Cell Lines BV173 Research Articles

Glyoxalase-I Inhibition Eliminates Hypoxia-Adapted CML Cells Resistant to Imatinib, Dasatinib, and INNO-406

The development of ABL tyrosine kinase inhibitor (TKI), imatinib mesylate and second-generation ABL TKIs such as dasatinib and nilotinib have dramatically improved the efficacy of chronic myeloid leukemia (CML) treatment, but “total cell kill” may not be possible with TKIs alone because they do not appear to eliminate CML stem cells. Although the CML stem cell niche has not yet been clearly identified, it may be a hypoxic environment, like that of normal hematopoietic stem cells, which mainly exists in the low oxygen-consuming region in the bone marrow (BM). Notably, most cancer cells that adapt to hypoxia are resistant to a variety of cell-death stimuli. Thus, we hypothesized that CML cells, in particular CML stem cells, adapt to hypoxia for their persistence. To test this notion, we first examined the oxygenated status of CML cells in vivo by transplanting K562 cells into NOD/SCID/gcnull (NOG) mice. Six weeks later, pimonidazole, which specifically accumulates in tissues with <1.3% O2, was administered and the mice were sacrificed. The K562 cells had mainly engrafted into the epiphysis, which is presumed to be a particularly hypoxic environment, and these engrafted cells were positively stained by antibody to pimonidazole. This suggests that the engrafted leukemic cells survived in hypoxic conditions. To investigate the role that adaptation to hypoxia plays in CML-cell persistence, we generated two hypoxia-adapted cell lines that can survive in hypoxic conditions and identified their unique characteristics compared to their parental cells. For this, the CML-derived cell lines K562, KCL-22, BV173 and MYL were cultivated in 1.0% O2 for two weeks. Two subclones that survived were obtained from K562 and KCL22 and were denoted as K562/HA and KCL22/HA, respectively. Both cell lines proliferated continuously for months in 1.0% O2 conditions in RPMI1640 medium containing 10% FCS but lacking any additional nutrient supplies, although their growth was slower than that of their parental counterparts under 20% oxygen in the same media. We found that, compared to their parental counterparts, K562/HA and KCL22/HA had higher glyoxalase-I (Glo-I) expression and kinase activity. Glo-I is an enzyme that detoxifies methylglyoxal, a cytotoxic byproduct of glycolysis. Interestingly, BCR-ABL phosphorylation was suppressed in both hypoxia-adapted cell lines. However, the phosphorylation of AKT, STAT and ERK was unchanged in these hypoxia-adapted cells, which suggests the presence of alternative hypoxia-specific activation pathways. When NOG mice were inoculated with equivalent numbers of K562/HA and K562 cells, K562/HA engrafted more rapidly than K562 and K562/HA-engrafted mice died earlier than K562-engrafted mice. Thus, adaptation to hypoxia is crucial for the engraftment and proliferation of CML cells in the BM. Notably, compared to their parental counterparts, K562/HA and KCL22/HA cells were less sensitive to TKIs (imatinib, dasatinib, and INNO-406) and alkylating agents (daunorubicin and busulfan). However, they were more sensitive to Glo-I inhibitors such as S-p-bromobenzylglutathione cyclopentyl diester (BBGC), 2-crotonyloxymethyl- 4,5,6-trihydroxycylohex-2-enone (COTC), and methyl-gerfelin. Thus, Glo-I enzymatic activity plays an important role in CML-cell adaptation to hypoxic environments, which means Glo-1 may be an alternative molecular target for therapies aiming to eliminate those CML cells that adapt to hypoxia and thereby acquire resistance to conventional chemotherapies, including those based on TKIs. Indeed, when K562/HA-transplanted mice were treated with 100mg/kg/day BBGC for 8 days or left untreated, the treated mice survived longer than the untreated mice. In contrast, the survival of mice transplanted with the parental cell line K562 did not change whether they were treated with BBGC or left untreated. Thus, Glo-1 enzymatic activity plays an important role in CML-cell survival under hypoxic environmental conditions and Glo-I inhibitors could be promising agents for the elimination of CML (stem) cells that persist in the hypoxic BM milieu

STI571 reduces NER activity in BCR/ABL-expressing cells

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.

Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death

Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

Comprehensive Highly Multiplexed Single-Tube Liquid Bead Microarray Assay for Constitutively Activated Tyrosine Kinases in Chronic Myeloproliferative Disorders.

Chronic Myeloproliferative disorders (CMPDs) are malignancies characterized by excessive proliferation of one or more of the myeloid lineages driven by constitutively activated tyrosine kinases (CATKs). CATKs are increasingly serving as targets for cancer-specific therapy and newer and more potent TK inhibitors are becoming available for clinical use. CATKs in CMPDs may result from karyotypically evident or cryptic chromosomal translocations leading to TK gene fusions or from karyotypically silent gain-of-function point mutations. More than ten CATK genes have been shown to result in CMPDs by imparting a demonstrable positive growth drive. CATK inhibitors important in the treatment of CMPDs have different inhibitory profiles, making it critical to identify the specific mutation involved in patients receiving targeted therapy. Of these TK genes, ABL, PDGFRB, PDGFRA, FGFR1, ETV6, JAK2 and MPL are the most frequently involved. Since most of these TK genes can participate in chromosome translocations with a large number of partner genes, the resulting number of possible CATKs is rather large, making detection and identification a challenging task. To date, two classic (B2A2 and B3A2) and seven less common fusions of the ABL and BCR genes have been described. In addition, over twenty non-BCR-ABL mutations have been shown to have pathogenetic relevance in CMPDs. We have developed a simple, high-throughput assay with a fast turnaround time that solves the complex problem of detecting and identifying multiple translocations and point mutations. Two steps are involved in this expandable assay. The first is a multiplex RT-PCR reaction that amplifies fusion transcripts and point mutations described in CMPDs from peripheral blood or bone marrow. This PCR reaction employs internal specific primers targeted to each of eight BCR-ABL mutations (E1A2, E1A3, B2A2, B3A2, E6A2, E8A2, E15A2, E19A2) eight non-BCR-ABL mutations (FIP1L1-PDGFRA, BCR-PDGFRA, BCR-FGFR1, MYO18A-FGFR1, CDK5RAP2-PDGFRA, ETV6-PDGFRB, RABAPTIN-5-PDGFRB, H4-PDGFRB) and two gain of function mutations (JAK2V617F and MPLW515L) and a biotinylated common primer set to increase amplification efficiency. The second is a liquid bead microarray (Luminex) which uses optically encoded beads to detect probes specific for multiple known translocations in a single tube. To date, we have selectively detected four BCR-ABL and three non-BCR-ABL mutations from cloned patient samples and the cell lines SupB15 (E1A2 p190 BCR-ABL fusion), K562 (B3A2 p210 BCR-ABL fusion) and BV173 (B2A2 p210 BCR-ABL fusion). Dilution studies using these cell lines demonstrate detection down to between 1/1000 and 1/100000 cell equivalents without significant cross-talk. [Display omitted] [Display omitted]

IKZF1 (Ikaros) Deletions Are a Hallmark of BCR-ABL1 Positive Acute Lymphoblastic Leukemia.

Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature 2007; 446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

Cytotoxic Mannich bases of 6-(3-aryl-2-propenoyl)-2(3 H)-benzoxazolones

A series of 12 new Mannich bases with chalcone core structure were synthesized as potential antineoplastic agents, via N-aminomethylation of two parent 6-(3-aryl-2-propenoyl)-2(3 H)-benzoxazolones. The newly synthesized compounds as well as the chalcone prototypes were evaluated for cytotoxicity in the human pre-B-cell leukemia cell line BV-173 using the MTT-dye reduction assay. The tested compounds exhibited concentration-dependent cytotoxic effects at low micromolar concentrations. Ten of the Mannich bases characterized by significant activity in BV-173 were further evaluated against the chronic myeloid leukemia cell line K-562 and were found to suppress the growth of these cells at relatively higher concentrations as compared to the former tumor model. Selected Mannich bases induced programmed cell death in BV-173 at a concentration of 2.5 μM as evidenced by the encountered DNA-laddering. Taken together our data suggest that the presented heterocyclic chalcone derived Mannich bases necessitate detailed pharmacological evaluation in order to define further the structure activity relationships, in a larger spectrum of tumor models and to elucidate the mechanisms implicated in the observed cytotoxic effects.

