cyclin-d1 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic lymphocytic leukemia and chronic myeloid leukemia are the most common types of adult leukemia. However, it is rare for the same patient to suffer from both. Richter's transformation to diffuse large B-cell lymphoma is frequently observed in chronic lymphocytic leukemia. Purine analog therapy and the presence of trisomy 12, and CCND1 gene rearrangement have been linked to increased risk of Richter's transformation. The coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma in the same patient is extremely rare, with only nine reported cases. Here, we describe the first reported case of concurrent chronic myeloid leukemia and diffuse large B-cell lymphoma in a background of chronic lymphocytic leukemia.. A 60-year-old Saudi man known to have diabetes, hypertension, and chronic active hepatitis B was diagnosed as having Rai stage II chronic lymphocytic leukemia, with trisomy 12 and rearrangement of the CCND1 gene in December 2012. He required no therapy until January 2016 when he developed significant anemia, thrombocytopenia, and constitutional symptoms. He received six cycles of fludarabine, cyclophosphamide, and rituximab, after which he achieved complete remission. One month later, he presented with progressive leukocytosis (mostly neutrophilia) and splenomegaly. Fluorescence in situ hybridization from bone marrow aspirate was positive for translocation (9;22) and reverse transcription polymerase chain reaction detected BCR-ABL fusion gene consistent with chronic myeloid leukemia. He had no morphologic or immunophenotypic evidence of chronic lymphocytic leukemia at the time. Imatinib, a first-line tyrosine kinase inhibitor, was started. Eight months later, a screening imaging revealed new liver lesions, which were confirmed to be diffuse large B-cell lymphoma.. In chronic lymphocytic leukemia, progressive leukocytosis and splenomegaly caused by emerging chronic myeloid leukemia can be easily overlooked. It is unlikely that chronic myeloid leukemia arose as a result of clonal evolution secondary to fludarabine treatment given the very short interval after receiving fludarabine. It is also unlikely that imatinib contributed to the development of diffuse large B-cell lymphoma; rather, diffuse large B-cell lymphoma arose as a result of Richter's transformation. Fludarabine, trisomy 12, and CCND1 gene rearrangement might have increased the risk of Richter's transformation in this patient.

Topics: Antineoplastic Agents; Chromosomes, Human, Pair 12; Cyclin D1; Cyclophosphamide; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Rearrangement; Hematopoietic Stem Cell Transplantation; Humans; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocyte Count; Lymphoma, Large B-Cell, Diffuse; Male; Middle Aged; Rituximab; Treatment Outcome; Trisomy; Vidarabine

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3'-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Base Sequence; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Disease Models, Animal; Disease Progression; DNA Breaks, Double-Stranded; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Gene Ontology; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes; Mice, Inbred C57BL; MicroRNAs; RNA, Messenger; Up-Regulation

In the present study, we studied downstream signals of BCR-ABL with regard to Src family kinases and YAP, a transcription cofactor and an effector of the Hippo pathway. We first checked the phosphorylation status of YAP and found that it was constitutively phosphorylated at tyrosine 357 in CML-derived cell lines (TCC-S and K562) but not in AML-derived cell lines (HL-60 and KG-1a). Treatment with imatinib or RK-20449 inhibited cell growth and decreased tyrosine phosphorylation of YAP in both CML lines. Expression of Survivin or Cyclin D1 was decreased in TCC-S, but not in either HL-60 or KG-1a. Furthermore, we established BCR-ABL stable transfectant and control empty vector transfectant from TF-1, a factor-dependent human erythroleukemia cell line, to verify our results obtained with CML cell lines. YAP was phosphorylated at Y357 constitutively in BCR-ABL stable transfectant but not in control transfectant, and treatment with imatinib or RK-20449, a Src family kinase-specific inhibitor, inhibited cell growth, YAP tyrosine phosphorylation, and expression of Cyclin D1 in BCR-ABL stable transfectant. These results suggest that BCR-ABL induces tyrosine phosphorylation of YAP presumably through Src family kinases, which results in expression of Survivin and Cyclin D leading to leukemogenesis in CML cells.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Fusion Proteins, bcr-abl; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Protein Kinase Inhibitors; src-Family Kinases; Survivin; Transcription Factors; Tyrosine; YAP-Signaling Proteins

