cyclin-d1 and Leukemia--Myeloid

TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in the growth of leukaemia cells have been reported, the effect of TYRO3 has not been determined. Therefore, we examined the effects of TYRO3 knockdown on the growth of leukaemia cell lines.. Three human leukaemia cell lines (AA derived from pure erythroid leukaemia, OCI/AML2, and K562), which express TYRO3 protein were used in this study. To induce TYRO3 knockdown, small interfering RNA (siRNA) against TYRO3 was transfected using an electroporation system. Cell growth was assessed by a colorimetric assay. The expression levels and activation of various signalling proteins were examined by immunoblotting. Changes in comprehensive gene expression after TYRO3 knockdown were examined by microarray analysis.. TYRO3 knockdown suppressed cell growth in the leukaemia cell lines tested. Additionally, the knockdown suppressed phosphorylation of signal transducer and activator of transcription-3 in AA cells, and extracellular signal-regulated kinase (ERK) 1/2 in AA and OCI/AML2 cells; both are downstream molecules of TYRO3 signalling. TYRO3 knockdown also suppressed the expression of survivin in all the cell lines. TYRO3 knockdown potently suppressed TYRO3 mRNA expression but not that of AXL and MERTK. Furthermore, TYRO3 knockdown suppressed cyclin D1 mRNA expression, which is a downstream molecule of ERK.. TYRO3 plays a role in leukaemia cell growth and is a potential therapeutic target for leukaemia.

Topics: Axl Receptor Tyrosine Kinase; c-Mer Tyrosine Kinase; Cell Line, Tumor; Cyclin D1; Gene Knockdown Techniques; Humans; Leukemia, Myeloid; Phosphorylation; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; RNA, Small Interfering; STAT3 Transcription Factor

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Biomimetic Materials; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cyclin D1; Cytochromes c; Drug Synergism; Gene Expression Regulation, Leukemic; Heterocyclic Compounds, 4 or More Rings; Humans; Leukemia, Myeloid; Myeloid Cell Leukemia Sequence 1 Protein; Peptide Fragments; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.

Topics: Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; Immunophenotyping; K562 Cells; Leukemia, Myeloid; Phosphorylation; RNA Interference; RNA, Small Interfering; U937 Cells

Resistance to chemotherapy is still a challenge for the treatment of acute myeloid leukemia. Combination use of histone deacetylase inhibitors (HDACIs) and proteasome inhibitors may provide a potential way to overcome drug resistance. One of the HDACIs, valproic acid (VPA), and a proteasome inhibitor, bortezomib (BOR), were assessed. Co-exposure of cells to VPA and BOR inhibited proliferation, arrested the cell cycle in G0-G1 phase and induced apoptosis in both HL60 and HL60A cells. These events were accompanied by the inhibition of cyclin D1 and human telomerase reverse transcriptase (hTERT) as well as telomerase activity. Moreover, synergism of proliferation inhibition was found in HL60A, superior to the additivity in HL60. The effects of combination treatment on cell cycle arrest and telomerase activity inhibition in HL60A were also more striking than those in HL60. In summary, our findings provide an insight into future clinical applications of the VPA-BOR combination regimen for AML, especially in those cases which are resistant to conventional chemotherapy.

Topics: Acute Disease; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Proliferation; Cell Survival; Cyclin D1; Drug Synergism; Gene Expression; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Inhibitory Concentration 50; Leukemia, Myeloid; Proteasome Inhibitors; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Telomerase; Valproic Acid

With the aim of determining the differential expression of WNT and FZD genes, before and after induction of apoptosis in BCR-ABL positive cells, we treated the myeloid cell line K562 and control cell line HL60 with imatinib mesylate and etoposide, and analyzed relative mRNA expression levels of WNT, FZD and sFRP genes under normal and apoptotic conditions by real-time RT-PCR. We observed marked increase in mRNA levels of FZD4, FZD5, FZD7 and WNT5b, correlating with apoptotic activity and independent of the agent or cell line used. Our results suggest the involvement of non-canonical Wnt signaling in executing programmed cell death in myeloid cell lines.

