1. Impaired Ferritin mRNA Translation in Primary Erythroid Progenitors: Shift to Iron-Dependent Regulation by the v-ErbA Oncoprotein
by Wolfgang Mikulits, Matthias Schranzhofer, Anton Bauer, Helmut Dolznig, Lioba Lobmayr, Anthony A. Infante, Hartmut Beug, and Ernst W. Müllner
Blood
Volume 94(12):
December 15, 1999
©1999 by American Society of Hematology

2. Iron-dependent translational control of ferH and eALAS mRNA in LMH-2A hepatoma cells and AEV-transformed ebls (HD3E22).
Iron-dependent translational control of ferH and eALAS mRNA in LMH-2A hepatoma cells and AEV-transformed ebls (HD3E22). Cells were incubated with the iron chelator desferrioxamine (DES; 50 μmol/L) or iron-loaded transferrin (TF; 1 mg/mL iron-saturated chicken ovotransferrin) for 24 hours before harvesting. (A) Cytoplasmic extracts were separated in linear 15% to 40% sucrose gradients47 and the RNA was isolated from 18 fractions analyzed by Northern blotting. Fraction 1 corresponds to the top and fraction 18 to the bottom of the gradient. The filters were hybridized with 32P-labeled probes specific for chicken ferH and, in case of HD3E22 ebls, subsequently with a chicken eALAS cDNA.49 The lowest panel displays the profile obtained by staining of the RNA with methylene blue (mb). The emergence of a constant molar ratio between 28S and 18S RNA (upper and lower band, respectively) around fraction 9 indicates the assembly of 80S initiation complexes and marks the boundary between the ribosome-free and polyribosome-bound compartment. Signals between gradients from the same cell type are directly comparable, because (1) the same aliquot of RNA from each fraction was blotted, (2) both filters were hybridized with the very same probe, and (3) exposed for the same time. Thus, the sum of signals over all the fractions corresponds to total cytoplasmic mRNA levels. (B) Quantitative analysis of hybridization signals from Northern blotting by laser densitometry. (Open symbols) Cells depleted of iron by Des; (solid symbols) cells under high Tf. To facilitate comparison between different experiments, the RNA content of each fraction was expressed as a percentage of the total amount of RNA contained in the gradient. (C) Ferritin protein expression in HD3E22, LMH-2A, and HeLa cells was determined by Western blotting as described in Materials and Methods. Numbers below each pair of lanes represent the factors of iron-dependent upregulation of ferritin expression (fold induction).
Wolfgang Mikulits et al. Blood 1999;94:
©1999 by American Society of Hematology

3. Iron-independent reduction of ferH mRNA translation in proliferating and differentiating primary ebls.
Iron-independent reduction of ferH mRNA translation in proliferating and differentiating primary ebls. The SCF-dependent erythroid progenitors were grown from chicken bone marrow as described.25 Maturation was induced by removal of self-renewal factors (SCF and IGF-1) and addition of avian Epo (high-titer anemic serum) and insulin to the medium (see Materials and Methods). Self-renewing SCF-ebls and cells induced to differentiate for 48 or 96 hours were treated with low (0.03 mg/mL) or high levels of iron-saturated Tf (1 mg/mL) or high ferric ammonium citrate concentrations (FeCit; 50 μg/mL, lowest panel at 48 hours) for 24 hours. Sucrose gradient analysis and densitometrical quantitation were performed as described in the legend to Fig 1. (○) ferH mRNA; (•) eALAS mRNA.
Wolfgang Mikulits et al. Blood 1999;94:
©1999 by American Society of Hematology

4. Retroviral expression of v-ErbA or v-ErbB in primary ebls and the consequences for ferritin biosynthesis.
Retroviral expression of v-ErbA or v-ErbB in primary ebls and the consequences for ferritin biosynthesis. Bone marrow-derived, primary ebls expressing either the v-ErbA or the v-ErbB oncoprotein (see Materials and Methods) were kept for 24 hours under self-renewing conditions in the presence of 50 μmol/L Des to achieve iron depletion or were incubated with 1 mg/mL Tf to induce full iron saturation. (A) Distribution of ferH mRNA as determined by sucrose gradient analysis (for details, see legend to Fig 1). (Insets) The ribosome-bound mRNA compartment (fractions 9-18) is shown with an extended ordinate to highlight the difference between v-ErbA and v-ErbB expressing cells. (Open symbols) Des; (solid symbols) Tf. (B) Ferritin protein levels as detected by Western blotting in self-renewing primary ebls (SCF), v-ErbA– or v-ErbB–expressing ebls (v-ErbA, v-ErbB), and AEV-transformed red blood cells (HD3E22) under iron scarcity (Des) or high iron abundance (Tf). In case of SCF-progenitors, low Tf (0.03 mg/mL) instead of Des was used to induce IRP-activity. Numbers below each pair of lanes represent the factors of iron-dependent upregulation of ferritin expression (fold induction).
Wolfgang Mikulits et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Iron-dependent modulation of IRP-binding activity to the ferH-IRE in primary versus transformed ebls.
Iron-dependent modulation of IRP-binding activity to the ferH-IRE in primary versus transformed ebls. (A) IRP-IRE-binding activity was determined in vitro by a gel retardation assay as outlined in Materials and Methods. Aliquots of the cell extracts from self-renewing ebls used for immunoblotting (see Fig 3) were incubated with an excess of radiolabeled ferH-IRE transcripts and the resulting RNA-protein complexes were separated in nondenaturing polyacrylamide gels. Where indicated, maximal IRP1-IRE binding activity was estimated by treatment of the extracts with 2-mercaptoethanol (2-ME) in vitro. The amount of radioactivity in the IRP-IRE complexes was quantified by phosphoimaging. Numbers below the pair of corresponding lanes represent the fold induction of IRP activity. (B) Levels of IRP1 (left panel) and IRP2 (right panel) in transformed versus primary ebls cultivated under different iron supply, as detected by immunoblotting. (C) IRP1 versus IRP2 binding activity to the ferH-IRE in extracts of transformed and primary ebls treated for 24 hours with Des. Gel retardation assays in combination with antibody-mediated supershifts were performed as described in detail under Materials and Methods. As a control (no extract), lysis buffer was incubated with 3 μL antibody and the radioactive ferH-IRE probe. -IRP1 and -IRP2, IRP1 and IRP2 specific antisera, respectively; IgG, nonspecific antibody.
Wolfgang Mikulits et al. Blood 1999;94:
©1999 by American Society of Hematology

6. Functional inactivation of the v-ErbA oncoprotein switches AEV-transformed ebls from iron-dependent to iron-independent ferritin expression.
Functional inactivation of the v-ErbA oncoprotein switches AEV-transformed ebls from iron-dependent to iron-independent ferritin expression. AEV-transformed ebls (HD3E22) were cultivated in Epotest medium either under proliferation conditions (IGF-1) or induced to terminal differentiation by supplementation with 2 U/mL human Epo, 1.4 nmol/L insulin, 1 μmol/L each of the steroid antagonists ICI and ZK , the ErbB inhibitor PD , and the c-Kit antagonist EXBW 50 for up to 96 hours (see Materials and Methods). Twenty-four hours before harvesting for Western blot analysis, ebls were switched to identical media containing either Des (50 μmol/L) or Tf (1 mg/mL). Numbers below each pair of lanes represent the fold induction of iron-dependent ferritin expression.
Wolfgang Mikulits et al. Blood 1999;94:
©1999 by American Society of Hematology