Human Lymphoblastic Cell Line Research Articles

Identification of Glycan Structure Alterations on Cell Membrane Proteins in Desoxyepothilone B Resistant Leukemia Cells

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

Mitochondrial permeability and toxicity of diethylhexyl and monoethylhexyl phthalates on TK6 human lymphoblasts cells

Phthalates are ubiquitous compounds used in the manufacturing industry. Some are known endocrine disruptors, acting as xenoestrogens, others induce reproductive toxicity and damage to DNA among other effects. Studies on apoptosis induction and mitochondrial damage capacity of phthalates on the immune system are limited. This study aims to determine cell viability inhibition and apoptosis induction of diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) on the human TK6 lymphoblast cell line at concentrations found in the environment. Key hallmark events, such as mitochondrial membrane permeability, generation of reactive oxygen species (ROS) and activation of caspase 3 and 7 were measured. Concentrations that inhibit viability of 50% (IC50) of the cells were determined at 24, 48 and 72h with doses ranging from 10 to 500μM. Changes in mitochondrial membrane permeability, ROS generation and activation of caspases 3 and 7, were measured as part of the cell death mechanism. The IC50 at 24h was approximately 250μM for both phthalates; at 48h were 234 and 196μM for DEHP and MEHP, respectively and at 72h IC50s were 100 and 80μM for DEHP and MEHP, respectively. Overall the longer the time of exposure the lower the IC50’s for both compounds. Both compounds affected mitochondrial membrane potential, promoted ROS generation and activated caspases 3 and 7. MEHP is more toxic, promotes higher level of ROS production and caspases activation. Our findings suggest that DEHP and MEHP have the capacity to induce apoptosis in cells of the immune system at concentrations found in the environment.

Expression level of miR‐34a rather than P53 gene status correlates with mutability in related human lymphoblast cell lines

The P53 gene is a tumor suppressor gene and can prevent mutation and tumor induction though apoptosis and DNA repair when it is activated by genotoxic stress. miR-34a expression is regulated by the P53 gene and might be required for cell response to DNA damage. TK6 cells are human lymphoblast cells with normal P53 function while WTK1 and NH32 cells derived from the same progenitor as TK6 cells are P53-deficient. Previous mutation research showed an unexpected result that NH32 cells were much less mutable than WTK1 cells, although the P53 gene in both the cell lines is not functional. To explore the possible mechanisms involved in the different mutability of the cell lines and relationship between P53 and miR-34a, we investigated the expression levels of miR-34a in the cells. The basal and X-ray-induced expression levels of miR-34a in TK6 and NH32 cells were much higher than those in WTK1 cells. The miR-34a was also able to be up-regulated to respond to X-ray exposure without a functional P53 gene in both of the NH32 and WTK1 cells. In addition, the expression levels of miR-34a in these three cell lines are inversely correlated well with their mutability: higher levels of miR-34a correspond with less mutable cells. These results suggest that alteration of miR-34a expression is at least partially independent of P53 regulation and its expression levels are closely related to cells' mutability regardless of P53 status.

Phosphatidylserine Metabolism in Human Lymphoblastic Cells Exposed to Chromium (VI)

Hexavalent chromium (Cr(VI)) compounds are widely found in different working environments. These compounds can cause apoptosis in human cells, but the mechanisms underlying chromium-induced apoptosis are not clear. A marker of apoptosis is the exposure of phosphatidylserine on cell membrane and the modification of phosphatidylserine metabolism. The aim of this study was to verify whether chromium could cause phosphatidylserine exposure and modification of its metabolism in human lymphoblastic leukemia cell line (MOLT-4). Phosphatidylserine exposure was evaluated by annexin V binding whereas phosphatidylserine metabolism was studied measuring the incorporation of [³H]serine. Cell treatment with Cr(VI) increases phosphatidylserine exposure and cell apoptosis, but decreases the incorporation of [³H]serine into phosphatidylserine in a dose- and time-dependent manner. The Cr(VI)-induced apoptosis also through modification of phosphatidylserine exposure and metabolism.

