Human T-lymphoblastic Leukemia Cell Research Articles

Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

C9Mab-1: An Anti-Mouse CCR9 Monoclonal Antibody for Immunocytochemistry.

C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.

Changes in intracellular activation-related gene expression and induction of Akt contribute to acquired resistance toward nelarabine in CCRF-CEM cell line

Drug resistance is a major problem in treatment with nelarabine, and its resolution requires elucidation of the underlying mechanisms. We established two nelarabine-resistant subclones of the human T-cell lymphoblastic leukemia cell line CCRF-CEM. The resistant subclones showed changes in the expression of several genes related to nelarabine intracellular activation and inhibition of apoptosis. Activation of the Akt protein upon nelarabine treatment was observed in both subclones. The combination treatment with nelarabine and PI3K/Akt inhibitors was shown to inhibit cell growth. Cross-resistance was observed with ara-C and not with vincristine, daunorubicin, or etoposide treatment. Thus, changes in the expression of cellular activation-related genes, inhibition of apoptosis, and induction of Akt may be involved in the development of nelarabine resistance in the CCRF-CEM cell model. The use of different classes of chemotherapeutic agents and combination therapy with PI3K/Akt pathway inhibitors may be used to overcome resistance to nelarabine.

ANTIOXIDANT AND CYTOTOXIC ACTIVITY OF A SERIES OF O- AND S-CONTAINING MACROCYCLES

The significant interest in the search and study of antioxidants, both natural and synthetic, is explained by the possibility of preventing these harmful effects of free radicals in the human body. The similarity of macroheterocyclic derivatives to endogenous biomolecules makes them promising for research as antioxidant agents. By the interaction of 1,3-oxoheterocycloalkanes with ethyl 2-diazo-3-oxobutanoate, previously unexplored polyfunctional oxygen and sulfur-containing macroheterocycles are obtained. The relative antioxidant activity of new macroheterocycles in two model systems: generating active forms of oxygen and modulating lipid peroxidation reaction, was revealed active forms of oxygen and described by the method of recording luminol-dependent chemiluminescence lipid peroxidation reaction. The in vitro cytotoxic activity of the studied compounds was studied on Jurkat (human T-lymphoblastic leukemia cells), HepG2 (human liver carcinoma cells), HEK293 (human embryonic kidney cells) cell lines. The data obtained indicate practical significance. So, reagents 1-7 in the active forms of oxygen generating system have anti- and prooxidative properties, and in the system modeling lipid peroxidation reaction, they show only prooxidant activity. The influence of heterocycles 1–7 on the processes of free radical oxidation was determined by the intensity of the maximum flash (Imax, у.е.) and the luminosity of light (S, у.е.). Compounds with maximum anti- (4) and prooxidative (6) properties in the active forms of oxygen generating system and reagent 3 with the highest prooxidant activity in the lipid peroxidation reaction modeling system do not possess cytotoxic activity in vitro on cell lines HEK293 and HepG2 cultured in DMEM medium (Biolot, Russia), Jurkat in RPMI medium (Biolot, Russia) in the presence of 10% fetal calf serum (Invitrogen, USA), 2 mM L-glutamine and 50 μg / ml gentamicin sulfate.

Synthesis of C60 Fullerene-Quadricyclane Hybrid Compound and Its Preliminary In Vitro Antitumor Activity in Combination with Cisplatin.

