cyclin-d1 and Acute-Disease

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

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

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

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