Abstract 1342: Effects of the plasticizer metabolite 2 ethyl-1-hexanol on human lymphoblast cells

Abstract The study main objective was to determine the toxicity of 2-ethyl-1-hexanol (EH) and to compare them to its precursor di-ethylhexyl phthalate (DEHP) on the TK6 human lymphoblast cell line. EH is an organic alcohol produced in cells as result of the biotransformation of DEHP. Release of EH from lung cancer cells was also reported recently. Environmental human exposure to the parent DEHP can result through interaction with personal care products, paint lacquer, inks, rubber, dry cleaning and plasticizers such as PVC. Analyzed effects in this study included doses that inhibit cell growth (IC50), damage to the mitochondrial membrane, generation of Reactive Oxygen Species (ROS), and activation of caspase 3 and 7. TK6 cells were cultured on 12.5cm2 flasks with modified RPMI 1640 culture media with 10% FBS, and incubated at 37°C with 5% CO2. IC50 was determined by trypan blue exclusion and mitochondrial membrane permeability applying the Mito PT JC-1 assay from Immunochemistry. For ROS determination the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA) was applied. For caspases 3 and 7 analysis the Magic RedTM assay from Immunochemistry was applied. Results indicated that IC50s of DEHP and EH on TK6 cells were 234 μM and 75nM respectively. Cells where then exposed to the identified IC50 for 48 hours to measure effects on mitochondria and ROS generation. ROS results indicated that EH generated an average of 96.31 Fluorescence Standard Units (FSU), the negative control Dimethyl Sulphoxide (DMSO) generated 58.22 FSU, the compound used as positive control cis-Di ammine platinum (II) dichloride (Cisplatin) generated 1309.67 FSU and for DEHP the ROS analysis resulted on 793.27 FSU. The level of ROS generated between the parent and this metabolite are not consistent with the degree of toxicity observed. Determination and comparison of the mitochondrial membrane permeabilization as well as Caspases 3 and 7-activation assays of EH are still developing. The preliminary results, however indicates a wide difference in citotoxicity between the parent compound and its metabolite clearly supporting the need for prevention strategies reducing exposure to these controversial plasticizer and therefore potential health effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2011-1342

VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines

Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC(50) values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods.

Inhibition of BCL11B Expression Leads to Apoptosis of Malignant T Cells but Not CD34+ Cells.

AbstractAbstract 3755Altered expression, mutation, disruption, or rearrangement of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) has been associated with T-cell malignancies. BCL11B overexpression occurs primarily in T-cell malignancies, suggesting that it may be a target gene for cancer therapy. Our previous study showed that the inhibition of BCL11B expression by siRNA led to apoptosis in human T-cell acute lymphoblastic leukemia cell lines (Jurkat, huT78, and Molt-4), but not in normal, mature T cells. To confirm the potential of BCL11B siRNA as a therapeutic agent and to determine its safety, the present study further analyzed the effects of BCL11B suppression on hemopoietic stem/progenitor cells, CD34+ cells were collected from three samples of umbilical cord blood using magnetic-activated cell sorting. BCL11B expressions in mRNA level of umbilical cord blood CD34+ cells were analyzed by the real-time quantitative PCR with TaqMan technique. For nucleofection of CD34+ cells, BCL11B-siRNA935 and the U-001 program were used of a Nucleofection Device II. Mock-transfected cells nucleofected without siRNA were used as a negative control. To evaluate the role of differentiation and proliferation in CD34+ cells after BCL11B-siRNA935 transfection, erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) from BCL11B siRNA935-, mock-CD34+ cells were performed using methylcellulose assays. Colonies were assessed 14 days after plating. The BCL11B mRNA expression level in the CD34+ cells was significantly lower than that in Molt-4 cells and in peripheral blood mononuclear cells from eight healthy individuals. There was no significant difference between BCL11B siRNA935-transfected and mock-transfected CD34+ cells with respect to the formation of BFU-E, CFU-GM, or CFU-Meg (P > 0.05). We speculate that the BCL11B gene, because of its very low expression level, may not play a major role in proliferation and differentiation of hematopoietic stem/progenitor cells, but we cannot rule out another anti-apoptotic mechanism. Nevertheless, BCL11B gene silencing alone does not affect the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. In conclusion, our findings provide evidence for an anti-apoptotic function of BCL11B in T-cell malignancies, but not in hemopoietic stem/progenitor cells, suggesting that BCL11B siRNA is safe and may be considered a new targeted therapeutic strategy for T-cell malignancies.* National Natural Science Foundation(30771980)& Guangdong Science and Technology Project (2007B030703008) Grants Disclosures:Li:National Natural Science Foundation (30771980) and Guangdong Science and Technology Project (2007B030703008, 2009B050700029) Grants: Research Funding.

