gamma 375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum

Background: Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous gamma-chain variant fibrinogen in the C terminal region. Of interest, substitution of gamma R375W induced hypofibrinogenemia and HERSD, whereas gamma R375G induced dysfibrinogenemia. Objectives: To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells. Methods: The fibrinogen gamma-chain expression vectors coding gamma 375W and gamma 375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy. Results: The medium/cell lysate fibrinogen ratio of gamma 375W-CHO cells was markedly lower than that of the normal cells and gamma 375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only gamma 375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER. Conclusion: These results demonstrated that assembled and non-secreted gamma 375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients' hepatocytes affected with HERSD.ArticleTHROMBOSIS RESEARCH. 133(1):101-107 (2014)journal articl

E13.5 pregnant wild-type mice were IP injected with saline or etoposide (0.5 mg/kg) on 3 consecutive days, and FL-HSCs were harvested for expression analysis 24 h after the final injection (E16.5).

<p>(A) Number of chimeric mRNAs detected by RNA-seq in <i>Atm</i>^-/- mice and wild-type littermates. Numbers in parenthesis indicate in-frame fusion mRNAs. (B) Heat map of selected genes associated with signal transduction, enriched using the DAVID software. Cont. DMSO; treated, ETO; etoposide-treated. Red indicates upregulated and green indicates downregulated expression in color ramp for heatmap</p

Dysregulation of the DNA Damage Response and <i>KMT2A</i> Rearrangement in Fetal Liver Hematopoietic Cells

<p>(A) Dose-dependence of γH2AX positivity in fetal liver hematopoietic stem cells (FL-HSCs), analyzed 4 h after IP injection of etoposide at the indicated doses. Etoposide was IP injected into E13.5 pregnant mice. (B) DNA double-strand breaks were detected according to γH2AX positivity, and cell-cycle distribution was monitored by propidium iodide (PI) incorporation. Etoposide (10 mg/kg) was IP injected into E13.5 pregnant mice, and samples were analyzed at the indicated time points. A two-dimensional dot blot is shown. FL-HSC: fetal liver hematopoietic stem cells; mBM: maternal bone marrow mono nuclear cells. (C) The kinetics of γH2AX positivity in the samples shown in B are expressed as a line graph. Bold line indicates FL-HSC, and broken line indicates mBM. (D) Percentage of apoptotic cells in the samples shown in B.</p

FL-HSCs of <i>Atm</i>-knockout mice were pulse-treated with etoposide for 4 h <i>in vitro</i>; metaphase spreads were generated 24 h after pulse treatment.

<p>(A) Representative metaphase spreads. Arrows indicate chromosomal or chromatid breaks. (B) Number of chromosomal breaks and chromatid breaks, shown as a table. (C) Sequence electropherogram of <i>Kmt2a</i>-<i>Ptp4a2</i> fusion mRNA.</p

Fetal etoposide concentration after intraperitoneal (IP) etoposide injection.

<p>Etoposide (10 mg/kg) was IP injected into E13.5 pregnant mice, and fetal livers were collected at the indicated time points.</p