This paper describes the authors’ preliminary studies directed toward the possibility of the practical implementation of the idea to design efficient antitumor drugs based on hybrid molecules composed of fullerene C60 and quadricyclanes. The essence of the proposed idea is that these hybrid molecules are able to cleave DNA owing to the fullerene moiety they contain and to simultaneously thermally affect tumor cells via cleavage of the carbon–carbon bond in quadricyclanes under the action of Pd and Pt ions. As a result, testing of the cytotoxic activity in vitro for a number of fullerene C60 hybrids with the norbornadiene or quadricyclane moieties against the human T-lymphoblastic leukemia cells (Jurkat cells) in combination of the known cisplatin drug, which was taken as the source of Pt ions, showed a statistically reliable dose-dependent increase in the number of dead cells in each group, which were formed according to the amount of cisplatin added, in comparison with the control, that is, cells treated with cisplatin or quadricyclane fullerene derivatives alone. Indeed, the difference between the percentages of viable cells after treatment with either cisplatin alone or cisplatin in combination with methanofullerene 5 ranged from ∼10% (for Pt (0.015 mkM), 5 (0.015 mkM)) to ∼55% (for Pt (0.03 mkM), 5 (0.045 mkM)).

Modulation of multidrug resistant in cancer cells by EGCG, tannic acid and curcumin

Background: Cancer is one of the most common life-threatening diseases worldwide; many patients develop multidrug resistance after treatment with anticancer drugs. The main mechanism leading to multidrug resistance is the overexpression of ABC transporters in cancer cells. Chemosensitizers are needed to inhibit the activity of ABC transporters, resulting in higer intracellular concentration of anticancer drugs. Some secondary metabolites have been reported to be chemosensitizers by inhibiting ABC transporters. Epigallocatechin gallate (EGCG), tannic acid, and curcumin were employed in this study. Different assays were used to detect whether they have the ability to inhibit P-gp activity and overcome multidrug resistance in cancer cells overexpressing P-gp. Hypothesis/Purpose: CEM/ADR 5000 and Caco-2 cell lines, which overexpress P-gp, are multidrug resistant cell lines. We first detected whether the combination of polyphenols (EGCG, tannic acid, curcumin) and doxorubicin, an anticancer drug, is synergistic or not. To further understand the potential mechanism, EGCG, tannic acid, and curcumin were tested to check whether they have the ability to inhibit P-gp activity. When P-gp activity is inhibited, the intracellular concentration of doxorubicin is higher, resulting in enhanced cytotoxicity of doxorubicin. Study design: The P-gp overexpressing human colon cancer cell line Caco-2 and human T-lymphoblastic leukemia cell line CEM/ADR 5000 were used in this study. Two-drug combinations (doxorubicin + polyphenol) and three-drug combinations (doxorubicin + polyphenol + digitonin) were tested to examine potential synergism. The potential mechanism leading to synergism would be the inhibition of P-gp activity. A Rhodamine 123 assay and Calcein-AM assay in Caco-2 and CEM/ADR 5000, respectively, were used to detect P-gp inhibition by EGCG, curcumin, and tannic acid. Methods: MTT assay was used to determine the cytotoxicity of doxorubicin, polyphenols and digitonin alone, and then their combinations. Furthermore, Rhodamine 123 and Calcein-AM were used to detect the effects of polyphenols on the activity of P-gp. Results: The results demonstrated that a combination of non-toxic concentrations of each polyphenol with doxorubicin synergistically sensitized Caco-2 and CEM/ADR 5000 cells. Furthermore, three-drug combinations (doxorubicin + polyphenol + digitonin) were much more effective. In addition, the activity of P-gp in Caco-2 and CEM/ADR 5000 cells was measured. Consistent with the combination results, tannic acid and curcumin decreased the activity of P-gp both in Caco-2 and CEM/ADR 5000. EGCG, which weakly affected the activity of P-gp in CEM/ADR 5000, only had an effect on P-gp under higher concentration in Caco-2 cells. Conclusion: Our results show that EGCG, curcumin, and tannic acid, when combined with doxorubicin, can exert synergism, mediated by a reduced activity of P-gp. This study suggests that polyphenols, by modulating the activity of P-gp, may be used as chemosensitisers.