Single Agent Activity of the Cyclin-Dependent Kinase (CDK) Inhibitor Dinaciclib (SCH 727965) In Acute Myeloid and Lymphoid Leukemia Cells

AbstractAbstract 3981Dinaciclib (SCH 727965) is a selective and potent inhibitor of CDK 1, 2, 5 and 9 (IC50 < 5 nM) that has demonstrated in vitro and in vivo anti-tumor activity against a variety of tumor cell lines and human tumor xenograft models. The concentration of dinaciclib required to achieve these effects (< 100 nM) is achieved in clinical trials, and dinaciclib was found to have a more favorable therapeutic index, in preclinical murine models, than other CDK inhibitors. We have studied the effect of dinaciclib on human acute myelogenous (AML) and acute lymphoblastic (ALL) leukemia cell lines, including HL-60, K562 and Molt-4, and primary leukemia cells.Dose response curves (0.0004-10 μM) were generated for different exposure times (2, 24 and 72 h), and data from cell proliferation assay (WST-1) were used to calculate the IC50 values. Short 2 h exposure to dinaciclib followed by 24 h culture without drug resulted in different responses between the cell lines (IC50 values of 0.13 μM, 2.17 μM and ND; and viability at 10 μM 62%, 76% and 95%, for HL-60, Molt-4, and K562, respectively). With longer exposure times (24 and 72 h), the IC50 was similar between the cell lines (IC50 24 h values of 0.017, 0.015, and 0.019 μM for HL-60, Molt-4, and K562, respectively). However, even in the presence of the highest drug concentration tested (10 μM), approximately 5–25% of cells remained metabolically active after 24 h culture, and in a colony forming assay were able to proliferate and form colonies after removal of the drug. Longer 72 h exposure to dinaciclib (0.2-10 μM) completely inhibited cell proliferation in all cell lines and prevented colony formation.Next, we examined the effect of dinaciclib (2-200 nM) on cell cycle in HL-60 and K562 cells (2, 6, 9, 24 h). While lower drug concentrations and shorter exposures resulted in a minor increase in the proportion of cells in the G2 phase, a considerable increase of cells in the sub-G1 phase was observed with prolonged exposures and higher drug concentrations, most prominently in HL-60 cells (4h 200 nM 38%; 6h 20 nM 53% or 200 nM 71%, and 24 h 20 nM 84%), which is consistent with cell viability assay data. These findings were also confirmed by Annexin V/PI staining. To characterize the molecular mechanisms behind the induction of cell cycle arrest and apoptosis by dinaciclib, we measured the changes in protein expression of Mcl-1, phosphorylation of retinoblastoma (Rb) protein, and cleavage of PARP by Western blotting. Dinaciclib treatment in a dose- and time-dependent manner (6 and 24 h; 10–500 nM) significantly decreased the expression of anti-apoptotic protein Mcl-1, Rb phosphorylation at Ser 811/817, and induced cleavage of the PARP protein in the three cell lines tested. For HL-60 cells, even 2 h exposure to dinaciclib was able to induce these effects when cells were examined 4 h after treatment; however, both Mcl-1 and p-Rb returned to baseline 24 h later, suggesting that the cells were able to recover. Using HL-60 cells, we were also able to demonstrate that a decrease in Mcl-1 correlates with the decrease in phosphorylation of the carboxy-terminal domain of RNA polymerase II, suggesting that dinaciclib successfully inhibits CDK9 which may lead to transcriptional down-regulation of Mcl-1. Dinaciclib treatment also down-regulated the expression of XIAP, Bcl-xl, and phosphorylation of Bad at Ser 112 (the pro-survival form of Bad), while Bak and Bax levels remained unaffected. The cleavage of PARP correlated with the activation of the caspase-3 and -9, suggesting the involvement of the intrinsic pathway of apoptosis.We confirmed our findings in primary leukemia cells. Dinaciclib was able to induce growth inhibition in all 7 primary AML samples (IC50 for 24 h exposure ranging from 0.008 to 0.017 μM) and apoptosis (Annexin V/PI staining). Treatment with dinaciclib also resulted in down-regulation of Mcl-1, cleavage of PARP, and dephosphorylation of Rb in all primary leukemia cells examined. In summary, dinaciclib potently inhibits the growth and induces apoptosis of human leukemia cells in vitro. Prolonged exposure times may be required for its maximum efficacy, and given its short half-life in humans (1.5 to 3.3 hours), this should be considered when designing the clinical studies for patients with acute leukemias. Disclosures:Sadowska:Merck & Co: Research Funding. Muvarak:Merck & Co: Research Funding. Lapidus:Merck & Co: Equity Ownership, Research Funding. Bannerji:Merck & Co: Employment, Equity Ownership. Gojo:Merck & Co.: Research Funding.

