The hematopoietic transcription factor AML1 (RUNX1) is negatively regulated by the cell cycle protein cyclin D3

The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFbeta for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation.     

Low-dose triptolide in combination with idarubicin induces apoptosis in AML leukemic stem-like KG1a cell line by modulation of the intrinsic and extrinsic factors

Leukemia stem cells (LSCs) are considered to be the main reason for relapse and are also regarded as a major hurdle for the success of acute myeloid leukemia chemotherapy. Thus, new drugs targeting LSCs are urgently needed. Triptolide (TPL) is cytotoxic to LSCs. Low dose of TPL enhances the cytotoxicity of idarubicin (IDA) in LSCs. In this study, the ability of TPL to induce apoptosis in leukemic stem cell (LSC)-like cells derived from acute myeloid leukemia cell line KG1a was investigated. LSC-like cells sorted from KG1a were subjected to cell cycle analysis and different treatments, and then followed by in vitro methyl thiazole tetrazolium bromide cytotoxicity assay. The effects of different drug combinations on cell viability, intracellular reactive-oxygen species (ROS) activity, colony-forming ability and apoptotic status were also examined. Combination index-isobologram analysis indicates a synergistic effect between TPL and IDA, which inhibits the colony-forming ability of LSC-like cells and induces their apoptosis. We further investigated the expression of Nrf2, HIF-1α and their downstream target genes. LSC-like cells treated with both TPL and IDA have increased levels of ROS, decreased expression of Nrf2 and HIF-1α pathways. Our findings indicate that the synergistic cytotoxicity of TPL and IDA in LSCs-like cells may attribute to both induction of ROS and inhibition of the Nrf2 and HIF-1α pathways.     

The homeodomain transcription factor Prep1 (pKnox1) is required for hematopoietic stem and progenitor cell activity

Most of the hypomorphic Prep1^i/i embryos (expressing 3-10% of the Prep1 protein), die between E17.5 and P0, with profound anemia, eye malformations and angiogenic anomalies [Ferretti, E., Villaescusa, J.C., Di Rosa, P., Fernandez-Diaz, L.-C., Longobardi, E., Mazzieri, R., Miccio, A., Micali, N., Selleri, L., Ferrari G., Blasi, F. (2006). Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype. Mol. Cell. Biol. 26, 5650-5662]. We now report on the hematopoietic phenotype of these embryos. Prep1^i/i fetal livers (FL) are hypoplastic, produce less common myeloid progenitors colonies (CFU-GEMM) in cytokine-supplemented methylcellulose and have an increased number of B-cells precursors that differentiate poorly. Prep1^i/i FL is able to protect lethally irradiated mice only at high cell doses but the few protected mice show major anomalies in all hematopoietic lineages in both bone marrow (BM) and peripheral organs. Prep1^i/i FL cells compete inefficiently with wild type bone marrow in competitive repopulation experiments, suggesting that the major defect lies in long-term repopulating hematopoietic stem cells (LTR-HSC). Indeed, wt embryonic expression of Prep1 in the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), cKit⁺Sca1⁺Lin⁻AA4.1⁺ (KSLA) cells and B-lymphocytes precursors agrees with the observed phenotype. We therefore conclude that Prep1 is required for a correct and complete hematopoiesis.     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.