Synthesis, screening and pro-apoptotic activity of novel acyl spermidine derivatives on human cancer cell lines

The polyamines putrescine, spermidine, and spermine are polycationic, alkyl polyamines which play a significant role in eukaryotic cell proliferation. The polyamine metabolism and function are dysregulated in tumor cells making them an attractive therapeutic target by employing polyamine analogs. These analogs have a high degree of similarity with the structure of polyamines but not with their function. Multidrug resistance is a major factor in the failure of many chemotherapeutic drugs which necessitates further research and exploration of better novel alternatives. In the present study, Twenty-six novel acylspermidine derivatives were synthesized and evaluated for their anti-proliferative and pro-apoptotic activities on human breast cancer cells and T-lymphoblastic leukemia cells. The cell proliferation and apoptosis assays using WST-1 and annexin-V/7AAD staining respectively suggest that Compound 1 (C19H41N3O2), Compound 7(C25H51N3O2) and Compound 8 (C29H59N3O) significantly reduced cancer cell viability in a dose- and time-dependent manner. Interestingly, compounds 7, 8 and 9 had slight or no effect on cell proliferation of non-cancerous cells. These studies speculate that these novel acylspermidine derivatives could be promising candidates in designing an anti-proliferative drug, targeting both solid and blood cancer cells.

Repairing effects of interleukin 11 (IL-11) towards high dose methotrexate-induced rat small intestinal mucositis and its impacts on T-lymphoblastic leukemia cell line.

To investigate the efficacy of interleukin 11 (IL-11) towards the high dose methotrexate (HDMTX)-concurrent rat small intestinal mucositis and its impacts on the proliferation of the human T-lymphoblastic leukemia (CEM) cell line. 95 Wistar rats were randomly divided into five groups, the normal control group (A), the methotrexate (MTX) control group (B), the IL-11-pre-treated high-dose group (C), the post-IL-11-treatment high-dose group (D) and the post-IL-11-treatment low-dose group (E). After the intraperitoneal injection of MTX in the groups B-E, the rats were sacrificed at 1, 3, 5 and 7 days. The mortality, morphological and ultrastructural changes of small intestine of each group were observed. The cells were then cultured in vitro, and the MTT method was used to investigate the effects of different concentration of IL-11 on CEM proliferation and also on HDMTX-induced mucositis. IL-11 could reduce the intestinal histopathological score, increase the height of small intestinal villi, promote the proliferation of intestinal lacunar cells and reduce the mortality rate of rats. The IL-11 pre-treatment group exhibited the best efficacies, demonstrating significant difference with the control group (P<0.01). In addition, the proliferation of CEM was not promoted, indicating that IL-11 could not inhibit HDMTX. IL-11 could reduce the severity of HDMTX-induced intestinal mucositis, and improve the survival rate of experimental rats, and could be safely used as the adjuvant treatment of HDMTX in childhood leukemia.

Abstract 97: Establishment of a new in vivo model for human T-cell lymphoblastic leukemia (T-ALL) suitable for evaluation of the tumor stromal component

