Search

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Öhlinger, Müllner, Fritz, Werning, Baron-Stefaniak, Jungbauer, Baron and Salzer.)

Premature infants commonly receive adult packed red blood cells (pRBCs) during their hospital stay. As adult erythrocytes differ substantially from those of preterm infants, transfusion of adult pRBCs into preterm infants can be considered inappropriate for the physiology of a preterm infant. An absence of standardisation of transfusion protocols makes it difficult to compare and interpret pertinent clinical data, as reflected by unclear associations between pRBC transfusion and complications related to prematurity, such as bronchopulmonary dysplasia, neurodevelopmental impairment, retinopathy of prematurity, or necrotising enterocolitis. The difficulty in interpreting clinical data is further increased by differences in study designs that either overestimate pRBC-associated complications of prematurity or have not yet been designed to directly link pRBC transfusions to their respective complications. Thus, neonatal transfusion practice has become an ongoing difficulty, in which differences in transfusion guidelines hinder the ability to generate comparable clinical data, and heterogeneity in clinical data prevents the implementation of standardised transfusion protocols. To overcome these issues, novel approaches with biochemical-clinical translational designs could enable clinicians to gather causal evidence instead of circumstantial correlation., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography-mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.

Ascorbic acid (AA; or vitamin C) is an important physiological antioxidant and radical scavenger. Some mammalian species, including homo sapiens , have lost the ability to synthetize AA and depend on its nutritional uptake. Erythrocytes from AA-auxotroph mammals express high amounts of the glucose transporter GLUT1. This isoform enables rapid uptake of glucose as well as dehydroascorbate (DHA), the fully oxidized form of AA. Here, we explored the effects of DHA uptake on the redox metabolism of human erythrocytes. DHA uptake enhanced plasma membrane electron transport (PMET) activity. This process is mediated by DCytb, a membrane bound cytochrome catalyzing extracellular reduction of Fe 3+ and ascorbate free radical (AFR), the first oxidized form of AA. DHA uptake also decreased cellular radical oxygen species (ROS) levels. Both effects were massively enhanced in the presence of physiological glucose concentrations. Reduction of DHA to AA largely depleted intracellular glutathione (GSH) and induced the efflux of its oxidized form, GSSG. GSSG efflux could be inhibited by MK-571 ( IC 50 = 5 μM), indicating involvement of multidrug resistance associated protein (MRP1/4). DHA-dependent GSH depletion and GSSG efflux were completely rescued in the presence of 5 mM glucose and, partially, by 2-deoxy-glucose (2-DG), respectively. These findings indicate that human erythrocytes are physiologically adapted to recycle AA both intracellularly via GLUT1-mediated DHA uptake and reduction and extracellularly via DCytb-mediated AFR reduction. We discuss the possibility that this improved erythrocyte-mediated AA recycling was a prerequisite for the emergence of AA auxotrophy which independently occurred at least twice during mammalian evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Eigenschink, Savran, Zitterer, Granitzer, Fritz, Baron, Müllner and Salzer.)