AMD3100 Inhibits Chemotaxis towards SDF-1 and CXCR4-Mediated Stroma-Contact in a Dose-Dependent Manner, Resulting in Increased Susceptibility to Imatinib.

Besides new efforts in the treatment with tyrosine-kinase-inhibitors like imatinib were made, Chronic myelogenous leukemia (CML) still remains chronic with a progression of the disease by time. New ways of treatment have to be found to eradicate the malignant clone in the bone marrow.In our study we are evaluating whether an inhibition of the SDF-1-CXCR4-axis by the selective CXCR4 receptor antagonist AMD3100 results in increased cell kill of the BCR-ABL+ cell line BV-173 when combined with treatment with imatinib. AMD3100 significantly blocks binding of the FACS-anti-CXCR4-Mab to its corresponding receptor. Furthermore, AMD3100 significantly inhibits SDF-1 mediated chemotaxis towards 500ng/ml SDF-1 to 0.71±0.15 of spontaneous transmigration at the highest concentration (100μM) of AMD3100 whilst SDF-1 500ng/ml leads to 1.4±0.06 fold spontaneous transmigration (n=3). Even lower concentrations (10μM and 1μM) still significantly reduce transmigration to a level lower than spontaneous transmigration. Also migration into the SDF-1 producing stromal-cell line FBMD-1 was dose-dependently significantly reduced to 56.7% ± 8.6% migration of untreated controls at 100μM AMD3100 (n=4). Lower concentrations also significantly reduced migratory activity of BV-173. In a stroma-dependent co-culture AMD3100 dose-dependently increased susceptibility of BV-173 towards simultaneous treatment with Imatinib at a dose of 1.6μM. We saw 23% ± 8% PI+ cells in the control without AMD3100 vs. 40% ± 12% PI+ cells al 100μM AMD3100 (n=3; p=0.06). Incubation with 10μM and 1μM AMD3100 only resulted in a slight increase of PI+ cells to 28% ± 13% at 1μM and 29% ± 3 at 10μM respectively.The same assays on the AML-cell line HL-60 and on the BCR-ABL+ cell line SD-1 led to a significant reduction in SDF-1 dependent chemotaxis and stromal-dependent migration especially at the 100μM concentration but on a level with higher transmigration rates. Interestingly, for these cell lines we found lower CXCR4 surface expression (MFI: 478 ± 71 for the BV-173 vs. 29 ± 8 for the HL-60 and 17 ± 1.5 for the SD-1) going hand in hand with lower effects on migration rates following incubation with AMD3100 than we could show for the CML cell line BV-173.We conclude that co-treatment with AMD3100 and Imatinib leads to a sensitization of BCR-ABL+ leukaemia cells which is dependent on the baseline CXCR4 expression. This is a promising strategy to reduce residual BCR-ABL+ leukaemia cell load in CML patients.

Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

The recent discovery of a single point mutation in the JH2 pseudokinase domain of Janus kinase 2 (JAK2) in a considerable fraction of patients has shed light on the molecular pathomechanism in Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph- CMPDs). We established a robust and reliable method for detection of the JAK2 mutation in bone marrow cells derived from archival bone marrow trephines based on polymerase chain reaction and subsequent restriction site analysis. In a series of proven Ph- CMPDs classified according to World Health Organization criteria (n = 79), we detected the JAK2 mutation in 90% of polycythemia vera, 22% of cellular prefibrotic chronic idiopathic myelofibrosis, 60% of advanced chronic idiopathic myelofibrosis, and 27% of essential thrombocythemia. JAK2 mutation was not detected in Ph+ chronic myeloid leukemia (n = 5), acute myeloid leukemia (n = 10), acute lymphoblastic leukemia (n = 10), secondary erythrocytosis (n = 10), or normal bone marrow (n = 10). Restriction site analysis was also suitable for unfixed cell populations derived from peripheral blood and bone marrow aspirates. Besides providing support in the differential diagnosis of reactive versus neoplastic myeloproliferations, this newly developed assay reveals considerable overlaps between histologically different disease entities, indicating that additional genetic alterations might be responsible for the established differences of CMPD subentities.

Growth inhibition and apoptosis in human Philadelphia chromosome-positive lymphoblastic leukemia cell lines by treatment with the dual PPARα/γ ligand TZD18

Treatment of adult Philadelphia chromosome-positive lymphocytic leukemia is rarely successful. We report here the effects of TZD18, a novel dual ligand specific for peroxisome proliferator-activated receptor alpha and gamma (PPARalpha/gamma) on Ph(+) lymphocytic leukemia cell lines BV173, SD1, and SupB-15. Exposure of these cells to TZD18 resulted in growth inhibition in a dose- and time-dependent manner that was associated with G(1) cell cycle arrest. This effect was much stronger than that mediated by the PPARgamma ligand pioglitazone (PGZ), which also belongs to the thiazolidinediones (TZD) class of ligands. However, it may not be mediated through PPARgamma or PPARalpha activation because antagonists of PPARgamma and PPARalpha cannot reverse it. Study of the key regulators of cell cycle progression by Western blot analysis showed that the expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1), but not that of p21(cip1), was enhanced, whereas that of c-Myc, cyclin E, cyclin D2, and cyclin-dependent kinases 2 and 4 (CDK-2 and CDK-4) was decreased when these cells were treated with TZD18 (10 or 20 microM). Therefore, the up-regulation of p27(kip1) and the down-regulation of CDK-2 and CDK-4 may, at least in part, account for the G(1) cell cycle arrest. Furthermore, a remarkable induction of apoptosis was observed in the cells treated with this dual ligand. No obvious alteration of bcl-2 protein level occurred, but bax was up-regulated in these TZD18-treated cells. Activation of caspase 8 and caspase 9 by TZD18 was also observed. Importantly, NF-kappaB DNA-binding activity was markedly decreased by the TZD18 treatment. In addition, TZD18 enhanced the growth inhibitory effect of imatinib, a specific tyrosine kinase inhibitor therapeutically used in the treatment of Ph(+) leukemia. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic approach to aid in the treatment of Ph(+) lymphocytic leukemia.

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation.Disconnection between slp-65 transcripts and protein expressiontotalslp-65 protein+ (51 patients)slp-65 protein weak (19 patients)slp-65 protein- (49 patients)PCR+10851(9 TEL/AML+, 42 TEL/AML-)19 (9 TEL/AML+, 10 TEL/AML-)38 (15 TEL/AML+, 23 TEL/AML-)PCR-110011 (T-ALL)

Transcriptional Regulation of the Bach2 B-Cell Differentiation and Apoptotic Factor by the Bcr-Abl Oncoprotein.

Transformation by the Bcr-Abl oncoprotein of CML is mediated by the activation of a variety of signalling pathways, leading to transcriptional regulation of genes conferring the malignant phenotype of increased proliferation, altered adhesion and inhibition of apoptosis. We previously reported that expression of the BACH2 gene is downregulated by Bcr-Abl. Bach2 is a B-lymphoid specific transcription factor, which regulates somatic hypermutation and class switch recombination of Ig genes. It is also a pro-apoptotic factor, coupling oxidative stress to transcription repression. It is possible that in an environment of increased genomic instability, Bcr-Abl transformed cells may repress pro-apoptotic signals by suppressing BACH2 transcription. To determine the direct association between Bcr-Abl and decreased BACH2 transcription, we infected human B-lymphoid cells with a retroviral vector expressing both p210Bcr-Abl and eGFP genes. Infected cells were treated with imatinib, an Abl tyrosine kinase inhibitor, prior to quantification of BACH2 transcripts by Real Time RT/PCR. Ectopic expression of BCR-ABL significantly decreased BACH2 mRNA levels, and this effect was completely abolished by imatinib. To investigate whether this regulation was exerted at the transcriptional level, we identified the BACH2 transcription initiation site (TIS), and then cloned and characterised a 3.9 Kb genomic DNA fragment including the BACH2 promoter region. By generating luciferase reporter constructs of various lengths of the BACH2 promoter we found that a region of 725 bp upstream the TIS conferred maximum promoter activity in human B-lymphoid cells. The effect of Bcr-Abl on promoter activity was demonstrated by co-transfection of the reporter and p210Bcr-Abl constructs. BACH2-promoter activity was reduced up to 60% in the presence of Bcr-Abl. Furthermore, when co-transfected cells were incubated with different concentrations of imatinib, the Bcr-Abl-mediated promoter repression was abrogated in a dose dependent manner, confirming the dependence of the effect on the tyrosine kinase activity of the oncoprotein. In support of these data, no effect on promoter activity was seen when the BACH2 promoter was co-transfected with a kinase-dead BCR-ABL construct. Moreover, treatment with imatinib of the BCR-ABL+ cell line BV173 transfected with the reporter induced a nearly 2-fold upregulation in its activity. Bioinformatics inspection of the promoter sequence revealed potential sites for the Pax5 B-cell differentiation factor and the Foxo3a transcription factor, a regulator of pro-apoptotic genes. In co-transfection experiments of either factor with the BACH2 promoter, both demonstrated a significant inducing effect on its activity. Gel shift and chromatin immunoprecipitation showed direct binding of Pax5 within the BACH2 promoter in vitro and in vivo. Moreover, Western analysis showed elevated Pax5 levels in BCR-ABL+ cell lines after imatinib treatment, indicating that inhibition of Bach2 expression by Bcr-Abl is mediated at least in part by Pax5. As to Foxo3a, it has been reported to be constitutively phosphorylated and inactivated in BCR-ABL+ cells, processes which prevent its translocation to the cell nucleus. Altogether, our data suggest that Bcr-Abl transcriptional repression of Bach2 via Pax5 could lead to a differentiation arrest in transformed B-cells, and that Foxo3a may induce imatinib-mediated apoptosis through up-regulation of the Bach2 apoptotic function.

FoxO3a and BCR-ABL Regulatecyclin D2Transcription through a STAT5/BCL6-Dependent Mechanism

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

The influence of INK4 proteins on growth and self-renewal kinetics of hematopoietic progenitor cells

AbstractThis study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16INK4aexpression in the p16INK4a-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16INK4a and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34+ bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells fromp16INK4a−/− mice and wild-type mice. The results from p16INK4a−/− marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT ofp16INK4a. Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow ofp16INK4a−/− mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16INK4a−/− cell lines readily became growth factor–independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16INK4a, increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.

Preferential Expression of HMGI-C Isoforms Lacking the Acidic Carboxy Terminal in Human Leukemia

The high mobility group HMGI chromosomal proteins are an important component of chromatin. The HMGI-C protein consists of three amino terminal DNA binding domains (“AT hooks”), a linker region and an acidic carboxy domain. In mesenchymal tumors, chromosomal translocations of 12q13-15 result in fusion proteins containing the AT hooks and novel carboxy terminals. We have investigated the status of the HMGI-C gene in two cases of leukemia with anomalies of chromosome 12q and identified three novel isoforms (designated α, β and γ) derived from alternate splicing. One of the patients expressed all three isoforms, whereas the second patient expressed only the γ isoform; preferential expression of the HMGI-Cγ isoform was also detected in the leukemic cell lines ML3 and BV173. The results are consistent with a crucial role for truncation of the acidic carboxy domain of HMGI-C in abnormal growth.