In vitro antitumor activity of Gracilaria corticata (a red alga) against Jurkat and molt-4 human cancer cell lines

Gracilaria corticata is a red alga which can be collected from many sea coasts around the world such as China, India, Persian Gulf, etc. The Persian Gulf is a unique marine habitat infested with diverse seaweeds. The aim of the present study is to explore anticancer potential of the crude extracts from G. corticata which was collected from the Bushehr coast (South west of Iran). Here, different concentration of the aqueous extract from G. corticata was tested for probable antitumoral activity on Jurkat and molt4 human lymphoblastic leukemic cell lines. The cells were treated by different concentration of algal extract and the number of viable cells was determined by trypan blue. Also, cytotoxicity of the extract was evaluated by methyl thiazolyl tetrazolium (MTT) assay. The results showed that 9.336 and 9.726 µg/µl of algal extract were the most effective concentrations against Jurkat and molt-4 cells, respectively. The water crude extract of red alga G. corticata had significant anticancer activity and it might be a good candidate for further investigations in order to develop a natural compound as an anticancer agent which can be used for the production of potential anticancer drug and novel pharmaceutical leads.

Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes

Bisphenol A-glycidyl methacrylate (BisGMA) is monomer of dental filling composites, which can be released from these materials and cause adverse biologic effects in human cells. In the present work, we investigated genotoxic effect of BisGMA on human lymphocytes and human acute lymphoblastic leukemia cell line (CCRF-CEM) cells. Our results indicate that BisGMA is genotoxic for human lymphocytes. The compound induced DNA damage evaluated by the alkaline, neutral, and pH 12.1 version of the comet assay. This damage included oxidative modifications of the DNA bases, as checked by DNA repair enzymes EndoIII and Fpg, alkali-labile sites and DNA double-strand breaks. BisGMA induced DNA-strand breaks in the isolated plasmid. Lymphocytes incubated with BisGMA at 1 mM were able to remove about 50% of DNA damage during 120-min repair incubation. The monomer at 1 mM evoked a delay of the cell cycle in the S phase in CCRF-CEM cells. The experiment with spin trap—DMPO demonstrated that BisGMA induced reactive oxygen species, which were able to damage DNA. BisGMA is able to induce a broad spectrum of DNA damage including severe DNA double-strand breaks, which can be responsible for a delay of the cell cycle in the S phase.

ChemInform Abstract: Inhibition of Human Dihydrofolate Reductase by 2,4-Diaminoquinazolines Bearing Simple Substituents on the Aromatic Ring.

A series of thirty eight 2,4-diaminoquinazolines having diverse substitution patterns on the aromatic ring was evaluated for inhibitory activity against dihydrofolate reductase (DHFR) obtained from a human lymphoblast cell line. Many of these compounds were also evaluated as inhibitors of rat liver DHFR under the same experimental conditions. In most instances the results obtained with each enzyme were comparable indicating that the rodent enzyme is a suitable model for the human DHFR as far as the determination of I50 values is concerned. The results demonstrate that relatively simple 5-substituted- or 5,6-disubstituted-2,4-diaminoquinazolines can be potent DHFR inhibitors. The presence of a nonpolar substituent at position 7 or 8 was highly detrimental to inhibitory potency.

Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in Golabi-Ito-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

Severe hypocholesterolaemia is often neglected in haematological malignancies

Aim of the study It is generally believed that high levels of cholesterol (hypercholesterolaemia) are life-threatening, while low levels seem to be positive. Unfortunately this assumption is far from true, and can be indicative of an underlying serious medical condition in most of the cases (i.e. cancer). However, the biological role of severe hypocholesterolaemia is poorly understood. Here, the possible biological process is being investigated. Cholesterol plays a key role in cell proliferation, hence it has been suggested that low cholesterol levels are probably linked to the high cellular cholesterol demands from neoplastic cells. Summary of the methods We used serum and isolated T-lymphocytes from patients with acute lymphoblast leukaemia and human lymphoblast cell line to test this hypothesis. Results We found that patients with low serum cholesterol levels have instead high levels of cholesterol in lymphocytes. These data were supported with in vitro studies. In fact we have demonstrated that low cholesterol level in the culture medium was related to the neoplastic cellular growth, suggesting a greater use by lymphoma cells for their proliferation. Therefore by inhibiting cholesterol synthesis by mevastatin, in vitro, we showed that cholesterol levels did not change significantly in culture medium and the cellular growth was inhibited. Concluding statement Following these preliminary results, blood cholesterol levels could be potentially considered a good biological marker to follow up the neoplastic process.

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.

Distinct Signaling Pathways After Higher or Lower Doses of Radiation in Three Closely Related Human Lymphoblast Cell Lines

The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation. The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles. Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation. After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of gamma-irradiation.

BCR/ABL Requires Fanconi Anemia D2 (FANCD2) Protein to Transform Hematopoetic Stem Cells.

Abstract 3249Poster Board III-1Fanconi D2 protein (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) and phosphorylated on S222 (FANCD2-phosphoS222) in response to DNA double-strand breaks (DSBs). FANCD2-Ub interacts with RAD51 to facilitate homologous recombination repair (HRR), and FANCD2-phosphoS222 activates the S phase checkpoint. We detected an increased amount of FANCD2-Ub in CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) patients and in BCR/ABL-positive cell lines in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of reactive oxygen species (ROS) with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). In addition mitomycin C routinely used for diagnostic testing in Fanconi anemia, strongly elevated FANCD2-Ub in CD34+ CML cells. Therefore we postulate that FANCD2-Ub may play a role in BCR/ABL transformation. In support for this hypothesis, we observed that clonogenic potential of BCR/ABL-positive murine leukemia stem cells (LSCs)-enriched FANCD2-/- Sca1+Kit+lin- bone marrow cells was reduced by approximately 10-fold in comparison to BCR/ABL-positive FANCD2+/+ counterparts; non-transformed -/- and +/+ cells displayed similar clonogenic potential stimulated by SCF and GM-CSF. Restoration of FANCD2 expression “rescued” the impaired clonogenic activity of BCR/ABL-positive FANCD2-/- cells. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Negative effect of BCR/ABL kinase on FANCD2-/- cell growth was reversed by expression of exogenous FANCD2. The in vitro growth defect of BCR/ABL-positive FANCD2-/- cells was accompanied by delayed leukemogenesis in SCID mice. These results suggest that FANCD2, a key regulator of DNA damage response, may play an important role in the initiation and/or maintenance of BCR/ABL-positive leukemias. We showed before that CD34+ CML-CP and CML-BC cells contain higher number of ROS-induced DSBs in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that LSC-enriched (CD34+CD38-) CML-CP and CML-BC cells display more DSBs than normal counterparts. Thus, BCR/ABL-mediated leukemogenesis is associated with accumulation of an excess of ROS-induced DSBs, which if not repaired, may induce apoptosis. We hypothesize that FANCD2 is necessary to “protect” leukemia cells from potentially lethal effect of BCR/ABL-induced oxidative DNA damage (including DSBs) at early stages of transformation and possibly also during the progression to CML-BC. This suggestion is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DSBs in comparison to +/+ counterparts. This effect did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect of BCR/ABL-positive FANCD2-/- cells. Accumulation of excessive DNA damage (DSBs) and impairment of growth potential of BCR/ABL-positive FANCD2-/- cells could be prevented by expression of FANCD2 wild-type (proficient in DNA repair and S phase checkpoint) and S222A phosphorylation-deficient mutant (proficient in DNA repair, but deficient in S phase checkpoint), but not the K561R monoubiquitination-deficient mutant (deficient in DNA damage, but proficient in S phase checkpoint). Since FANCD2-Ub interacts with RAD51 to promote HRR and BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, it is plausible that elevated expression of FANCD2-Ub may facilitate DSB repair to protect leukemia cells from lethal effects of DSBs. In concordance, co-localization of FANCD2-Ub and RAD51 was readily detected in the nuclei of BCR/ABL-positive leukemia cells. In conclusion our results indicate that FANCD2 plays an important role during the induction and perhaps also progression of Philadelphia-chromosome positive leukemias due to its ability to facilitate the repair of numerous, potentially lethal DSBs. Disclosures:No relevant conflicts of interest to declare.