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Pantothenate kinase 2-associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration., Methods: Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry-Albright Dystonia Scale (BAD-Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level., Results: Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12-56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations., Interpretation: Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient-derived cell system with still underestimated diagnostic potential., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Recent studies indicate that erythrocytes actively modulate blood clotting and thrombus formation. The lipid mediator lysophosphatidic acid (LPA) is produced by activated platelets, and triggers a signaling process in erythrocytes. This results in cellular calcium uptake and exposure of phosphatidylserine (PS) at the cell surface, thereby generating activated membrane binding sites for factors of the clotting cascade. Moreover, erythrocytes of patients with a bleeding disorder and mutations in the scramblase TMEM16F show impaired PS exposure and microvesiculation upon treatment with calcium ionophore. We report that TMEM16F inhibitors tannic acid (TA) and epigallocatechin-3-gallate (EGCG) inhibit LPA-induced PS exposure and calcium uptake at low micromolar concentrations; fluoxetine, an antidepressant and a known activator of TMEM16F, enhances these processes. These effectors likewise modulate erythrocyte PS exposure and microvesicle shedding induced by calcium ionophore treatment. Further, LPA-treated erythrocytes triggered thrombin generation in platelet-free plasma which was partially impaired in the presence of TA and EGCG. Thus, this study suggests that LPA activates the scramblase TMEM16F in erythrocytes, thereby possibly mediating a pro-thrombotic function in these cells. EGCG as well as fluoxetine, substances with potentially high plasma concentrations due to alimentation or medical treatment, should be considered as potential effectors of systemic hemostatic regulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Few and inconsistent data exist describing the effect of storage duration on glycated hemoglobin (HbA1c) concentrations of red blood cells (RBCs), impeding interpretation of HbA1c values in transfused diabetic patients. Hence the aim of this study was to evaluate to what extent HbA1c concentrations of RBCs change during the maximum allowed storage period of 42 days., Study Design and Methods: Blood was drawn from 16 volunteers, leukofiltered, and stored under standard blood banking conditions. HbA1c concentrations of RBCs were measured on Days 1 and 42 of storage using three different validated devices (ion-exchange high-performance liquid chromatography Method A1 and A2, turbidimetric immunoassay Method B)., Results: Mean HbA1c concentrations of RBCs on Day 1 were 5.3 ± 0.3% (Method A1), 5.4 ± 0.4% (Method A2), and 5.1 ± 0.4% (Method B). HbA1c concentrations increased to 5.6 ± 0.3% (A1, p < 0.0001), 5.7 ± 0.3% (A2, p = 0.004), and 5.5 ± 0.4% (B, p < 0.0001) on Day 42, respectively, corresponding to a 1.06-fold increase across all methods. Glucose concentrations in the storage solution of RBCs decreased from 495 ± 27 to 225 ± 55 mg/dL (p < 0.0001), confirming that stored RBCs were metabolically active., Conclusion: These results suggest a significant, albeit minor, and most likely clinically insignificant increase in HbA1c concentrations during storage of RBCs for 42 days., (© 2018 The Authors. Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.)

Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/chorein protein - likely a crucial organizer at the erythrocyte cytoskeleton/membrane interface - showed a strong decrease in chlorpromazine-induced endovesiculation. The responses of ChAc erythrocytes to effectors of endovesiculation were similar to that of control erythrocytes, yet at drastically reduced levels. This suggests a more rigid and less dynamic interaction at the membrane-cytoskeleton interphase of ChAc erythrocytes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.

Recent data from a series of laboratories has pinpointed the relevant role of translation control on the regulation of gene expression. In particular, an analysis of T cell activation has led to demonstrate that during this physiological transition about 20% of the regulated mRNAs are controlled at the translation level. Furthermore, modulating the host mRNA translation is one of the mechanisms used by infectious agents to achieve a productive infection. In the present review, we summarize the current knowledge on the translation machinery, the translational control mechanisms during the immune response, as well as the mechanisms used by different pathogens to avoid, inhibit or postpone the host response; and suggest that the analysis on genome-wide screening of the host-pathogen interactions, identifying translationally regulated mRNAs, might unravel new drug targets in infectious diseases.

Erythropoiesis strictly depends on signal transduction through the erythropoietin receptor (EpoR)-Janus kinase 2 (Jak2)-signal transducer and activator of transcription 5 (Stat5) axis, regulating proliferation, differentiation, and survival. The exact role of the transcription factor Stat5 in erythropoiesis remained puzzling, however, since the first Stat5-deficient mice carried a hypomorphic Stat5 allele, impeding full phenotypical analysis. Using mice completely lacking Stat5--displaying early lethality--we demonstrate that these animals suffer from microcytic anemia due to reduced expression of the antiapoptotic proteins Bcl-x(L) and Mcl-1 followed by enhanced apoptosis. Moreover, transferrin receptor-1 (TfR-1) cell surface levels on erythroid cells were decreased more than 2-fold on erythroid cells of Stat5(-/-) animals. This reduction could be attributed to reduced transcription of TfR-1 mRNA and iron regulatory protein 2 (IRP-2), the major translational regulator of TfR-1 mRNA stability in erythroid cells. Both genes were demonstrated to be direct transcriptional targets of Stat5. This establishes an unexpected mechanistic link between EpoR/Jak/Stat signaling and iron metabolism, processes absolutely essential for erythropoiesis and life.

Stem cell factor (SCF)-induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of erythroblasts.

Erythropoiesis requires erythropoietin (Epo) and stem cell factor (SCF) signaling via their receptors EpoR and c-Kit. EpoR, like many other receptors involved in hematopoiesis, acts via the kinase Jak2. Deletion of EpoR or Janus kinase 2 (Jak2) causes embryonic lethality as a result of defective erythropoiesis. The contribution of distinct EpoR/Jak2-induced signaling pathways (mitogen-activated protein kinase, phosphatidylinositol 3-kinase, signal transducer and activator of transcription 5 [Stat5]) to functional erythropoiesis is incompletely understood. Here we demonstrate that expression of a constitutively activated Stat5a mutant (cS5) was sufficient to relieve the proliferation defect of Jak2(-/-) and EpoR(-/-) cells in an Epo-independent manner. In addition, tamoxifen-induced DNA binding of a Stat5a-estrogen receptor (ER)* fusion construct enabled erythropoiesis in the absence of Epo. Furthermore, c-Kit was able to enhance signaling through the Jak2-Stat5 axis, particularly in lymphoid and myeloid progenitors. Although abundance of hematopoietic stem cells was 2.5-fold reduced in Jak2(-/-) fetal livers, transplantation of Jak2(-/-)-cS5 fetal liver cells into irradiated mice gave rise to mature erythroid and myeloid cells of donor origin up to 6 months after transplantation. Cytokine- and c-Kit pathways do not function independently of each other in hematopoiesis but cooperate to attain full Jak2/Stat5 activation. In conclusion, activated Stat5 is a critical downstream effector of Jak2 in erythropoiesis/myelopoiesis, and Jak2 functionally links cytokine- with c-Kit-receptor tyrosine kinase signaling.

We characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species by sequestering iron in the mature erythrocyte. Moreover, our data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir.

Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA+ RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA+ RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.

Terminal erythropoiesis is accompanied by extreme demand for iron to ensure proper hemoglobinization. Thus, erythroblasts must modify the "standard" post-transcriptional feedback regulation, balancing expression of ferritin (Fer; iron storage) versus transferrin receptor (TfR1; iron uptake) via specific mRNA binding of iron regulatory proteins (IRPs). Although erythroid differentiation involves high levels of incoming iron, TfR1 mRNA stability must be sustained and Fer mRNA translation must not be activated because iron storage would counteract hemoglobinization. Furthermore, translation of the erythroid-specific form of aminolevulinic acid synthase (ALAS-E) mRNA, catalyzing the first step of heme biosynthesis and regulated similarly as Fer mRNA by IRPs, must be ensured. We addressed these questions using mass cultures of primary murine erythroid progenitors from fetal liver, either undergoing sustained proliferation or highly synchronous differentiation. We indeed observed strong inhibition of Fer mRNA translation and efficient ALAS-E mRNA translation in differentiating erythroblasts. Moreover, in contrast to self-renewing cells, TfR1 stability and IRP mRNA binding were no longer modulated by iron supply. These and additional data stemming from inhibition of heme synthesis with succinylacetone or from iron overload suggest that highly efficient utilization of iron in mitochondrial heme synthesis during normal erythropoiesis alters the regulation of iron metabolism via the IRE/IRP system.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free culture systems delineated in this chapter meet these requirements, employing primary hematopoietic cells derived from murine fetal liver and human umbilical cord blood, which show physiological self-renewal responses to cytokine/hormone combinations, which in vivo are involved in stress hematopoiesis. We describe the expansion and sustained proliferation of multipotent (mouse) and erythroid (mouse and human) progenitors, responding to physiological signals. Moreover, both mouse and human erythroid progenitors can be induced to undergo synchronous terminal differentiation by addition of high levels of erythropoietin. If fetal liver cells from p53-/- mice are used, respective multipotent and erythroid cells undergo immortalization without an obvious Hayflick crisis, but otherwise retain their primary cell characteristics. Finally, both primary and immortal mouse progenitors can be subjected to genetic manipulation via retroviral constructs with high efficiency.

Outgrowth, long-term self-renewal, and terminal maturation of human erythroid progenitors derived from umbilical cord blood in serum-free medium can be modulated by steroid hormones. Homogeneous erythroid cultures, as characterized by flow cytometry and dependence on a specific mixture of physiologic proliferation factors, were obtained within 8 days from a starting population of mature and immature mononuclear cells. Due to previous results in mouse and chicken erythroblasts, the proliferation-promoting effect of glucocorticoids was not unexpected. Surprisingly, however, androgen had a positive effect on the sustained expansion of human female but not male erythroid progenitors. Under optimal conditions, sustained proliferation of erythroid progenitors resulted in a more than 10(9)-fold expansion within 60 days. Terminal erythroid maturation was significantly improved by adding human serum and thyroid hormone (3,5,3'-triiodothyronine [T3]) to the differentiation medium. This resulted in highly synchronous differentiation of the cells toward enucleated erythrocytes within 6 days, accompanied by massive size decrease and hemoglobin accumulation to levels comparable to those in peripheral blood erythrocytes. Thus, obviously, different ligand-activated nuclear hormone receptors massively influence the decision between self-renewal and terminal maturation in the human erythroid compartment.

The expansion and differentiation of hematopoietic progenitors is regulated by cytokine and growth factor signaling. To examine how signal transduction controls the gene expression program required for progenitor expansion, we screened ATLAS filters with polysome-associated mRNA derived from erythroid progenitors stimulated with erythropoietin and/or stem cell factor. The putative proto-oncogene nucleoside diphosphate kinase B (ndpk-B or nm23-M2) was identified as an erythropoietin and stem cell factor target gene. Factor-induced expression of nm23-M2 was regulated specifically at the level of polysome association by a phosphoinositide 3-kinase-dependent mechanism. Identification of the transcription initiation site revealed that nm23-M2 mRNA starts with a terminal oligopyrimidine sequence, which is known to render mRNA translation dependent on mitogenic factors. Recently, the nm23-M2 locus was identified as a common leukemia retrovirus integration site, suggesting that it plays a role in leukemia development. The expression of Nm23 from a retroviral vector in the absence of its 5'-untranslated region caused constitutive polysome association of nm23-M2. Polysome-association and protein expression of endogenous nm23-M2 declined during differentiation of erythroid progenitors, suggesting a role for Nm23-M2 in progenitor expansion. Taken together, nm23-m2 exemplifies that cytokine-dependent control of translation initiation is an important mechanism of gene expression regulation.

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.

Background: Erythropoietin (Epo) is required for correct execution of the erythroid differentiation program. Erythropoiesis requires Bcl-X(L), a major late target of Epo-receptor signaling. Mice lacking Bcl-X(L) die around embryonic age E12.5, forming normal erythroid progenitors but lacking functional red cells. Recently, serum-free culture conditions for expansion of murine red cell progenitors were developed, yielding cells capable of in vivo-like terminal differentiation into enucleated erythrocytes, in response to Epo/insulin. Here we address whether Epo function during terminal maturation involves a cytokine-independent "default program," requiring only apoptosis inhibition through Epo-dependent upregulation of Bcl-X(L)., Results: Exogenous expression of Bcl-X(L) or Bcl-2 in primary murine erythroblasts or clonal erythroblast lines derived from p53(-/-) mice allowed these cells to undergo terminal erythroid maturation, in the complete absence of cytokines. A potential autocrine Epo loop was ruled out by respective neutralizing antibodies. Importantly, sustained proliferation of Bcl-X(L)-expressing immature erythroblasts still required respective factors (Epo, stem cell factor [SCF], and the glucocorticoid receptor ligand dexamethasone [Dex]). Epo-independent differentiation in these Bcl-X(L)- or Bcl-2-expressing, primary erythroblasts was thus triggered by removal of the renewal factors SCF and Dex. This initiated the maturation-specific expression cascade of erythroid transcription factors, followed by differentiation divisions (characterized by a short G1 phase and decrease in cell size), hemoglobin accumulation, and enucleation., Conclusions: During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X(L). This allows "default" terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals.

In primary chicken erythroblasts (stem cell factor [SCF] erythroblasts), transferrin receptor (TfR) messenger RNA (mRNA) and protein were hyperexpressed as compared to nonerythroid chicken cell types. This erythroid-specific hyperexpression was abolished in transformed erythroblasts (HD3E22 cells) expressing the v-ErbA and v-ErbB oncogenes of avian erythroblastosis virus. TfR expression in HD3E22 cells could be modulated by changes in exogenous iron supply, whereas expression in SCF erythroblasts was not subject to iron regulation. Measurements of TfR mRNA half-life indicated that hyperexpression in SCF erythroblasts was due to a massive stabilization of transcripts even in the presence of high iron levels. Changes in mRNA binding activity of iron regulatory protein 1 (IRP1), the primary regulator of TfR mRNA stability in these cells, correlated well with TfR mRNA expression; IRP1 activity in HD3E22 cells and other nonerythroid cell types tested was iron dependent, whereas IRP1 activity in primary SCF erythroblasts could not be modulated by iron administration. Analysis of avian erythroblasts expressing v-ErbA alone indicated that v-ErbA was responsible for these transformation-specific alterations in the regulation of iron metabolism. In SCF erythroblasts high amounts of TfR were detected on the plasma membrane, but a large fraction was also located in early and late endosomal compartments, potentially concealing temporary iron stores from the IRP regulatory system. In contrast, TfR was almost exclusively located to the plasma membrane in HD3E22 cells. In summary, stabilization of TfR mRNA and redistribution of Fe-Tf/TfR complexes to late endosomal compartments may contribute to TfR hyperexpression in primary erythroblasts, effects that are lost on leukemic transformation.

Normalization of mRNA profiling data remains an open issue, which turns critical when comparing divergent samples or mRNA populations with different complexities. To address this question, we generated samples with different RNA amounts and complexities by subcellular fractionation of cytoplasmic RNA into the mutually exclusive ribosome-free and polysome-bound RNA pools. For each of the 563 mRNAs analyzed, the hybridization signal corresponding to the cytoplasmic sample equals the sum of signals from the ribosome-free plus the polysome-bound targets (cytoplasmic mRNA = ribosome-free mRNA + polysome-bound mRNA). This intuitive equation was fulfilled only after data normalization following "spiking" of the samples with an exogenous RNA. This is the first demonstration that spiking allows one to correct not only for differences in reaction efficiencies but also to reflect the variations in amount and complexity between the initial mRNA populations.

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

In general, translation efficiency of ferritin mRNAs is modulated by variations in iron supply. In primary avian erythroblasts undergoing short-term proliferation, however, ferritin heavy chain (ferH) mRNA is repressed at all iron levels. Yet, expression of v-ErbA oncoprotein is sufficient to reinduce ferH mRNA utilization at physiological iron concentrations. Since overexpression of the receptor tyrosine kinase c-Kit and erythropoietin receptor (EpoR) stimulates long-term proliferation of primary erythroblasts like v-ErbA, we analyzed the impact of cooperation between c-Kit and EpoR on the regulation of iron storage. Whereas endogenous c-Kit in combination with exogenous EpoR had no significant effect, ectopic overexpression of both receptors abolished translational repression of ferH mRNA upon iron administration. Thus, high-intensity signaling through c-Kit plus EpoR pathways mimics the v-ErbA-mediated regulatory phenotype., (Copyright 2000 Academic Press.)

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.

Studies using genetically modified mice and ex vivo tissue culture of erythroid progenitors converge to show that generation of mature erythroid cells depends on the interplay between specific transcriptional regulators and intracellular signals controlled by cytokines and growth factors. These studies also show that terminal differentiation in the erythroid lineage is unusual since the acquisition of the phenotypic traits of mature cells occurs while the cells are still actively dividing. Furthermore, under specific stress conditions, a massive and sustained self-renewal of committed erythroid progenitors can take place to replenish the pool of terminally differentiated cells. We review here how the erythroid genetic program and its interplay with specific cytokines, growth factors and hormones controls survival, proliferation and differentiation of erythroid progenitors both in normal and stress conditions. Special emphasis is laid on our present understanding of the differences in cell cycle control, which result either in self-renewal of erythroid progenitors or in the particular cell divisions which accompany terminal differentiation. Finally, we discuss how deregulation of the various aspects of the physiological control of erythroid progenitor survival, proliferation and differentiation can lead to erythroblast transformation and erythroleukemia.

In immortalized cells of the erythroid lineage, the iron-regulatory protein (IRP) has been suggested to coregulate biosynthesis of the iron storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity, IRP binds to an iron-responsive element (IRE) located in ferritin and eALAS mRNA leaders, causing a block of translation. In contrast, IRP-IRE interaction is reduced under high iron conditions, allowing efficient translation. We show here that primary chicken erythroblasts (ebls) proliferating or differentiating in culture use a drastically different regulation of iron metabolism. Independently of iron administration, ferritin H (ferH) chain mRNA translation was massively decreased, whereas eALAS transcripts remained constitutively associated with polyribosomes, indicating efficient translation. Variations in iron supply had minor but significant effects on eALAS mRNA polysome recruitment but failed to modulate IRP-affinity to the ferH-IRE in vitro. However, leukemic ebls transformed by the v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an iron-dependent reduction of IRP mRNA-binding activity, resulting in mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the respective normal cellular homologues (c-ErbA/TRalpha, c-ErbB/EGFR). It turned out that v-ErbA, a mutated class II nuclear hormone receptor that arrests erythroid differentiation, caused the change in ferH mRNA translation. Accordingly, inhibition of v-ErbA function in these leukemic ebls led to a switch from iron-responsive to iron-independent ferH expression.

Tight regulation of iron metabolism is crucial to avoid formation of deleterious radicals and is mainly executed at the post-transcriptional level. The regulatory loops are exerted by trans-acting iron regulatory proteins (IRPs) and cis-acting stem-loop motifs, termed iron-responsive elements (IREs), located in the untranslated regions (UTRs) of target mRNAs. Iron scarcity induces binding of IRPs to a single IRE in the 5'-UTR of ferritin, eALAS, aconitase and SDHb mRNAs, which specifically suppresses translation initiation. Simultaneous interaction of IRPs with multiple IREs in the 3'-UTR of transferrin receptor (TfR) mRNA selectively causes its stabilization. The pattern is reverted under iron overload: IRP-mRNA binding affinity is reduced, which results in efficient protein synthesis of target transcripts harboring IREs in the 5'-UTR and rapid degradation of TfR mRNA. Although multiple evidences support this model, several studies reported massive alterations in the regulation of iron homeostasis under specific physiological conditions, raising the possibility for additional regulatory events. Intensive analysis of the palindromic IRE consensus sequence revealed the critical elements for the formation of a functional structure and demonstrated the consequences of IRE mutations in IRP binding. Recent investigations indicated the involvement of naturally occurring IRE mutations of the ferritin L subunit in the hyperferritinemia-cataract syndrome, a hereditary disorder. This review summarizes the apparent links between iron-dependent post-transcriptional control and its abnormalities, governed by the properties of a single mRNA stem-loop structure.

Distributional changes of individual mRNAs between free ribonucleoprotein particles (mRNP) and ribosome-bound transcripts are used to assess translational control. Simultaneous analysis of many mRNA species is required to estimate the overall contribution of translation to the regulation of gene expression. To this purpose, total cytoplasmic RNA was fractionated in sucrose step gradients and poly(A)+ RNA was prepared from mRNP and ribosome-bound fractions. Since direct, simultaneous analysis of a profusion of mRNAs is not feasible, distribution of their in vitro translation products was examined after separation in 2-dimensional gels, followed by computer-based analysis of autoradiographs. When this analysis was applied to antigenically stimulated T cells, 36% of in vitro translation products showed a greater than 10-fold increase in intensity, suggesting transcriptional activation of the corresponding mRNAs. In comparison, 7.9% of individual mRNAs (54 of 685 species) were translationally activated. They were redistributed from free mRNP to ribosome-associated fractions; 4.7% (32 species) were translationally repressed, as indicated by the opposite pattern. The differential recruitment of 12.6% of mRNA species demonstrates specificity and the general significance of translational control during T cell activation, which implies that translation may play a similar role in regulating gene expression in a variety of physiological processes.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

We report the cloning and functional characterization of the iron responsive element (IRE) of human ferritin light (L) chain mRNA from a cDNA library of primary human T lymphocytes. Comparison of this palindromic cDNA element to the IRE predicted from the reported genomic sequence revealed significant differences, resulting in a stem-loop structure with lower stability than the IRE of the heavy (H) chain mRNA. Nevertheless, the L subunit IRE mediated efficient binding of the iron regulatory protein (IRP) in a manner comparable to that of human ferritin H chain mRNA in vitro. In accordance with previous observations on H form transcripts, the cis-acting regulatory IRE motif of human ferritin L chain mRNA was capable of repressing translation under iron deprivation but permitted mobilization of the transcripts into polysomes following iron repletion in vivo.

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G1-S transition and stays high throughout G2 until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G1. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cell but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G1 and presumably also G2.

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.

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.

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.

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.

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.