Abstract T-cell lymphoblastic leukemia (T-ALL) is a hematological hematopoietic T cell precursor disorder with a not well defined etiology, although it would be linked to NOTCH signaling defects. CUTLL1 (Columbia University T-cell Lymphoblastic Lymphoma 1) is a human T-ALL cell line displaying biological responses to NOTCH inhibition, thus mimicking the disease´s etiology. The absence of human T-ALL cell lines with verifiably clinical aspects turns the establishment of an in vivo model an asset for preclinical assays; further, it serves to study the interaction between disease-mimicking cells and their surrounding niche. We have established an in vivo model for T-ALL in immunosuppressed NOD.CB17-Prkdc scid/J mice using CUTLL1 cells. Tumors developed after subcutaneous inoculation of a minimum dose of 2x10E7 cells and became visible 2.5 weeks post-inoculation into the right flank (809.1±67.33 mmE3). Administration of larger cell doses (4x10E7) did not affect the lapse at which tumors appear, but originated larger tumors (3830±1571 mmE3, p&amp;lt;0.05). Tumors were histological characterized indicating high neoplastic proliferation infiltrating the tumor stroma with high content of fibroblast-like cells and microvessels. Tumor progression is modulated by microenvironmental factors, among them stromal cells regulating neoplastic cells. Since Mesenchymal stromal cells (MSCs) are incorporated into the tumor microenvironment, we evaluated the participation of MSCs as stromal component in this model. We administered MSCs into lymphoma-bearing mice and measured MSCs tumor stroma homing. MSCs tumor homing ability of pre-labeled DiR/CMDiI+ MSCs injected intravenously (1x10E6 cells) was measured by infrared detection and microscopic examination. MSCs levels at the tumor stroma reached highest values at smallest tumor volume (3,967x10E5 p/sec/cm2/sr /1004,372mm3) indicating a negative correlation between tumor size and MSCs homing ability and pointing at the high homing MSCs capacity into a niche of hematological neoplastic cells. Microenvironmental tumor factors may directly affect cell migration and promote tumor progression. We evaluated MSCs migratory in vitro capacity observing that MSCs are able to respond to chemotactic stimuli derived from CUTLL1 cells. Cell recruitment by tumor-derived factors may affect tumor progression and our data suggests that tumor recruited MSCs may be involved in tumor development in this model. This is the first report to our knowledge, of a xenomodel using the clinically representative cell line CUTLL1. The establishment of an in vivo model with T-ALL clinical aspects such as Notch signaling defects, serves as a relevant model and allows the study of stromal and neoplastic cell interaction within an in vivo tumor microenvironment. Citation Format: Mariana Amorós, M. Florencia Cayrol, M. Celeste Díaz Flaque, Luciana Gutiérrez, Osvaldo Podhjacer, Leandro Cerchietti, Graciela Cremaschi, Marcela F. Bolontrade. Establishment of a new in vivo model for human T-cell lymphoblastic leukemia (T-ALL) suitable for evaluation of the tumor stromal component. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 97. doi:10.1158/1538-7445.AM2014-97

Reduced drug incorporation into DNA and antiapoptosis as the crucial mechanisms of resistance in a novel nelarabine-resistant cell line.

BackgroundNine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis.MethodsIn the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting.ResultsCEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells.ConclusionsAn ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.

Molecular imaging of human tumor cells that naturally overexpress type 2 cannabinoid receptors using a quinolone-based near-infrared fluorescent probe.

Cannabinoid CB2 receptors (CB2R) hold promise as therapeutic targets for treating diverse diseases, such as cancers, neurodegenerative diseases, pain, inflammation, osteoporosis, psychiatric disorders, addiction, and immune disorders. However, the fundamental role of CB2R in the regulation of diseases remains unclear, largely due to a lack of reliable imaging tools for the receptors. The goal of this study was to develop a CB2R-targeted molecular imaging probe and evaluate the specificity of the probe using human tumor cells that naturally overexpress CB2R. To synthesize the CB2R-targeted probe (NIR760-Q), a conjugable CB2R ligand based on the quinolone structure was first prepared, followed by bioconjugation with a near-infrared (NIR) fluorescent dye, NIR760. In vitro fluorescence imaging and competitive binding studies showed higher uptake of NIR760-Q than free NIR760 dye in Jurkat human acute T-lymphoblastic leukemia cells. In addition, the high uptake of NIR760-Q was significantly inhibited by the blocking agent, 4-quinolone-3-carboxamide, indicating specific binding of NIR760-Q to the target receptors. These results indicate that the NIR760-Q has potential in diagnostic imaging of CB2R positive cancers and elucidating the role of CB2R in the regulation of disease progression.

