Biomedical subjects

E W Müllner

Publications and source records attributed to E W Müllner.

27 records · Page 2Linked to original sources

PubMed1995

Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control.

Detailed knowledge is available about the molecular makeup of the cell cycle clock in dividing cells. However, comparatively little is known about cell cycle regulation during terminal differentiation. Here we describe a primary cell system in which this question can be addressed. Normal avian erythroid progenitors undergo continuous self-renewal in suspension culture in the presence of growth factors and hormones, allowing us to obtain large cell numbers (10(10)-10(11)). By replacing these "self-renewal factors" with erythropoietin and insulin, the cells can be induced to synchronous, terminal differentiation. During the first 72 h, the cells undergo five cell divisions. Thereafter, they arrest in G1 and complete their maturation into RBC without further divisions. Sixteen to 24 h after induction of differentiation, the cell cycle length decreased from about 20 to 12 h. This shortened doubling time was due to a drastic reduction of G1 (from 12 to 5 h), while S- and G2-phase lengths were not affected. At the same time, the differentiating cells underwent an extensive and concerted switch in their gene expression pattern. During the subsequent four cell divisions, the cell volume decreased from about 300 to less than 70 femtoliters, but the rate of protein synthesis normalized to cell volume remained constant. Interestingly, the shortening of G1 was accompanied by a rapid down-regulation of D-type cyclins and their partner, cyclin-dependent kinase type 4 (cdk4), while expression of S- and G2-M-associated cell cycle regulators (cyclin A and cdk1/cdc2) remained high until the cells arrested in G1 72-96 h after differentiation induction. We conclude that concerted reprogramming of progenitor gene expression during erythroid differentiation is accompanied by profoundly altered cell cycle progression involving the loss or alteration of cell size control at the restriction point.

Animals↗

PubMed1994

Post-transcriptional repression of thymidine kinase expression during cell cycle and growth stimulation.

In vertebrates, endogenous thymidine kinase (TK) gene expression is strictly growth-dependent. Here we report that in continuously cycling Ltk-mouse fibroblasts, stably transfected with a vector expressing human TK cDNA from a constitutive promoter, enzyme activity rises 8-fold at the G1/S phase transition and declines again in G2. The mechanism did not involve changes in protein stability. When hTK was put under the control of a hormone-inducible promoter, production of high mRNA levels following addition of dexamethasone did not result in any enzyme activity in resting NIH-3T3tk- cells. After growth stimulation with serum, TK activity rose together with the onset of DNA synthesis only in the simultaneous presence of the hormone.

3T3 Cells↗

PubMed1994

Different regulation of thymidine kinase during the cell cycle of normal versus DNA tumor virus-transformed cells.

We compared the cell cycle regulation of thymidine kinase (TK) after centrifugal elutriation in normal human and mouse cells (primary cells, diploid fibroblasts) with its expression in cells transformed with different DNA tumor viruses. Normal cells showed a rise of TK enzyme activity near the G1/S boundary, which peaked in S phase, and in G2 returned approximately to the level of G1. Conversely, in cells derived from viral transformation, TK activity remained high throughout S and G2 phases, although it was induced to a comparable extent at the onset of DNA replication. In addition, transformed cells exhibited much more enzyme activity during all phases of the cell cycle. The observed differences in expression were due neither to different rates of protein turnover nor to differences in enzyme stability. Enzyme activity was always totally paralleled by the protein level. In all normal cells, the pattern of TK mRNA variation during the cell cycle was similar to that of enzyme activity. In all transformed lines, however, mRNA levels were higher and did not fluctuate throughout the cell cycle. Recently we showed (Ogris et al., 1993) that the E2F binding site present in the TK promoter is a target for trans activation of the TK gene by polyoma virus large T antigen. Using cells expressing this antigen under the control of a hormone-inducible promoter, we were able to switch TK cell cycle expression from the normal to the transformed status. Obviously, DNA tumor viruses suppress transcriptional down-regulation of the endogenous DNA precursor pathway enzyme TK during the eukaryotic cell cycle, maybe to improve conditions for their own replication.

Animals↗

PubMed1994

Insights into erythroid differentiation obtained from studies on avian erythroblastosis virus.

Analysis of the oncogenes v-erbB and v-erbA and their normal proto-oncogene counterparts has revealed several novel aspects of erythroid differentiation. A new erythroid progenitor capable of extended self-renewal has been described, tyrosine kinase receptors and steroid hormone receptors have been found to cooperate in controlling self-renewal, and dramatic alterations in the cell cycle have been found to accompany induction of terminal differentiation.