Topics: Antineoplastic Agents; Benzamides; Caspase 3; Cell Line, Tumor; Cyclin D1; Etoposide; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Piperazines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Wnt Proteins

Treatment of human promyelocytic leukemia cell HL60 with 12-o-tetradecanoylphorbol 13-acetate (TPA) induces growth arrest, differentiation towards the monocyte/macrophage lineage, and expression of cell cycle-regulating genes cyclin D1 and p21Waf1. First, we demonstrated that p21Waf1 expression was increased by TPA in other leukemia cell lines also, including THP-1, U937, and KG-1, which differentiate into monocytes/macrophages by TPA. Secondly, we demonstrated the signal transduction pathways of cyclin D1 and p21Waf1 expressions in TPA-treated HL60 cells. Induction of cyclin D1 expression in TPA-treated HL60 cells was inhibited with protein kinase C (PKC) inhibitor bisindolylmaleimide I and mitogen activated protein kinase kinase (MEK) inhibitor PD98059. Induction of p21Waf1 expression in TPA-treated HL60 cells was inhibited with PKC inhibitor bisindolylmaleimide I and Gö6976, MEK inhibitor PD98059, and p38 mitogen-actibated protein kinase (MAPK) inhibitor SB202190. Thus, cyclin D1 and p21Waf1 expressions are considered to be induced via PKC and extracellular signal-regulated kinase/mitogen-activated protein kinase (MAPK/ERK) pathways in TPA-treated HL60 cells. The upregulation of p21Waf1 seems to play a critical role in TPA-induced cell differentiation by suppressing cyclin dependent kinase activity , while the upregulation of cyclin D1 seems to be compensated by p21Waf1.

Topics: Cell Differentiation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cycloheximide; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; U937 Cells

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

Expression of cell cycle-regulating genes was studied in human myeloid leukemia cell lines ML-1, ML-2 and ML-3 during induction of differentiation in vitro. Myelomonocytic differentiation was induced by phorbol ester (12-o-Tetradecanoyl-phorbol-13-acetate, TPA), tumor necrosis factor alpha (TNFalpha) or interferon gamma (INFgamma), or their combination. Differentiation (with the exception of TNFalpha alone) was accompanied by inhibition of DNA synthesis and cell cycle arrest. Inhibition of proliferation was associated with a decrease in the expression of cdc25A and cdc25B, cdk6 and Ki-67 genes, and with increased p21(Waf1/Cip1) gene expression, as measured by comparative RT-PCR. Expression of the following genes was not changed after induction of differentiation: cyclin A1, cyclin D3, cyclin E1 and p27(Kip1). Surprisingly, cyclin D1 expression was upregulated after induction by TPA, TNFalpha with IFNgamma or BA. Cyclin D2 was upregulated only after induction by BA. The results of the expression of the tested genes obtained by comparative RT-PCR were confirmed by quantitative real-time (RQ) RT-PCR and Western blotting. Quantitative RT-PCR showed as much as a 288-fold increase of cyclin D1 specific mRNA after a 24h induction by TPA. The upregulation of cyclin D1 in differentiating cells seems to be compensated by the upregulation of p21(Waf1/Cip1). These results, besides others, point to a strong correlation between the expression of cyclin D1 and p21(Waf1/Cip1) on the one hand and differentiation on the other hand in human myeloid leukemic cells and reflect a rather complicated network regulating proliferation and differentiation of leukemic cells.

Topics: Acute Disease; Antineoplastic Agents; Blotting, Western; Carcinogens; Cell Cycle; Cell Differentiation; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Primers; Humans; Interferon-gamma; Leukemia, Myeloid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Wnt5a is a member of the Wnt family of secreted glycoproteins that play essential organizing roles in development. Similar to other Wnt members, Wnt5a can upregulate cell proliferation and has been proposed to have oncogenic function. Here we report that Wnt5a signals through the noncanonical Wnt/Ca++ pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation in a cell-autonomous manner. Wnt5a hemizygous mice develop myeloid leukemias and B cell lymphomas that are clonal in origin and display loss of Wnt5a function in tumor tissues. Furthermore, analysis of human primary leukemias reveals deletion of the WNT5A gene and/or loss of WNT5A expression in a majority of the patient samples. These results demonstrate that Wnt5a suppresses hematopoietic malignancies.