The apoptotic and anti-proliferative activity of Origanum majorana extracts on human leukemic cell line

Scientists are constantly searching for phytochemicals and compounds with anti-cancer and antioxidant activity. In this study, the anti-proliferative activity of plant extracts from Origanum majorana (marjoram) was tested on human lymphoblastic leukemia cell line Jurkat. Cytotoxicity was examined using non-radioactive cytotoxicity assay and the IC 50 was calculated. At non-cytotoxic concentrations, the viability of cells decreased with increase of concentration of plant extract. The anti-proliferative effect was also found to be dose-dependent. Analysis via flow cytometry shows that marjoram extracts stimulated apoptosis. Induction of apoptosis was caused by an up-regulation of p53 protein levels and down-regulation of Bcl-2α. Marjoram exhibited a strong scavenging activity (SC 50 = 0.03 mg dry weight). The conclusions from this study suggest that marjoram extracts exhibit anti-proliferative effect and high antioxidant activity. For that it merits further investigation as a potential therapeutic agent.

Erratum: Increased OPRM1 DNA Methylation in Lymphocytes of Methadone

Correction to: Neuropsychopharmacology advance online publication, 23 July 2008; doi:10.1038/npp.2008.108 In this article, on page 1, right column, second paragraph, line 11, an error occurred in the sentence beginning with ‘In human lymphoblast cell lines, average overall DNA….’ The sentence shouldread as follows: ‘In human lymphoblast cell lines, average overall DNA methylation at the monoamine oxidase A gene promoter was found….

Monoubiquitination of the Fanconi Anemia D2 (FANCD2) Protein Regulates the Transforming Potential of BCR/ABL

We showed before that BCR/ABL-mediated leukemogenesis is associated with the elevation of reactive oxygen species (ROS), which in addition to enhancing signaling pathways may increase the number of oxidative DNA lesions including DNA double-strand breaks (DSBs) [Blood, 2005, 2006]. CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain higher number of DSBs induced by ROS in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cell-enriched populations display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Thus, BCR/ABL-positive leukemia cells appear to accumulate an excess of ROS-induced DSBs, which may cause apoptosis if not repaired. We reported before that homologous recombination repair (HRR), involving RAD51 protein, plays a pivotal role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS [Blood, 2005]. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 by FANCL ubiquitinase and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 (FANCD2-Ub) interacts with RAD51 to facilitate HRR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint. We detected an increased amount of FANCD2-Ub in BCR/ABL-positive leukemia cell line and CD34+ CML-CP and CML-BC cells in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of ROS with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). Therefore we postulate that BCR/ABL kinase-dependent ROS-induced FANCD2- Ub may play a role in leukemic transformation. This hypothesis is supported by impaired transformation potential of BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. Restoration of the expression of FANCD2 protein in FANCD2−/− cells rescued the transforming potential of BCR/ABL kinase. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. Again BCR/ABL kinase did not exert a negative effect on the proliferation of FANCD2-reconstituted cells. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied by delayed leukemogenesis in SCID mice. Growth potential of BCR/ABL-positive FANCD2−/− could be rescued by co-expression of FANCD2 wild-type and S222A mutant, but not the K561R mutant. This observation supports our hypothesis that K561 monoubiquitination, but not S222 phosphorylation of FANCD2 might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, elevated expression of FANCD2-Ub may facilitate this process. This speculation is supported by the observation that although BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, the former cells accumulate more DSBs evaluated by neutral comet assay and immunostained gamma-H2AX nuclear foci. This effect could be reversed by the expression of FANCD2 S222A mutant, but not K561R mutant, again implicating FANCD2-Ub in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that FANCD2-Ub, but not FANCD2-phosphoS222 may play an important role in BCR/ABL-dependent leukemogenesis probably due to its ability to interact with RAD51 and facilitate HRR of the numerous ROS-induced DSBs.