. Moreover, caspase-6 down-regulation suppressed 7A6 localization to mitochondria. Caspase-6 regulates 7A6 localization to mitochondria during anti-FAS-induced apoptosis of Jurkat cells.

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t1/2<1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells.

Synergistic Lethality with Gemcitabine and Arsenic Trioxide Against Human T Cell Lymphoma Jurkat Cells

Abstract There is still lack suitable agent that significantly changes the poor prognoses of T-cell non-Hodgkin's lymphoma (NHL), so that part of the patients who are far beyond the standard of first-line could not received effective treatment, therefore, it is urgent that seeking for a salvage treatment for patients with refractory malignant lymphoma, our destination is combination strategy aimed to higher anti-tumor activity with lower toxicity.GEM is a deoxycytidine analog with anti-metabolite activity in tumors through destroying the replication of cells and inhibiting ribonucleotide reductase as a prodrug. Recently, many clinical trials show that gemcitabine alone or in combination with other chemotherapy drugs can improve overall response rate and survival outcomes in malignant lymphoma patients compared with other chemotherapy regimens, such as vinorelbine and liposomal doxorubicin, busulfan and melphalan, which are active agents in all types of malignant lymphoma.ATO as a traditional medicine has been evaluated its good efficacy in a variety of hematologic malignancies, including myeloproliferative diseases, multiple myeloma, lymphoma. Currently, many clinical trials represented that the addition of ATO to standard treatment regimens can improve survival outcomes and may allow a reduction in cytotoxic chemotherapy exposure in other malignancies. The basis of related research found that ATO play killing effect on the cancer cells through multiple apoptotic pathways and many mechanisms. ATO was shown to bear relevant cytotoxic activity on lymphoma cell lines. The safety profile of ATO for the effective treatment and its toxicity is easily under control, which make it a preferred solution for combined chemotherapy scheme.Thus, in this study we found that the combination of ATO and GEM can lead to synergistic the cytotoxic effect, exert an enhanced effect on inhibiting Akt signaling and Bcl-2, up-regulating cleaved caspase-3, and inhibit the cell proliferation and self-renewal ability, and induce G0/G1 cell cycle arrest and apotosis in Jurkat cells, which provide preclinical evidence and a theoretical basis for combination therapy with ATO and GEM as a salvage therapy in treating relapsed or refractory aggressive non-Hodgkin's lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Detection and significance of BRAF gene in mature T/NK cell lymphoma

we aimed to investigate the mutation and expression of BRAF gene in mature T/NK cell lymphoma tissues and cell lines, explore the correlation between gene alterations and clinicopathological features and clinical outcomes of mature T/NK cell lymphoma. Firstly, we detected common mutant sites of BRAF (locus 1 799 mutation in exon 15 and loci 463, 465 and 468 mutation in exon 11) in lymphoma Jurkat, Hut-78 and YTS cell lines, normal peripheral blood lymphocytes, different types of mature T/NK cell lymphoma and reactive hyperplasia lymph nodes by direct sequencing. Then we measured the expression of BRAF in Jurkat, Hut-78, YTS cells and normal peripheral blood lymphocytes by real time-PCR and Western-blot detection. We also used immunohistochemistry (IHC) to detect the expression of BRAF in mature T/NK cell lymphoma tissues and reactive hyperplasia lymph nodes, and to analyze the correlation between the expression of BRAF and clinocopathological features and clinical outcomes. We did not find common BRAF mutation in mature T/NK cell lymphoma tissues and cell lines, and the relatively expression of BRAF gene mRNA in normal peripheral blood lymphocytes, YTS, Hut-78 and Jurkat cells were 1.000, 5.207±0.013, 8.412±0.615 and 36.720±1.797, respectively, and protein expressions were 0.051±0.003, 0.102±0.013, 0.113±0.017 and 0.304±0.010, respectively, and the expression of BRAF in peripheral T cell lymphoma Jurkat cells was significantly higher than that of Hut-78, YTS cells and normal lymphocytes (P<0.05). Only 6 of 58 peripheral T cell lymphomas (10.3%) had positive BRAF expression, and were the subgroups of peripheral T cell lymphoma-unspecified type. The statistical data did not show any correlation between positive expression of BRAF and gender, age, clinical stage, location, lactate dehydrogenase in the 21 cases of peripheral T cell lymphoma-unspecified type (P<0.05), but the positive rate of BRAF in the effective treatment group (8.3%) was significantly lower than that of the invalid group (55.6%, P<0.05). The expression of BRAF gene may become a marker of malignant biological characteristics and clinical therapeutic target of peripheral T cell lymphoma.