Antisense Oligodeoxyribonucleotides Suppress Hematologic Cell Growth Through Stepwise Release of Deoxyribonucleotides

AbstractAntisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have re-examined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3′ sequences of the ODNs determine their effect. At concentrations of 10 μmol/L (for phosphorothioate ODNs) or 25 μmol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3′ positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5′ monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2′deoxycytidine 5′-monophosphate] is not cytotoxic), on their concentration (d-GMP [2′-deoxyguanosine 5′-monophosphate], TMP [thymidine 5′-monophosphate], and d-AMP [2′-deoxyadenosine 5′-monophosphate] are cytotoxic at concentrations between 5 and 10 μmol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3′ hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.

Antisense Oligodeoxyribonucleotides Suppress Hematologic Cell Growth Through Stepwise Release of Deoxyribonucleotides

Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have re-examined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3′ sequences of the ODNs determine their effect. At concentrations of 10 μmol/L (for phosphorothioate ODNs) or 25 μmol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3′ positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5′ monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2′deoxycytidine 5′-monophosphate] is not cytotoxic), on their concentration (d-GMP [2′-deoxyguanosine 5′-monophosphate], TMP [thymidine 5′-monophosphate], and d-AMP [2′-deoxyadenosine 5′-monophosphate] are cytotoxic at concentrations between 5 and 10 μmol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3′ hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.

An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism.

The specificity of antisense oligonucleotides targeted to the mRNA breakpoint region of the Bcr-Abl oncogene, found in leukaemic cells from patients with chronic myeloid leukaemia, remains controversial due to non-specific effects. To prevent protein binding of oligonucleotides we designed and tested a methylphosphonate oligonucleotide with an attached 3' soluble phosphodiester tail. Growth of chronic myeloid leukaemia (CML) cell lines BV173, KCL-22 and cells of CML patients tested was inhibited by the b2a2 type antisense Bcr-Abl oligonucleotide and not with controls. Also the growth of control CD34+ cells of two healthy donors, control cell lines and cells from AML patients was only moderately affected or not affected. Bcr-Abl protein studies in combination with growth-determination experiments indicated that the antisense methylphosphonate Bcr-Abl oligonucleotide tested is a potent inhibitor of the growth of CML cells but works in a non-antisense manner.

Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl.

Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.

Sequence specificity on the growth suppression and induction of apoptosis of chronic myeloid leukemia cells by BCR-ABL anti-sense oligodeoxynucleoside phosphorothioates.

Sequence specificity of inhibitory effects of various BCR-ABL anti-sense oligodeoxynucleoside phosphorothioates (AS PS-ODN) on the proliferation of the chronic myeloid-leukemia cell line BV173 was examined. We confirmed that 26, 18, and 16mer B2A2 AS PS-ODN had strong inhibitory effects on the proliferation of BV173 cells with B2A2 mRNA expression, and that B3A2 AS PS-ODN were equally inhibitory when cultures were initiated at lower cell concentrations. However, at higher cell concentrations, the inhibitory effects by B3A2 AS PS-ODN were reduced and B2A2 AS PS-ODNs could suppress the proliferation of BV173 cells with much more relative sequence specificity. The 26mer B2A2 AS PS-ODN induced apoptosis of BV173 cells following reduction of BCR-ABL mRNA expression and p210 protein synthesis. Various sense (S), reverse order, and random sequences had no inhibitory effects except 16mer B2A2 S and B3A2 S that revealed significant suppressive effects. Furthermore, 26mer B3A2 AS also reduced B2A2 mRNA expression and p210 protein synthesis, while 16mer S sequences did not. These results suggest that B2A2 AS may be cross-reactive with B3A2 AS on the growth suppression of CML cells under certain culture conditions, possibly due to their partial hybridization to the ABL portion of the target mRNA, although other non-sequence-specific mechanisms are also possible.