Topics: Animals; B-Lymphocytes; Calcium; Cell Division; Cells, Cultured; Cyclin D1; Flow Cytometry; Hematopoietic System; Humans; Interleukin-7; Leukemia, Myeloid; Loss of Heterozygosity; Lymphoid Tissue; Lymphoma, B-Cell; Mice; Mice, Knockout; Proto-Oncogene Proteins; Signal Transduction; Transplantation, Heterologous; Wnt Proteins; Wnt-5a Protein

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.

Topics: Acute Disease; Burkitt Lymphoma; Carrier Proteins; Cell Cycle Proteins; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; DNA, Neoplasm; Gene Expression Regulation, Leukemic; Genes, p16; Genes, Retinoblastoma; Humans; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Myeloid; Loss of Heterozygosity; Molecular Probe Techniques; Neoplasm Proteins; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Retinoblastoma Protein; Sequence Deletion; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19(INK4D) expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19(INK4D) induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2. (Blood. 2000;95:1264-1273)

Topics: Animals; Apoptosis; Cell Cycle; Cell Differentiation; Clone Cells; Cyclin D1; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Humans; Interleukin-4; Interleukin-6; Leukemia, Experimental; Leukemia, Myeloid; Macrophages; Mice; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Recombinant Proteins; Transcription Factors; Transfection; Tumor Cells, Cultured

We previously reported that injection of recombinant granulocyte colony-stimulating factor (G-CSF) suppressed the development of leukemia in mice transplanted with C2M-A5 (C2M) myeloid leukemia cells and that the anti-leukemic effect of G-CSF was ascribed to the induction of apoptosis of C2M cells. These observations make a striking contrast with other previous reports on the biological activities of G-CSF. In the present study, in order to further clarify the G-CSF-induced apoptosis of C2M cells, we studied the effects of G-CSF on the cell cycle as well as the molecular events involving D-type cyclines and their cyclin-dependent kinases (cdk) in G-CSF-treated C2M cells. Cell cycle analysis revealed that G-CSF treatment of C2M cells resulted in accelerated entry from the first gap (G1) phase into the DNA synthesis (S) phase. Western blotting disclosed that G-CSF treatment resulted in down-regulation of cyclin D2 and cdk2 and up-regulation of cyclin D1 and cdk4. The reciprocal relationship between the up-regulation of cyclin D1 and down-regulation of cyclin D2 was closely associated with accelerated entry into S phase and subsequent apoptosis of C2M cells. These results suggest that G-CSF-induced apoptosis of C2M cells might be ascribed to imbalanced cell cycle progression due to deregulated expression of D-type cyclins and their cdks.

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; CHO Cells; Cricetinae; Cyclin D1; Cyclin-Dependent Kinases; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Mice; Mice, Inbred C3H; S Phase; Up-Regulation

To study the involvement of cyclins in cell-cycle progression, changes of mRNA levels for three G1 cyclins (cyclin C, D1 and E) and cyclin A were studied in a leukemia cell line, HEL cells, before and after incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, the cyclin D1 mRNA level markedly increased in the HEL cells when the cells were growth-arrested by TPA, while the amounts of cyclin E and A mRNAs decreased to an almost undetectable level in HEL cells after incubation with TPA. The similar marked increment of the cyclin D1 mRNA level was observed in other leukemia cell lines, CMK and HL-60 cells, after incubation with TPA. The cyclin C mRNA was not detected in HEL cells before and after incubation with TPA.

Topics: Base Sequence; Blotting, Northern; Cell Cycle; Cell Division; Cloning, Molecular; Cyclin D1; Cyclins; Dimethyl Sulfoxide; Esophageal Neoplasms; Humans; Kinetics; Leukemia, Myeloid; Molecular Sequence Data; Molecular Weight; Oligonucleotide Probes; Oncogene Proteins; Open Reading Frames; Polymerase Chain Reaction; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured