Biomedical subjects

Carol Stocking

Publications and source records attributed to Carol Stocking.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of nucleocytoplasmic trafficking of the HuR ligand APRIL and its influence on CD83 expression.

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and are able to sensitize even naïve T cells. Mature DC are characterized by expression of CD83, a surface molecule that is proposed to be involved in efficient T cell activation. It has been recently shown that CD83 mRNA is transported from the nucleus to the cytoplasm in a HuR- and CRM1-dependent manner. Therefore we here investigated the impact of two known protein ligands of HuR, pp32 and APRIL, on CD83 expression. Both pp32 (ANP32A) and APRIL (ANP32B) are shuttle proteins, and it has been reported earlier that these HuR ligands can act as adaptors that link HuR and the CRM1-specific nuclear export pathway. By employing RNA interference (RNAi) technology we demonstrate that pp32 is dispensable for CD83 expression, whereas APRIL contributes to the nuclear export and subsequent translation of CD83 mRNA. Furthermore, we have determined the nuclear import signal (NLS) as well as the nuclear export signal (NES) of human APRIL. Moreover, we analyzed the status of phosphorylation of endogenous APRIL and identified threonine 244 to be an as yet unrecognized phosphate acceptor. Finally, we were able to show that phosphorylation of this specific amino acid residue regulates the nuclear export of APRIL. In sum, we report here the signal sequences in APRIL that mediate its intracellular trafficking and provide evidence that this protein ligand of HuR is an important player in the post-transcriptional regulation of CD83 expression by affecting the nucleocytoplasmic translocation of CD83 mRNA.

Active Transport, Cell Nucleus↗

PubMed2006

Importance of receptor usage, Fli1 activation, and mouse strain for the stem cell specificity of 10A1 murine leukemia virus leukemogenicity.

Murine leukemia viruses (MuLV) induce leukemia through a multistage process, a critical step being the activation of oncogenes through provirus integration. Transcription elements within the long terminal repeats (LTR) are prime determinants of cell lineage specificity; however, the influence of other factors, including the Env protein that modulates cell tropism through receptor recognition, has not been rigorously addressed. The ability of 10A1-MuLV to use both PiT1 and PiT2 receptors has been implicated in its induction of blast cell leukemia. Here we show that restricting receptor usage of 10A1-MuLV to PiT2 results in loss of blast cell transformation capacity. However, the pathogenicity was unaltered when the env gene is exchanged with Moloney MuLV, which uses the Cat1 receptor. Significantly, the leukemic blasts express erythroid markers and consistently contain proviral integrations in the Fli1 locus, a target of Friend MuLV (F-MuLV) during erythroleukemia induction. Furthermore, an NB-tropic variant of 10A1 was unable to induce blast cell leukemia in C57BL/6 mice, which are also resistant to F-MuLV transformation. We propose that 10A1- and F-MuLV actually induce identical (erythro)blastic leukemia by a mechanism involving Fli1 activation and cooperation with inherent genetic mutations in susceptible mouse strains. Furthermore, we demonstrate that deletion of the Icsbp tumor suppressor gene in C57BL/6 mice is sufficient to confer susceptibility to 10A1-MuLV leukemia induction but with altered specificity. In summary, we validate the significance of the env gene in leukemia specificity and underline the importance of a complex interplay of cooperating oncogenes and/or tumor suppressors in determining the pathogenicity of MuLV variants.

Animals↗

PubMed2006

Novel bis(1H-indol-2-yl)methanones as potent inhibitors of FLT3 and platelet-derived growth factor receptor tyrosine kinase.