Amides derived from heteroaromatic amines and selected steryl hemiesters

The current interest of the team has been focused on investigation of novel amides with potential cytotoxicity. The presented series of compounds was synthesized from selected steryl hemiesters and heteroaromatic amines. The synthetic protocol was designed in a simple and economic way, and divided into several general methodologies applicable to the compounds synthesized. The cytotoxicity was tested on cells derived from human T-lymphoblastic leukemia, breast adenocarcinoma and cervical cancer, and compared with tests on normal human fibroblasts. Most of the lanosterol-based compounds (3–5 and 7–10) showed medium to good cytotoxicity, while only two derivatives of cholesterol (18 and 19) showed medium cytotoxicity on human T-lymphoblastic leukemia cell line. The compounds 8 and 9 displayed the reasonable cytotoxicity among this series of amides, tested on the cell lines of T-lymphoblastic leukemia [14.5±0.4μM (8) and 18.5±3.9μM (9)], breast adenocarcinoma [19.5±2.1μM (8) and 23.1±4.0μM (9)] and cervical cancer [24.8±5.3μM (8) and 29.1±4.7μM (9)]. Only the compound 8 was adequately less active on normal human fibroblasts (40.4±11.1μM).

Development and validation of a general approach to predict and quantify the synergism of anti-cancer drugs using experimental design and artificial neural networks

The combination of two or more drugs using multidrug mixtures is a trend in the treatment of cancer. The goal is to search for a synergistic effect and thereby reduce the required dose and inhibit the development of resistance. An advanced model-free approach for data exploration and analysis, based on artificial neural networks (ANN) and experimental design is proposed to predict and quantify the synergism of drugs. The proposed method non-linearly correlates the concentrations of drugs with the cytotoxicity of the mixture, providing the possibility of choosing the optimal drug combination that gives the maximum synergism. The use of ANN allows for the prediction of the cytotoxicity of each combination of drugs in the chosen concentration interval. The method was validated by preparing and experimentally testing the combinations with the predicted highest synergistic effect. In all cases, the data predicted by the network were experimentally confirmed.The method was applied to several binary mixtures of cisplatin and [Cu(1,10-orthophenanthroline)2(H2O)](ClO4)2, Cu(1,10-orthophenanthroline)(H2O)2(ClO4)2 or [Cu(1,10-orthophenanthroline)2(imidazolidine-2-thione)](ClO4)2. The cytotoxicity of the two drugs, alone and in combination, was determined against human acute T-lymphoblastic leukemia cells (CCRF-CEM). For all systems, a synergistic effect was found for selected combinations.

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

BackgroundAlthough honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 – a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done.ResultsWe found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent.ConclusionsThese observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.

Noncytotoxic and Antitumour-Promoting Activities of Garcinia Acid Esters fromGarcinia atroviridisGriff. ex T. Anders (Guttiferae)

The in vitro antitumour-promoting, cytotoxic, and antioxidant activities of two ester derivatives of garcinia acid, that is, 2-(butoxycarbonylmethyl)-3-butoxycarbonyl-2-hydroxy-3-propanolide (1) and 1′,1′′-dibutyl methyl hydroxycitrate (2), that had been previously isolated from the fruits of Garcinia atroviridis Griff. ex T. Anders (Guttiferae), were examined. Based on the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation, compound 1 (IC50: 70 μM) showed much higher (8-fold) antitumour-promoting activity than compound 2 (IC50: 560 μM). In addition, both compounds were nontoxic towards CEM-SS (human T-lymphoblastic leukemia) cells (CD50: >100 μM), Raji (human B-lymphoblastoid) cells (CD50: >600 μM), and brine shrimp (LD50: >300 μM). Although the antitumour-promoting activity of compound 1 is moderate compared with the known antitumour promoter genistein, its non-toxicity suggests the potential of compound 1 and related structures as chemopreventive agents. The weak antioxidant activity displayed by both compounds also suggested that the primary antitumour-promoting mechanism of compound 1 did not involve oxidative-stress quenching.

Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells

Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, we find that in human and murine T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to ∼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, ∼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription.