Topics: Antineoplastic Agents; Caspase 3; Cell Line, Tumor; Cyclin D1; Glycolysis; Humans; Imatinib Mesylate; Lactic Acid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Signaling System; Oxidative Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-myc; STAT3 Transcription Factor

Tyrosine kinase inhibitors (TKI) have become a first-line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplasm Recurrence, Local; NF-kappa B; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction

Approximately 5-10% of chronic myeloid leukemia (CML) patients are found to have structural or numerical additional chromosomal abnormality (ACAs) in addition to the characteristic t(9;22)(q34;q11.2) BCR/ABL1 at the time of diagnosis. The prognostic significance of such additional chromosomal abnormalities has been controversial. Translocation t(11;14)(q13;q32) CCND1-IGH is typically associated with mantle cell lymphoma or a subset of plasma cell myeloma and is exceedingly rare in myeloid neoplasm. Here we report a unique case describing a patient found at diagnosis of chronic phase CML to have both the Philadelphia chromosome as well as t(11;14)-a rare cytogenetic combination. The patient was treated with imatinib with appropriate hematologic response but persistent disease by FISH and RT-PCR. She was switched to dasatinib and eventually achieved cytogenetic remission in both translocations, but still with persistent RT-PCR evidence of BCR-ABL1 fusion. As cyclin D1 is a regulatory subunit of cyclin-dependent kinases CDK4 and CDK6 and is required for the cells to progress through the G1 phase of the cell cycle, overexpression of cyclin D1 will likely promote cells into cell cycle. This may further augment proliferation in addition to upregulated ABL1 kinase activity in the index case. It may also contribute to the resistance to imatinib, as imatinib only targets on BCR-ABL fusion. Therefore, the addition of t(11;14)(q13;q32) may have significant implication in patient management.

Topics: Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Fusion Proteins, bcr-abl; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; Philadelphia Chromosome; Prognosis; Translocation, Genetic

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzamides; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p15; Down-Regulation; Drug Combinations; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Gene Expression; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; RNA, Small Interfering

The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.

Topics: Bone Morphogenetic Protein Receptors; Cell Differentiation; Cell Proliferation; Chemokine CCL2; Cyclin D1; DNA Repair; Down-Regulation; Gene Expression Profiling; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Signal Transduction; Stem Cells; Transcriptome; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Benzamides; Benzofurans; Benzophenones; Bone Marrow Cells; Cell Line, Tumor; Colony-Forming Units Assay; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Fusion Proteins, bcr-abl; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Genes, bcl-2; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Necrosis; Neoplasm Proteins; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Resting Phase, Cell Cycle; STAT5 Transcription Factor

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of β-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3β (glycogen synthase kinase 3β). Herein, we show that GSK3β is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3β activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3β complex formation. Specifically, GSK3β activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the β-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.

Topics: Antigens, CD34; Apoptosis; Benzamides; beta Catenin; Blotting, Western; Cell Nucleus; Cells, Cultured; Cyclin D1; Cytokines; Dasatinib; Drug Synergism; Fusion Proteins, bcr-abl; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; Indoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Maleimides; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Neoplastic Stem Cells; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Signal Transduction; Thiazoles

Cryptotanshinone (CPT), a diterpene quinone isolated from Salvia miltiorrhiza, is recently reported to have obvious anticancer activities against diverse cancer cells. However, the effect and regulatory mechanism of CPT remain unclear in human chronic myeloid leukemia (CML) cells. In this study, we investigated the antiproliferative activity of CPT on the multidrug resistant CML cells K562/ADM. Our results demonstrated that CPT decreased the cell viability of K562/ADM cells by inducing cell cycle arrest and apoptosis through suppressing the expression of cyclin D1 and Bcl-2. Further studies indicated that CPT mainly functions at post-transcriptional levels, suggesting the involvement of eukaryotic initiation factor 4E (eIF4E). CPT significantly reduced the expression and activity of eIF4E in K562/ADM cells. Overexpression of eIF4E obvious conferred resistance to the CPT antiproliferation and proapoptotic activity as well as the cyclin D1 and Bcl-2 expressions. Knockdown of eIF4E significantly reduced the inhibitory effect of CPT in K562/ADM, confirming the participation of eIF4E during CPT function process. More importantly, the relative inhibitory efficiency of CPT positively correlated with the reductions on eIF4E in primary CML specimens. These results demonstrated that CPT played antitumor roles in K562/ADM cells by inhibiting the eIF4E regulatory system. Our results provide a novel anticancer mechanism of CPT in human CML cells.

Topics: Apoptosis; Cell Cycle Checkpoints; Cyclin D1; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Peptide Chain Initiation, Translational; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2

The cytotoxic effect of berbamine on chronic myeloid leukemia (CML) cell line KU812 was evaluated, and the mechanisms of its action were explored.. The effect of berbamine on the KU812 cell growth was determined by methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry was used to profile cell cycle alteration upon berbamine treatment. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the transcripts of transforming growth factor-β (TGF-β) receptors (TβRs), Smad3, c-Myc, cyclin D1, p21(Cip1)(p21), and p27(Kip1)(p27). Changes in the protein levels of total Smad3, phosphorylated Smad3, the downstream targets of Smad3, and specific apoptosis-related factors were evaluated by Western blotting.. Berbamine inhibited KU812 cell proliferation in a dose- and time-dependent manner, and the half maximal inhibitory concentration (IC₅₀) values for treatments of 24, 48, and 72 h were 5.83, 3.43, and 0.75 μg/ml, respectively. Berbamine induced G₁ arrest as well as apoptosis in KU812 cells. Transcriptions of Smad3 and p21 were up-regulated, while those of TβRI, TβRII, c-Myc, cyclin D1 and p27 were not changed significantly. The protein levels of both total Smad3 and phosphorylated Smad3 were both up-regulated after berbamine treatment, together with decreased c-Myc and cyclin D1 and increased p21. Meanwhile, the levels of the anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, were decreased, whereas pro-apoptotic Bax was increased.. Berbamine suppresses KU812 cell proliferation through induction of cell cycle arrest in G₁ and apoptosis. It activates Smad3 without additional stimulation of TGF-β, and alters the levels of the Smad3 downstream targets, including c-Myc, cyclin D1 and p21. Our findings suggest that berbamine is a promising drug in the treatment of advanced stage patients with CML.

Topics: Apoptosis; Benzylisoquinolines; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Gene Expression Regulation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Receptors, Transforming Growth Factor beta; Smad3 Protein

Dasatinib (BMS-354825) is a novel, oral, potent, multi-targeted kinase inhibitor of Bcr-Abl and Src family kinases (SFK) and is a promising cancer therapeutic agent. Preclinical data indicate that dasatinib is 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl, and that dasatinib is active against 18 of 19 Bcr-Abl mutations known to cause imatinib resistance. Phase I clinical data show that dasatinib is well tolerated and highly effective for the treatment of imatinib-resistant/imatinib-intolerant chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia. However, the molecular mechanism of action of dasatinib is not fully understood. In this study, we confirm that dasatinib inhibits tyrosine phosphorylation of SFKs, including Src, Hck, and Lyn, in K562 human CML cells. Significantly, downstream signal transducer and activator of transcription 5 (Stat5) signaling is also blocked by dasatinib as shown by decreases in levels of phosphorylated Stat5 and Stat5 DNA-binding activities. In addition, dasatinib down-regulates expression of Stat5 target genes, including Bcl-x, Mcl-1, and cyclin D1. Consistent with these results, blockade of Stat5 signaling by dasatinib is accompanied by inhibition of cell proliferation and induction of apoptosis. Surprisingly, Stat5 DNA-binding activities are enhanced with increasing cell density, which is associated with resistance to apoptosis by dasatinib. Our findings indicate that inhibition of Stat5 signaling downstream of Bcr-Abl/SFKs contributes to the action of dasatinib, and, conversely, that increasing cell density up-regulates Stat5 activation and confers resistance to dasatinib. Moreover, the level of phosphorylated Stat5 in CML cells represents a mechanistically relevant biomarker for monitoring inhibition of Bcr-Abl signaling by dasatinib in CML patients using convenient immunocytochemical assays.

Topics: Apoptosis; bcl-X Protein; Cell Count; Cell Survival; Cyclin D1; Dasatinib; DNA, Neoplasm; Down-Regulation; Drug Resistance, Neoplasm; HL-60 Cells; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Phosphotyrosine; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Signal Transduction; src-Family Kinases; STAT5 Transcription Factor; Thiazoles

Celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, has been shown to possess antitumor activity in a variety of cancer cells. However, the antitumor activity of celecoxib in hematopoietic tumors, especially in chronic myeloid leukemia (CML), has not been well established. This study was designed to investigate the effect of celecoxib on growth and apoptosis in a human CML cell line (K562 cells) or in primary CML cells, and to examine the synergistic actions of celecoxib and hydroxyurea or imatinib on K562 cell proliferation and apoptosis. Celecoxib significantly inhibited the growth of both K562 and primary CML cells and induced apoptosis in a dose-dependent fashion. The IC50 of celecoxib was 46 microM for inhibition of K562 cell proliferation. The effect of celecoxib on growth inhibition was accompanied by the downregulation of cyclin D1 and cyclin E and p-Rb expression, the upregulation of P16(INK4a) and P27KIP expression, and a G1-S phase arrest of the cell cycle. The pro-apoptotic effect of celecoxib was determined to be mediated by caspase-3 activation. When K562 cells were pretreated with DEVD-fmk, a specific inhibitor of caspases, the apoptotic activity of celecoxib was, in part, abrogated. Importantly, we demonstrated for the first time that K562 cells were Cox-2-positive both at the mRNA and protein levels. We noted the following observations: (i) we detected Cox-2 mRNA in K562 cells by reverse transcription-PCR (RT-PCR) and protein expression by western blot analysis; (ii) Cox-2 expression in K562 cells was stimulated by IL-1beta, a specific inducing agent of Cox-2 expression; (iii) primary CML cells from CML patient bone marrow also exhibited Cox-2 protein expression. Furthermore, Cox-2 expression was downregulated at higher doses of celecoxib (80-160 microM), suggesting a Cox-2-dependent mechanism was involved in the drug's effects of growth inhibition and induction of apoptosis. In addition, a synergistic effect was observed when cells were exposed to low-dose celecoxib (40 microM) and hydroxyurea (10 mM) or a combination of celecoxib (40 microM) and imatinib (0.2 microM). These findings provide the basis for uncovering the mechanism of celecoxib's antitumor effects and developing a new therapeutic strategy for treating CML.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Bone Marrow Cells; Caspase 3; Caspase Inhibitors; Caspases; Celecoxib; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; G1 Phase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Humans; Hydroxyurea; Imatinib Mesylate; Interleukin-1; Intracellular Signaling Peptides and Proteins; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Piperazines; Pyrazoles; Pyrimidines; S Phase; Sulfonamides

The eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E. HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E's dysregulation. In regard to these results and our previous finding that approximately 200 homeodomain proteins contain eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through which this family of proteins implements their effects on growth and development.

Topics: Active Transport, Cell Nucleus; Acute Disease; Amino Acid Sequence; Bone Marrow Cells; Cell Nucleus; Cyclin D1; Cytoplasm; Eukaryotic Initiation Factor-4E; Homeodomain Proteins; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Molecular Sequence Data; Ornithine Decarboxylase; Sequence Homology, Amino Acid; Transcription Factors; Transcription, Genetic

Cyclins are very important components of the cell cycle machinery because their levels regulate cell proliferation. They have also been found to be prognostic factors in various cancers. We studied the expression of the positive cell cycle regulators (D cyclins) and the cell proliferation marker (Ki-67) in human acute myeloid (AML), chronic myeloid (CML), acute lymphoblastic (ALL) and chronic lymphocytic (CLL) leukemia [mainly by comparative reverse transcription polymerase chain reaction (RT-PCR)]. Both leukemic and normal cells were positive for cyclin D3 expression. Significant differences were found in the expression of cyclin D1, which was the highest in leukocytes (CD19 + ) of CLL patients whereas lower expression was found in CML, AML and ALL patients and normal bone marrow and peripheral blood leukocytes (P < 0.001). The higher expression of cyclin D1 in leukocytes of CLL patients compared to CML patients was confirmed by quantitative real-time RT-PCR with a TaqMan probe in a subset of CLL and CML patients. Differences in cyclin D1 expression between CLL and CML patients were also confirmed on protein levels by western blotting. Expression of the proliferative marker Ki-67 was high in CML, ALL and AML cells and low in CD19-positive CLL cells. The results demonstrate that the level of cyclin D1 negatively correlates with the proliferation properties of leukemic cells. We did not find any significant relationship between cyclin D1 expression in cells of CML and AML patients and their clinical outcome.

Topics: Acute Disease; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reverse Transcriptase Polymerase Chain Reaction

Chronic myeloid leukaemia invariably progresses from a drug-sensitive to a drug-resistant, aggressive acute leukaemia. The mechanisms responsible for this are unknown, although loss of p53 has been reported in approximately 25% of cases. Elevated expression of Bcr-Abl is also associated with disease progression. We have shown that cells expressing high levels of Bcr-Abl also express elevated levels of p53 and the cell cycle inhibitor, p21WAF-1. Despite this, cells continue to cycle and are drug resistant. As p21WAF-1 inhibitory activity is associated with nuclear localization, we investigated its localization in Bcr-Abl-expressing cells, and found that it is predominantly cytoplasmic. We have also shown that it associates physically with the serine/threonine kinase AKT, but this association and the cytosolic location of p21WAF-1 are phosphinositide-3-kinase (PI3K) independent. Cytosolic p21WAF-1 has been reported to have a prosurvival role in other transformed cells. In Bcr-Abl-expressing cells, p21WAF-1 rapidly diminishes as the cells are sensitized to apoptosis, using the inhibitor STI571. It is possible therefore that p21WAF-1 could also have a positive, prosurvival role in these cells. This study suggests that, by retaining p21WAF-1 in a cytosolic location, Bcr-Abl can evade the cell cycle arrest normally induced by nuclear p21WAF-1 and therefore also enable the cells to negate an important feature of a tumour suppressor response.

Topics: Apoptosis; Benzamides; Biomarkers; Cell Cycle; Cell Line, Transformed; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytosol; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphatidylinositol 3-Kinases; Piperazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrimidines; Signal Transduction

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3beta were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.

Topics: Aminopeptidases; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Benzamides; Caspase 3; Caspases; Cell Division; Cyclin D1; Drug Resistance, Neoplasm; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hydroxamic Acids; Imatinib Mesylate; K562 Cells; Leucine; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitogen-Activated Protein Kinases; Phosphorylation; Piperazines; Protease Inhibitors; Pyrimidines; Serine; Signal Transduction

Accumulating evidence indicates that the activation of cellular oncogenes is a cause of some human cancers. ErbB-1, erbB-2 and abl oncogenes encoding tyrosine kinases, ras oncogenes encoding GTP binding proteins and myc oncogenes whose functions are not well understood are some examples. Therefore, agents which inhibit the activity of these oncogene products may provide new means to overcome certain human tumors. Herbimycin A and tyrphostins have been found and developed as inhibitors of tyrosine kinases and the effectiveness of these agents against tumors of Ph1-positive leukemia (CML, ALL) or squamous cell carcinomas has been reported. Although specific inhibitors of ras or myc oncogene products have not yet been described, recent studies on the processing of Ras proteins toward the cell membrane provide a strategy to search for inhibitors of ras functions.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Carcinoma, Squamous Cell; Catechols; Cyclin D1; Female; Genes, ras; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Neoplasms; Nitriles; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Rifabutin; Tyrphostins