Myeloid-Derived Growth Factor Protects Against Pressure Overload-Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca2+-ATPase Expression in Cardiomyocytes.

BACKGROUND: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. METHODS: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf-deficient (Mydgf-/-), and Mydgf bone marrow-chimeric or bone marrow-conditional transgenic mice with pressure overload-induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography-mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. RESULTS: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf-/- mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow-derived inflammatory cells attenuated pressure overload-induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein-coupled receptor agonist-induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf-/- mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf-/- mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload-induced hypertrophy and dysfunction. Pressure-overloaded Mydgf-/- mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. CONCLUSIONS: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells.

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in "expanded" groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.

The genomic risk of somatic gene therapy.

Gene vectors with an untargeted insertion profile have been explored in preclinical models and clinical trials for the transfer of potentially therapeutic genetic information into somatic cells that have a high replicative potential. The gene-modified cell population can be viewed as a genetic mosaic whose complexity depends upon the target cell type, the number of transduced cells, the average number of insertions per cell, the genetic stability and composition of the transgene, and the integration pattern of the vector. Selection by the environment encountered in the patient may support the preferential survival of clones with insertional deregulation of genes that are involved in the control of engraftment, proliferation or differentiation, in the worst case initiating oncogenic progression. Rapid scientific and technological progress has shed much light onto this dark side of untargeted vector integration. New approaches to unbiased and highly sensitive "integromics" promise a precise documentation of stable polyclonality, clonal fluctuation or clonal imbalance of gene-modified cell populations. Evidence has been obtained for a number of approaches to potentially reduce the genomic risk of gene therapy: targeting cells that lack sustained replicative potential, using vectors with a more neutral integration spectrum, reducing the number of vector copies per cell, designing gene expression cassettes that avoid long-distance enhancer interactions or fusion transcripts, and reducing, as far as possible, the risk of secondary mutations. The genomic risk of gene therapy can thus be prevented by the collective targeting of all contributing factors.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004-2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Multiple Genes Surrounding Bcl-xL, a Common Retroviral Insertion Site, Can Influence Hematopoiesis Individually or in Concert.

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS) Bcl-xL, which revealed seven genes located within ±150 kb from the RVIS for our study. We confirmed that Bcl-xL enhanced the competitiveness of HSPCs, whereas the Bcl-xL neighbor Id1 hindered HSPC long-term repopulation. This negative influence of Id1 could be counteracted by co-expressing Bcl-xL. Interestingly, >90% of early reconstituted myeloid cells were found to originate from transduced HSPCs upon simultaneous overexpression of Bcl-xL and Id1, which implies that Bcl-xL and Id1 can collaborate to rapidly replenish the myeloid compartment under stress conditions. To directly compare the competitiveness of HSPCs conveyed by multiple transgenes, we developed a multiple competitor competitive repopulation (MCCR) assay to simultaneously screen effects on HSPC repopulating capacity in a single mouse. The MCCR assay revealed that multiple genes within a CIS can have positive or negative impact on hematopoiesis. Furthermore, these data highlight the importance of studying multiple genes located within the proximity of an insertion site to understand complex biological effects, especially as the number of gene therapy patients increases.

Cell-intrinsic and vector-related properties cooperate to determine the incidence and consequences of insertional mutagenesis.

In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient gamma-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of approximately 10(-4). Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10(-6)). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

EVI1 phosphorylation at S436 regulates interactions with CtBP1 and DNMT3A and promotes self-renewal.

The transcriptional regulator EVI1 has an essential role in early development and haematopoiesis. However, acute myeloid leukaemia (AML) driven by aberrantly high EVI1 expression has very poor prognosis. To investigate the effects of post-translational modifications on EVI1 function, we carried out a mass spectrometry (MS) analysis of EVI1 in AML and detected dynamic phosphorylation at serine 436 (S436). Wild-type EVI1 (EVI1-WT) with S436 available for phosphorylation, but not non-phosphorylatable EVI1-S436A, conferred haematopoietic progenitor cell self-renewal and was associated with significantly higher organised transcriptional patterns. In silico modelling of EVI1-S436 phosphorylation showed reduced affinity to CtBP1, and CtBP1 showed reduced interaction with EVI1-WT compared with EVI1-S436A. The motif harbouring S436 is a target of CDK2 and CDK3 kinases, which interacted with EVI1-WT. The methyltransferase DNMT3A bound preferentially to EVI1-WT compared with EVI1-S436A, and a hypomethylated cell population associated by EVI1-WT expression in murine haematopoietic progenitors is not maintained with EVI1-S436A. These data point to EVI1-S436 phosphorylation directing functional protein interactions for haematopoietic self-renewal. Targeting EVI1-S436 phosphorylation may be of therapeutic benefit when treating EVI1-driven leukaemia.

Retroviral insertion site analysis in dominant haematopoietic clones.

Identification of retroviral vector insertion sites in single, dominating cell clones has become an important tool for the investigation of cellular signalling pathways involved in clonal expansion and malignant transformation. Also, recent severe adverse events in clinical trials resulting from retroviral vector-mediated insertional mutagenesis underline the need of well-designed safety studies including integration site analyses to estimate cost/benefit ratios in gene therapy. We have recently described a modified ligation-mediated PCR (LM PCR) method allowing preferential retrieval of insertion sites causally linked to clonal dominance of an affected clone. In the first part of the given work we focus on particularities of the LM PCR procedure to be taken into account when working with self-inactivating as compared to 'classical' retrovectors. In the following sections we focus on data acquisition, processing, organisation, and analysis. Thus the protocol presented here should be helpful in establishing and utilising databases of retroviral integration sites.

Retroviral integration site analysis in hematopoietic stem cells.

Stable transgene insertion into a host genome irrevocably and unambiguously marks individual cells and all their descendants, i.e., the respective cell clone. Based thereon, retroviral gene marking has become an important tool for investigating the in vivo fate of different cell types, both in animal models and in clinical gene transfer. Moreover, identification of (vector) insertion sites in malignant clones transformed because of insertional activation of proto-oncogenes after experimental as well as therapeutic retroviral gene transfer has resulted in new insights into oncogenic transformation of hematopoietic stem cells (HSCs). However, because of the high sensitivity of the PCR-based methods for insertion site detection, researchers are often confronted with large numbers of different insertion sites/cell clones whose contribution to the given state is hard to judge. A relatively simple ligation-mediated polymerase chain reaction (LM-PCR) method allows the preferential analysis of insertion sites in those cell clones that significantly contributed to the cell pool analyzed. In murine bone marrow transplantation models, we have shown that this method is very useful to analyze the impact of retroviral insertion sites on both malignant and benign clonal dominance of individual repopulating HSC.

Insertional mutagenesis by replication-deficient retroviral vectors encoding the large T oncogene.

Insertion sites of replication-deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor-suppressor proteins p53 and Rb by virtue of a chaperone-like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg-induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics ( approximately 8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D-derived monocytic leukemias showed hits in Pim2 and Max proto-oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector-mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.

Multimodal Lentiviral Vectors for Pharmacologically Controlled Switching Between Constitutive Single Gene Expression and Tetracycline-Regulated Multiple Gene Collaboration.

Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied. Doxycycline-dependent switching can be tracked via fluorescent markers coupled to constitutive and tetracycline-regulated GOIs. This article describes transduction of murine primary HPCs with different doses of multimodal LVs, distinct cytokine conditions, and their influence on the number and viability of cells co-expressing both collaborating GOIs upon doxycycline induction. A 2-week protocol is provided for multimodal LV production, titer determination, and evaluation of tetracycline responsive promoter background activity in a murine fibroblast cell line. The power of this model to assess the dose/time/order-controlled contribution of single and multiple genes into hematopoietic networks opens new routes in reprogramming, stem cell, and leukemia biology.

Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors.

Increasing evidence reveals that random insertion of gene transfer vectors into the genome of repopulating hematopoietic cells may alter their fate in vivo. Although most insertional mutations are expected to have few if any consequences for cellular survival, clonal dominance caused by retroviral vector insertions in (or in the vicinity of) proto-oncogenes or other signaling genes has been described for both normal and malignant hematopoiesis. Important insights into these side effects were initially obtained in murine models. Results from ongoing clinical studies have revealed that similar adverse events may also occur in human gene therapy. However, it remains unknown to what extent the outcome of insertional mutagenesis induced by gene vectors is related to (1) the architecture and type of vector used, (2) intrinsic properties of the target cell, and (3) extrinsic and potentially disease-specific factors influencing clonal competition in vivo. This review discusses reports addressing these questions, underlining the need for models that demonstrate and quantify the functional consequences of insertional mutagenesis. Improving vector design appears to be the most straightforward approach to increase safety, provided all relevant cofactors are considered.

Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors.

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin(-)), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells.

Comparison of Tetracycline-regulated Promoters in Lentiviral-based Vectors in Murine Transplantation Studies.

Tetracycline-regulated systems with efficient temporal and dose regulation of transgene expression are useful for development of new physiologic/pathophysiologic experimental models and gene therapy approaches. Lentiviral vectors with improved tetracycline-regulated promoters help to overcome the existing limitations such as basal activity in the drug absence, poor inducibility or unstable transgene expression. To compare conventional and improved tetracycline-regulated promoters in lentiviral based vectors in vivo, we investigated doxycycline-regulated gene transfer/expression levels in a long-term murine transplantation model and demonstrated that the lentiviral vector with the improved T11 promoter exhibited more efficient inducibility and higher gene transfer level. The time required to reverse transgene expression after doxycycline removal was increased for animals with higher gene expression levels and vector copy numbers. Examination of peripheral blood leukocytes and splenocytes revealed similar cell lineage distributions for transgene positive and negative cell populations from experimental and control mice, but increased variability in the percentages of myeloid and lymphoid cells was detected in transgene positive bone marrow cells. However, no indication of lineage bias in total bone marrow cells and no signs of hematopoietic disease were observed seven months after transplantation. Our results showed that the T11 tetracycline-regulated promoter enabled improved transgene expression in a murine transplantation model. The established system allows further development of tetracycline-regulated experimental models to investigate normal and malignant hematopoiesis.

The ability of Fos family members to produce phenotypic changes in epithelioid cells is not directly linked to their transactivation potentials.

Numerous studies have revealed distinct functions of Fos proteins in different mouse tissues and cell lines. Here, we perform a direct comparison of the features of exogenous c-Fos, Fra-1 and Fra-2 proteins expressed in murine tumor cells of epithelial origin, CSML0. Although transactivation potential of c-Fos is much stronger than that of Fra-1 and Fra-2, all three proteins are capable of modulating transcription of target genes. Moreover, there is a certain degree of specificity in the induction of the transcription of AP-1-responsive genes by different Fos proteins. For instance, c-Fos and Fra-1 but not Fra-2 activated genes of the urokinase system. Additionally, not only a strong transcriptional activator c-Fos, but also Fra-1 induced morphological alterations in CSML0 cells. N-terminal domain of Fra-1 was required for this function. On the other hand, Fra-2 failed to change morphology of CSML0 cells. We therefore conclude that c-Fos, Fra-1 and Fra-2 differently activate transcription of target genes and induce morphological changes in epithelioid carcinoma cells in a manner not directly linked to their transactivation potentials.