Cytotoxic Effect of Paclitaxel Incorporated in Nanoparticles Based on Lactic and Glycolic Acid Copolymer

Paclitaxel dosage form on nanoparticles of 200-300 nm based on lactic and glycolic acid copolymer was obtained by the co-precipitation method. The possibility of controlled release of paclitaxel at pH 7.4 for 24 h was studied in vitro. Studies on Jurkat/WT human T-lymphoblastic leukemia cells showed that incorporation of paclitaxel in the nanoparticles led to a 4-fold increase of its cytotoxicity (6.8×10(-6) M) in comparison with paclitaxel solution. The efficiency of compositions containing polysorbate-80 was comparable to that of non-modified nanoparticles containing paclitaxel.

Abstract 4543: Targeting the NADPH binding site of dihydrofolate reductase for cancer therapy

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate into tetrahydrofolate using NADPH as the cofactor. Folate-dependent one carbon metabolism is essential for the de novo synthesis of purines, pyrimidines and certain amino acids. Thus, DHFR plays an essential role in production and maintaining reduced folates for cellular proliferation, and consequently it has been a critical target for chemotherapy. Methotrexate (MTX), an antifolate that targets DHFR, has been used for decades in the clinic. However, both intrinsic and acquired resistance occurs due to decreased uptake, gene amplification and protein mutation. We have initiated studies to find an inhibitor that targets the cofactor NADPH binding site that would bypass these resistance mechanisms to MTX in contrast to analogs that target the dihydrofolate binding site. Fourteen NAD/NADP analogues were screened by the MTT assay in Chinese hamster ovary (CHO) cells which lack endogenous DHFR expression but were stably transfected with a DHFR-EGFP fusion construct driven by a CMV promoter (DG44 DHFR-EGFP). Two compounds, thionicotinamide adenine dinucleotide (NAD-S) and thionicotinamide adenine dinucleotide phosphate (NADP-S), had IC50 values of 1 μM. NAD-S and NADP-S were also found to be potent inhibitors of two other cancer cell lines, C85 (metastatic human colon cancer cell line) and CCRF-CEM (human T-cell lymphoblastic leukemia cell line). Surprisingly, both NAD-S and NADP-S decreased the levels of DHFR in CEM-CCRF cells. Pulse-chase experiments showed that the half life of DHFR is significantly reduced upon treatment of cells with 10 μM NADP-S. Furthermore, this NADP analog caused an increase in cells at the G1 phase (11% at 10 μM and 44% at 40 μM NADP-S treatment) that was reversible by removing NADP-S from the cell culture. Because NADP-S decreased DHFR levels, the combination of NADP-S with MTX was tested and found to significantly reduce colony formation, and was synergetic in vitro (Chou-Talalay analysis). We conclude that NADPS has a novel mechanism of action, i.e., it causes degradation of DHFR, likely by competing with NADPH binding, known to stabilize DHFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4543.

Pre T-cell receptor alpha (pTalpha) expression patterns and functional analysis in human T-cell lymphoblastic leukemia.

Background: The pTα/preTCR regulates the β-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56Lck. Methods: To define the relevance of pTα in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTα and p56Lck mRNA and protein expression as also the tyrosine-phosphorylation. The p56Lck specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC50 value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines. Results: pTα expression patterns were markedly different in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G1/0 phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56Lck and showed a corresponding P-Tyr signal. Conclusions: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTα mediated proliferation in this leukemic sub-type. In addition, p56Lck represents also independently of pTα a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases.

Pre T-Cell Receptor Alpha (pTα) Expression Patterns and Functional Analysis in Human T-Cell Lymphoblastic Leukemia

Background: The pTα/preTCR regulates the β-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56Lck.Methods: To define the relevance of pTα in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTα and p56Lck mRNA and protein expression as also the tyrosine-phosphorylation. The p56Lck specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC50 value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines.Results: pTα expression patterns were markedly different in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G1/0 phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56Lck and showed a corresponding P-Tyr signal.Conclusions: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTα mediated proliferation in this leukemic sub-type. In addition, p56Lck represents also independently of pTα a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases.