The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Delivery of daunorubicin to cancer cells with decreased toxicity by association with a lipidic nanoemulsion that binds to LDL receptors

A lipidic nanoemulsion termed LDE concentrates in neoplastic cells after injection into the bloodstream and thus can be used as a drug carrier to tumour sites. The chemotherapeutic agent daunorubicin associates poorly with LDE; the aim of this study was to clarify whether the derivatization of daunorubicin by the attachment of an oleyl group increases the association with LDE, and to test the cytotoxicity and animal toxicity of the new preparation. The association of oleyl-daunorubicin (oDNR) to LDE showed high yield (93 +/- 2% and 84 +/- 4% at 1:10 and 1:5 drug:lipid mass, respectively) and was stable for at least 20 days. Association with oDNR increased the LDE particle diameter from 42 +/- 4 nm to 75 +/- 6 nm. Cytotoxicity of LDE-oDNR was reduced two-fold in HL-60 and K-562 cell lines, fourteen-fold in B16 cells and nine-fold in L1210 cells when compared with commercial daunorubicin. When tested in mice, LDE-oDNR showed remarkable reduced toxicity (maximum tolerated dose > 253 mu mol kg(-1), compared with <3 mu mol kg(-1) for commercial daunorubicin). At high doses, the cardiac tissue of LDE-oDNR-treated animals had much smaller structural lesions than with commercial daunorubicin. LDE-oDNR is therefore a promising new preparation that may offer superior tolerability compared with commercial daunorubicin.

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)S) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M-r similar to 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2S from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. (C) 2008 Elsevier Inc. All rights reserved.

The Ruthenium Complex cis-(Dichloro)tetraammineruthenium(III) Chloride Presents Selective Cytotoxicity Against Murine B Cell Lymphoma (A-20), Murine Ascitic Sarcoma 180 (S-180), Human Breast Adenocarcinoma (SK-BR-3), and Human T Cell Leukemia (Jurkat) Tumor Cell Lines

The aim of present study was to verify the in vitro antitumor activity of a ruthenium complex, cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) toward different tumor cell lines. The antitumor studies showed that ruthenium(III) complex presents a relevant cytotoxic activity against murine B cell lymphoma (A-20), murine ascitic sarcoma 180 (S-180), human breast adenocarcinoma (SK-BR-3), and human T cell leukemia (Jurkat) cell lines and a very low cytotoxicity toward human peripheral blood mononuclear cells. The ruthenium(III) complex decreased the fraction of tumor cells in G0/G1 and/or G2-M phases, indicating that this compound may act on resting/early entering G0/G1 cells and/or precycling G2-M cells. The cytotoxic activity of a high concentration (2 mg mL(-1)) of cis-[RuCl(2)(NH(3))(4)]Cl toward Jurkat cells correlated with an increased number of annexin V-positive cells and also the presence of DNA fragmentation, suggesting that this compound induces apoptosis in tumor cells. The development of new antineoplastic medications demands adequate knowledge in order to avoid inefficient or toxic treatments. Thus, a mechanistic understanding of how metal complexes achieve their activities is crucial to their clinical success and to the rational design of new compounds with improved potency.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 pM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 pM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 mu M, respectively. The critical micellar concentration (CMC) of ODPC was 200 mu M. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (Delta H) variation of 7.3 kcal mol(-1). The presence of 25 mu M ODPC decreased T(c) and Delta H to 393 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 mu M destabilized the liposomes (36.3 degrees C. 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition. (C) 2010 Elsevier B.V. All rights reserved.

Increased Levels of Circulating Endothelial Progenitor Cells in Human T-cell Lymphotropic Virus Type I Carriers

Background and Aims. HTLV-I-transformed T cells secrete biologically active forms of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF). In addition, HTLV-I-transformed cells have a high capacity of adhesion to endothelial cells. Methods. We measured the circulating endothelial progenitor cells (EPCs) and mature endothelial cells (MECs) by flow cytometry in 27 HTLV-I carriers in comparison to 30 healthy, age- and gender-matched subjects. All subjects had HTLV-I positivity confirmed by Western blot and/or polymerase chain reaction (PCR). The numbers of different subpopulations of EPCs and MECSs were evaluated by four-color flow cytometry using a panel of monoclonal antibodies. All reactions were done in duplicate to confirm reproducibility of the results. Results. The median age of all 27 HTLV-I carriers enrolled in this study was 45 years (range: 27-65 years); 11(41%) were male and 16 (59%) were female. The median age of the 30 healthy subjects in the control group was 45.5 years (range: 20-63 years); 11 (36.6%) were male and 19 (63.4%) were female. The number of EPCs was significantly higher in HTLV-I carriers (median 0.8288 cells/mu L, range: 0.0920-3.3176 cells/mu L) as compared to control group (median 0.4905 cells/mu L, range: 0.0000-1.5660 cells/mu L) (p = 0.035). In contrast, the median of the MECs in the HTLV-I carriers was 0.6380 cells/mu L (range: 0.0473-5.7618 cells/mu L) and 0.4950 cells/mu L (range: 0.0000-4.0896 cells/mu L) in the control group, with no statistical difference (p = 0.697). Conclusions. We demonstrated that EPCs, but not MECs, are increased in the peripheral blood of HTLV-I carriers. (C) 2011 IMSS. Published by Elsevier Inc.

Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15: 17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer ""stem"" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), Fc gamma RIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) possibly through a methylation-dependent mechanism, indicating that C/EBP alpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. (Blood. 2009; 114: 5415-5425)

Apoptosis induction by (+)alpha-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58 mu M, for NB4 and NB4-R2, respectively. a-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24 h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis. (C) 2008 Elsevier Ltd. All rights reserved.

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P<0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. (C) 2011 Elsevier B.V. All rights reserved.

High expression of AURKA and AURKB is associated with unfavorable cytogenetic abnormalities and high white blood cell count in patients with acute myeloid leukemia

In the present study, we analyzed AURKA and AURKB gene expression in 70 acute myeloid leukemia (AML) patients. There was no difference between leukemic samples and bone marrow mononuclear cells (BMMCs, n = 8) or CD34(+) progenitors (n = 10) from healthy donors. High white blood cells (WBC) counts were observed in the AURKA(+) and AURKB(+) groups, but no significant differences regarding age, gender, platelet counts or frequency of FLT3-ITD mutations. AURKA, but not AURKB, expression was independently associated with high WBC counts (OR: 3.15, 95% CI 1.07-9.24, p = 0.03). Moreover, the majority of cases that overexpressed AURKA and AURKB presented unfavorable cytogenetic abnormalities (p < 0.001). In conclusion, we described a significant association between overexpression of AURKA/B and cytogenetics findings in AML, which may be relevant to new therapeutic approaches, based on Aurora kinase inhibitors. (C) 2010 Elsevier Ltd. All rights reserved.

Cancer/Testis Antigen Expression on Mesenchymal Stem Cells Isolated from Different Tissues

Background/Aims: The expression of cancer/testis antigens (CTAs) on additional normal tissues or stem cells may restrict their use as cancer targets. The objective of the present study was to evaluate the mRNA levels of some CTAs in a variety of tissues. Materials and Methods: mRNA of pericytes, fibroblasts and mesenchymal stem cells (MSCs) derived from adult and fetal tissues, human umbilical vein endothelial cells, MSC-derived adipocytes, selected normal tissues and control cancer cell lines (CLs) were extracted and quantitative polymerase chain reaction was performed for MAGED1, PRAME, CTAG1B, MAGEA3 and MAGEA4. Results: MAGED1 was expressed in all normal tissues and cells evaluated. CTAG1B was expressed at levels comparable to control CLs on MSCs derived from arterial, fetal skin, adipose tissue and saphenous vein, heart, brain and skin tissues. MAGEA4 was detected only in fibroblasts and differentiated adipocytes from MSCs, at levels comparable to the control CLs. Conclusion: The potential use of CTAs in immunotherapy should take into account the potential off-target effects on MSCs.

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Tetrasomy 8 in a patient with chronic lymphocytic leukemia

We report a case of a 47-year-old man diagnosed with chronic lymphocytic leukemia (CLL) with two extra copies of chromosome 8. Classical cytogenetic analysis by the immunostimulatory combination of DSP30 and interleukin 2 showed tetrasomy of chromosome 8 in 60% of the metaphase cells (48,XY,+8,+8[12]/46,XY[8]). Spectral karyotype analysis confirmed the abnormality previously seen by G banding. Additionally, interphase fluorescence in situ hybridization using an LSI CEP 8 probe performed on peripheral blood cells without any stimulant agent showed tetrasomy of chromosome 8 in 54% of analyzed cells (108 of 200). To our knowledge, tetrasomy 8 as the sole chromosomal abnormality in CLL has not been previously described. The prognostic significance of tetrasomy 8 in CLL remains to be elucidated. However, the patient has been followed up in the outpatient hospital since 2004 without any therapeutic intervention and has so far remained stable. (C) 2010 Elsevier Inc. All rights reserved.