Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

The immediately-early response gene 5 (IER5) has been reported to be induced by γ-ray irradiation and to play a role in the induction of cell death caused by radiation. We previously identified IER5 as one of the 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP)-induced transcriptional responses in AML cells, using microarrays that encompassed the entire human genome. However, the biochemical pathway and mechanisms of IER5 function in regulation of the cell cycle remain unclear. In this study, we investigated the involvement of IER5 in the cell cycle and in cell proliferation of acute myeloid leukemia (AML) cells. We found that the over-expression of IER5 in AML cell lines and in AML-derived ALDHhi (High Aldehyde Dehydrogenase activity)/CD34+ cells inhibited their proliferation compared to control cells, through induction of G2/M cell cycle arrest and a decrease in Cdc25B expression. Moreover, the over-expression of IER5 reduced colony formation of AML-derived ALDHhi/CD34+ cells due to a decrease in Cdc25B expression. In addition, over-expression of Cdc25B restored TMPP inhibitory effects on colony formation in IER5-suppressed AML-derived ALDHhi/CD34+ cells. Furthermore, the IER5 reduced Cdc25B mRNA expression through direct binding to Cdc25B promoter and mediated its transcriptional attenuation through NF-YB and p300 transcriptinal factors. In summary, we found that transcriptional repression mediated by IER5 regulates Cdc25B expression levels via the release of NF-YB and p300 in AML-derived ALDHhi/CD34+ cells, resulting in inhibition of AML progenitor cell proliferation through modulation of cell cycle. Thus, the induction of IER5 expression represents an attractive target for AML therapy

Intranasal oxytocin suppresses seizure-like behaviors in a mouse model of NGLY1 deficiency

Abstract NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1 −/− mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1 −/− mice. Seizure-like behaviors in Ngly1 −/− mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism

Cost-effectiveness analysis 3L of axicabtagene ciloleucel versus tisagenlecleucel and lisocabtagene maraleucel in Japan - Supplementary materials

Supplemental Table 1. Costs related to SCT using databaseSupplemental Figure 1. Tornado plots of parameter impact on incremental costsSupplemental Figure 2. Tornado plots of parameter impact on incremental QALYs</p

Over-expression of IER5 inhibited AML cell proliferation.

<p>A. The cell proliferation of U937 cells, transfected with IER5 cDNA or treated with TMPP, was measured by counting cells using a hemocytometer (upper panel). Cell counting was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. *<i>P</i><0.01 compared with untransfected control cells. Cell viability of the IER5 over-expressing U937 cells was assessed by counting of viable cells using trypan blue staining at 72 h, starting 3 days after DNA transfection (bottom panel). B. QRT-PCR analysis of <i>IER5</i> mRNA expression in untreated cells, <i>IER5</i> cDNA-transfected cells, and TMPP-treated U937 cells. QRT-PCR was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. The levels of the quantified RT-PCR products were normalized t<i>o GAPDH</i> expression in the same sample and were then expressed relative to the mRNA level of a normal control, which was assigned a value of 1. *<i>P</i><0.01 compared with untransfected control cells. The protein expression of IER5 in cells was analyzed after 3 days of culture (bottom panels). Blotting of Actin was used as a loading control. C. The cell cycle distribution of U937 cells that were transfected with control DNA, <i>IER5</i> cDNA, scrambled shRNA, IER5 shRNA #1, IER5 shRNA #1, or were treated with TMPP, was analyzed using flow cytometric analysis. The transfected or TMPP-treated U937 cells were harvested after 3 days. The fraction of cells in the G1, S and G2/M stage of the cell cycle was determined. Data are shown as means ± S.D. of triplicate cultures. The <i>IER5</i> mRNA expression of the cells is shown at bottom and was assessed using RT-PCR. The RT-PCR results are representative of three independent experiments. Data are shown as means ± S.D. of triplicate cultures. *<i>P</i><0.01 compared with control cells. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML cells. U937 cells were transfected with <i>IER5</i> cDNA or treated with TMPP (5 µM). Mitochondrial membrane potential was determined 3 days after transfection or TMPP treatment by staining of the cells with DiOC6 followed by flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.</p

IER5 inhibits the colony formation of ALDH^hi/CD34⁺ cells isolated from AML patients.

<p>A. Selection of ALDH^hi/CD34⁺ hematopoietic progenitor cells from the bone marrow of two healthy volunteers (#1 and #2) by FACS sorting. Region P denotes populations of ALDH^hi cells. Region R and S denote populations of CD34⁺ and CD34^- cells in the ALDH^hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. B. Selection of ALDH^hi/CD34⁺ hematopoietic progenitor cells from the bone marrow of two AML patients (M1 and M2) by FACS sorting. Region P denotes populations of ALDH^hi cells. Region R and S denote populations CD34⁺ and CD34^- cells in the ALDH^hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. C. ALDH^hi/CD34⁺ cells were purified from a healthy volunteer (#1) and an AML patient (M1), and were cultured in semisolid methylcellulose media. The ALDH^hi/CD34⁺ cells from each source were left untransfected or were transfected with <i>IER5</i> cDNA, or treated with TMPP (5 µM). After 14 days culture, the colony forming ability of the cells was analyzed (left upper panel) and the cells were viewed using phase-contrast microscopy. Original magnification ×4 (left bottom panels). Their mRNA expression of <i>IER5</i> and <i>Cdc25B</i> was assessed using RT-PCR and quantitative RT-PCR (right panels). Colonies formed by these ALDH^hi/CD34⁺ cells (3×10² to 5×10² cells/plate) were counted following plating in semisolid methylcellulose media. Colony formation was evaluated by determination of colony counts as a percentage of the corresponding control. The results are the means ± SD of three independent experiments. *<i>P</i><0.01 compared with untreated control cells. RT-PCR results representative of three independent experiments are shown. <i>GAPDH</i> mRNA expression is shown as an internal control. The ALDH^hi/CD34⁺ cells whose <i>IER5</i> mRNA expression was analyzed by quantitative RT-PCR were derived from an AML patient (M1). The levels of the quantified RT-PCR products were normalized t<i>o GAPDH</i> expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML-derived ALDH^hi/CD34⁺ cells. ALDH^hi/CD34⁺ cells were purified from an AML patient (M1), and were then transfected with <i>IER5</i> cDNA or were treated with TMPP (5 µM). The IER5-transfected or TMPP-treated cells were harvested after 3 days. The cell cycle distribution and the ΔΨm of the ALDH^hi/CD34⁺ cells was analyzed using flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.</p

IER5 expression inhibited the colony formation of AML ALDH^hi/CD34⁺ cells through the regulation of Cdc25B expression.

<p>A. ALDH^hi/CD34⁺ cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH^hi/CD34⁺ cells were either untransfected or were transfected with <i>IER5</i> cDNA or with shRNA-#1, or -#2. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH^hi/CD34⁺ cells (3×10² to 5×10² cells/plate) was assessed after plating in semisolid methylcellulose media. B. Analysis of the mRNA expression of <i>IER5</i> and <i>Cdc25B</i> in each colony using QRT-PCR and RT-PCR. C. The cells were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4. D. ALDH^hi/CD34⁺ cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH^hi/CD34⁺ cells were either untransfected or were transfected with <i>IER5</i> cDNA or <i>Cdc25B</i> cDNA. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH^hi/CD34⁺ cells (3×10² to 5×10² cells/plate) was assessed after plating in semisolid methylcellulose media. Colony formation was evaluated as a percentage of the corresponding control. E. Analysis of the mRNA expression of <i>IER5</i> and <i>Cdc25B</i> in each colony using QRT-PCR and RT-PCR. The levels of the QRT-PCR products were normalized t<i>o GAPDH</i> expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. RT-PCR results representative of three independent experiments are shown. <i>GAPDH</i> mRNA expression is shown as an internal control. The results are the means ± SD of three independent experiments. *<i>P</i><0.01 compared with untreated control cells. F. The cells transfected with <i>Cdc25B</i> or <i>IER5</i> cDNA were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4.</p

IER5 induced G2/M cell cycle arrest through direct binding to Cdc25B promoter in AML cells.

<p>A. U937 cells were transfected with control DNA, <i>IER5</i> cDNA, or were treated with TMPP (5 µM or 10 µM). The cells were harvested after 3 days, and the expression of the indicated cell cycle regulating proteins was analyzed by SDS-PAGE followed by Western blotting using the indicated specific antibodies. Blotting of Actin was used as a loading control. B. Schematic representation of the human <i>Cdc25B</i> promoter regions, indicating sites for Sp1, Sp3, and NF-YB motifs, amplified from the precipitated DNA by specific primer sets 1 and 2. C. ChIP analysis for IER5 in the <i>Cdc25B</i> promoter. D & E. IER5-dependent changes in Sp1, Sp3 and NF-YB binding (E), and the bindings of coactivators, DNMT1 and p300, to interact with Sp1 and NF-YB, respectively (F) in <i>Cdc25B</i> promoter. U937 cells were either untransfected or were transfected with the control DNA or <i>IER5</i> DNA. After 3 days culture, cells were cross-linked with 1% formaldehyde and ChIP was performed with either control antibody (IgG) or the anti-IER5 antibody. The precipitated DNA was then assayed by PCR using pairs (primer set 1 and 2) of oligonucleotides encompassing specific regions of the <i>Cdc25B</i> promoter. The values of ChIP efficiencies are given as % of input.</p