cyclin-d1 and herbimycin

Bcr-Abl tyrosine kinase inhibitor induces apoptosis and erythroid differentiation of K562 cells. During this erythroid differentiation, c-Myc and cyclin D1 transcripts are transiently downregulated. Accordingly, we studied the effect of cyclin D1 overexpression on erythroid differentiation. After treatment with 250 nM STI571, 90% of K562 and 25% of K562/D1 cells underwent erythroid differentiation. The basal expression of glycophorin A in K562/D1 cells was markedly diminished compared with that by parental cells. STI571 treatment failed to induce glycophorin A expression in K562/D1 cells. During STI571 treatment, ERK activity was downregulated in parental cells, while it was constantly activated in K562/D1 cells. These results suggest that ectopic expression of cyclin D1 causes the resistance of K562 cells to erythroid differentiation by modulating ERK regulation.

Topics: Benzamides; Benzoquinones; Cell Differentiation; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Erythroid Precursor Cells; Glycophorins; Humans; Imatinib Mesylate; K562 Cells; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Quinones; Rifabutin; Signal Transduction; Transfection

The ansamycin antibiotic herbimycin A is a potent tyrosine kinase inhibitor and reduces the growth rate of various types of mammalian cells. When quiescent Rat6 fibroblast cells were treated with herbimycin A, serum-induced expression of cyclin D1 was inhibited, and this was associated with inhibition of G1 phase progression. However, herbimycin A also inhibited serum-induced G1 progression in derivatives of the Rat6 fibroblast cell line that stably overexpress a human cyclin D1 cDNA (R6ccnD1#4 cells), without affecting the expression levels of G1 cyclins. We found that herbimycin A prevented serum-induced downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), thereby leading to inactivation of the protein kinase activity of CDK2. These results suggest that herbimycin A inhibits a tyrosine kinase(s) that plays a role in degradation of the p27(Kop1) protein.

Topics: Animals; Benzoquinones; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Line; Culture Media, Serum-Free; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; Fibroblasts; G1 Phase; Gene Expression Regulation; Humans; Lactams, Macrocyclic; Microtubule-Associated Proteins; Protein Serine-Threonine Kinases; Quinones; Rats; Recombinant Proteins; Rifabutin; S Phase; Transfection; Tumor Suppressor Proteins

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

Topics: Benzoquinones; Catalytic Domain; Cyclin D1; DNA-Binding Proteins; Enzyme Induction; Gene Expression Regulation, Leukemic; Genes, myc; Humans; K562 Cells; Lactams, Macrocyclic; Neoplasm Proteins; Proto-Oncogene Proteins c-myc; Quinones; Recombinant Fusion Proteins; Rifabutin; RNA; RNA, Messenger; RNA, Neoplasm; Telomerase; Transfection

The ansamycin antibiotics, herbimycin A (HA) and geldanamycin (GM), bind to a conserved pocket in heat shock protein 90 (Hsp90) and alter the function of this chaperone protein. Occupancy of this pocket results in the degradation of a subset of signaling molecules. These include proteins known to associate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family. In a variety of tumor cell lines, treatment with HA potently inhibited cellular proliferation by inducing G1 arrest. This arrest was accompanied by hypophosphorylation of the retinoblastoma gene product (RB) and rapid down-regulation of cyclin D- and E-associated kinase activities. Inhibition of kinase activity was found to result from loss in expression of cyclins D1, D3, and E, as well as the associated cyclin-dependent kinases, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, HA treatment also caused a late induction of p27(Kip1) protein. The loss of cyclin D preceded the other effects of HA, suggesting that it might be the primary cause of G1 arrest. To determine whether the effects of HA are mediated by selective inhibition of the cyclin D-RB pathway, HA was added to tumor cell lines lacking functional RB. HA treatment of Rb-negative tumor cell lines failed to elicit a G1 arrest. In addition, after release from synchronization with nocodazole, Rb-negative but not Rb-positive cell lines were able to progress through G1 into S phase in the presence of HA. Together, these findings suggest that induction of G1 arrest by HA results from down-regulation of cyclin D expression and its associated kinase activity. Furthermore, these findings imply that Hsp90 selectively regulates signaling pathways upstream of RB.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Blotting, Western; Breast Neoplasms; Cell Cycle Proteins; Cell Division; Colonic Neoplasms; Cyclin A; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Flow Cytometry; G1 Phase; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Microtubule-Associated Proteins; Mutation; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Quinones; Retinoblastoma Protein; Rifabutin; Signal Transduction; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.

Topics: Animals; Benzoquinones; Butadienes; Cells, Cultured; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Estrogens; Female; Flavonoids; Gene Expression Regulation; Lactams, Macrocyclic; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Pituitary Gland; Prolactin; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; Pyrazoles; Pyrimidines; Quinones; Rats; Rats, Inbred F344; Rifabutin; RNA, Messenger; Signal Transduction; src-Family Kinases; Steroids; Transcription, Genetic; Tyrosine

The ansamycin antibiotic, herbimycin A, is a potent tyrosine kinase inhibitor, and induces the erythroid differentiation of bcr-abl-possessing K562 cells. The growth of K562 cells was cytostatically reduced to less than 50% of the control level at 48 h by 0.5 microgram/ml of herbimycin A treatment. A total of 12% and 53% of the treated cells were benzidine-positive at 24 h and 48 h, respectively. The percentage of cells in the S phase decreased rapidly from 60% to 15% after 12 h of treatment. The reduction of S phase cells persisted until 24 h, whereas the G1 population conversely increased. Then underphosphorylated retinoblastoma gene product increased from 6 h to 24 h, but returned to baseline at 48 h. Most cell cycle controlling genes were unchanged by herbimycin A treatment. However, both cyclin D1 and c-myc were prominently down-regulated in the early phase of treatment, corresponding to the decline of the S phase population. Cyclin D1 was initially down-regulated to an undetectable level at 6 h, although its expression recovered gradually from 12 h and returned to baseline at 24 h. c-myc was also down-regulated from 1 h to 6 h. These data suggest that signals originating from bcr-abl kinase are at least partly transduced through both c-myc and cyclin D1, and that herbimycin A-induced erythroid differentiation occurs during or after the cessation of growth due to interference with these signals.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Cell Differentiation; Cyclin D1; Cyclins; Down-Regulation; Erythroid Precursor Cells; Humans; Lactams, Macrocyclic; Neoplasm Proteins; Oncogene Proteins; Proto-Oncogene Proteins c-myc; Quinones; Rifabutin; RNA, Messenger; Tumor Cells, Cultured

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