Alpharetrovirus↗

PubMed1993

Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells.

We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.

Animals↗

PubMed1992

In vivo and in vitro modulation of the mRNA-binding activity of iron-regulatory factor. Tissue distribution and effects of cell proliferation, iron levels and redox state.

The mRNA-binding protein, iron-regulatory factor (IRF) has a central role in iron metabolism. It coordinately increases transferrin-receptor mRNA stability and inhibits translation of ferritin and erythroid delta-aminolevulinate synthase mRNA by binding to specific mRNA structures, the iron-responsive elements (IRE). In gel-retardation assays, IRF had a broad tissue distribution, showing activity in cytosolic extracts from 12 mouse organs tested. In all these extracts, IRF could be further activated in vitro by 2-mercaptoethanol. In cultured mouse 3T6 fibroblasts, growth stimulation after low serum arrest increased IRF activity 10-fold, mainly through activation of existing inactive IRF. No change was observed during progression of 3T6 cells through the cell cycle. IRF activation by iron chelators has been postulated to result in the reduction of an intramolecular sulfhydryl group. In a search for redox conditions that regulate IRE binding of IRF, we studied several compounds in vitro or in vivo. Hemin, known to inactivate IRF in vivo, showed a similar, reversible effect in vitro, presumably by oxidizing IRF. However, this did not appear to be relevant for the mode of IRF regulation in vivo. Addition of protoporphyrin IX to intact cells induced IRF activity almost to the same extent as desferrioxamine. This effect was inhibited by iron salts, indicating that IRF is activated in vivo through depletion of a chelatable iron pool. In vitro activation by reductants other than 2-mercaptoethanol suggested some selectivity in their access to relevant sulfhydryl groups, but did not reveal which natural redox-sensitive compound might regulate IRF in vivo. However, in cultured cells, inactivation of free IRF by the sulfhydryl-specific oxidizing agent diamide was much more rapidly reversed than inactivation by iron salts. This indicates the direct involvement of a cellular reductant in setting IRF activity and suggests a rate-limiting IRF conformation that is reached only in the presence of iron, but not after diamide oxidation.

Animals↗

PubMed1990

The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution.

A post-transcriptional regulatory protein, termed iron regulatory factor (IRF), that binds specifically to the iron-responsive elements of ferritin and transferrin receptor mRNA, has recently been identified in the cytoplasm of human and mouse cells. Activation of this factor by low intracellular iron levels leads to inhibition of ferritin translation and an increase of TR mRNA stability. To investigate whether these feedback regulatory mechanisms are conserved during evolution, we analysed cytoplasmic extracts from 12 different species for a specific IRE-binding activity. We found mRNA-binding proteins in chicken, frog, fish and fly, which are equivalent to human and mouse IRF in gel-retardation assays with radiolabeled RNA transcripts. Competition experiments, molecular weight determinations, and modulation of the mRNA-binding activity in response to intracellular iron levels or reduction by beta-mercaptoethanol indicate that IRF has similar structural and functional properties in these different species.

Animals↗

PubMed1989

A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA.

Iron regulates human transferrin receptor (hTR) expression by modulating the stability of cytoplasmic hTR mRNA. This regulation requires a distinct secondary structure in the mRNA 3' untranslated region. We identified a specific cytoplasmic factor that binds simultaneously to four homologous palindromes within the regulatory domain. Iron chelator induced the RNA binding activity 25-fold in parallel with mRNA. Upon the addition of iron salts, a rapid decay of factor activity closely preceded hTR mRNA degradation, indicating a causal relation. Induction and decay occurred posttranscriptionally. Binding of the factor to hTR mRNA palindromes was competed by 5' regulatory sequences of ferritin mRNA, which are responsible for iron-dependent translational control. These results suggest that cellular iron maintains its homeostasis by coordinate regulation of hTR and ferritin expression via a common factor.

Animals↗

PubMed1988

A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm.

Expression of the human transferrin receptor (hTR) and its mRNA is strongly induced by iron deprivation. By measuring transcription elongation rates, levels of hTR-specific nuclear RNA, and mRNA half-lives, we found this regulation to occur posttranscriptionally in the cytoplasm. Analysis of hTR cDNA mutants with deletions in the 3' untranslated region revealed the existence of two distinct domains, both of which are essential for regulation in mouse L cells. The regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm. Expression of point and deletion mutants affecting the stem-loop confirmed the requirement for this secondary structure in regulation. The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene.

Base Sequence↗