FLT3 receptor tyrosine kinase is aberrantly active in many cases of acute myeloid leukemia (AML). Recently, bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. To optimize FLT3 activity and selectivity, 35 novel derivatives were synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. The most potent FLT3 inhibitors 98 and 102 show IC50 values of 0.06 and 0.04 microM, respectively, and 1 order of magnitude lower PDGFR inhibiting activity. The derivatives 76 and 82 are 20- to 40-fold PDGFR selective. Docking at the recent FLT3 structure suggests a bidentate binding mode with the backbone of Cys-694. Activity and selectivity can be related to interactions of one indole moiety with a hydrophobic pocket including Phe-691, the only different binding site residue (PDGFR Thr-681). Compound 102 inhibited the proliferation of 32D cells expressing wildtype FLT3 or FLT3-ITD similarly as FLT3 autophosphorylation, and induced apoptosis in primary AML patient blasts.

Acute Disease↗

PubMed2006

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line.

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.

Animals↗

PubMed2006

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia.

INTRODUCTION: The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. METHODS: The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. RESULTS: The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. CONCLUSION: These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

Animals↗

PubMed2006

Expression of Pit2 sodium-phosphate cotransporter during murine odontogenesis is developmentally regulated.

Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.

Ameloblasts↗

PubMed2005

Defining the oncogenic function of the TEL/AML1 (ETV6/RUNX1) fusion protein in a mouse model.

The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1(+)/Kit(hi)/CD11b(+) phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

Animals↗

PubMed2005

Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate.

Multiple genetic alterations are required to induce acute myelogenous leukemia (AML). Mutations in the extracellular domain of the KIT receptor are almost exclusively found in patients with AML carrying translocations or inversions affecting members of the core binding factor (CBF) gene family and correlate with a high risk of relapse. We demonstrate that these complex insertion and deletion mutations lead to constitutive activation of the KIT receptor, which induces factor-independent growth of interleukin-3 (IL-3)-dependent cells. Mutation of the evolutionary conserved amino acid D419 within the extracellular domain was sufficient to constitutively activate the KIT receptor, although high expression levels were required. Dose-dependent growth inhibition and apoptosis were observed using either the protein tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) or by blocking the phosphoinositide-3-kinase (PI3K)-AKT pathway. Our data show that the addition of kinase inhibitors to conventional chemotherapy might be a new therapeutic option for CBF-AML expressing mutant KIT.

Animals↗

PubMed2005

Monoclonal anti-idiotype antibody 6G6.C4 fused to GM-CSF is capable of breaking tolerance to carcinoembryonic antigen (CEA) in CEA-transgenic mice.

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF). Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P=0.005) and to CEA (P=0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.

Animals↗

PubMed2005

AML1/ETO promotes the maintenance of early hematopoietic progenitors in NOD/SCID mice but does not abrogate their lineage specific differentiation.

AML1-ETO is generated by the t(8;21) translocation found in approximately 12% of acute myelogenous leukemia. Studies to delineate the mechanism by which AML1-ETO induces leukemia have primarily relied on transformed human cell lines or murine model systems. The goal of this study was to determine the effect of AML1-ETO expression on primary human hematopoietic cells in vitro and in a xenograft model. We used a FMEV retroviral vector for the transfer of AML1/ETO into human CD34 + cells. The repopulation, self-renewal, and differentiation potential of infected cells were assessed in serum-free liquid culture, colony assays, and in transplanted NOD-SCID mice. High transcription levels were confirmed by real-time PCR. AML1-ETO expressing cells were expandable for up to 12 weeks and retained an immature morphology. The capacity for prolonged survival, however, did not abrogate maturation, as AML1-ETO cells gave rise to normal colonies in a CFU-assay. AML1/ETO-expressing cells also contributed to myeloid (CD15, CD33), B-lymphoid (CD20), NK-cell (CD56) and erythroid (GPA) lineages in xenografted NOD/SCID mice. Although able to engraft all major lineages, AML1/ETO transplanted cells were primarily found in less differentiated fractions as measured by cell surface markers CD34 and CD38. In spite of a good engraftment and prolonged observation period none of the NOD/SCID-mice developed an acute myelogenous leukemia. Our findings demonstrate that AML1/ETO promotes the maintenance of early human hematopoietic progenitors, but does not abrogate their physiologic differentiation. Furthermore, the leukemogenic potential of AML1/ETO expressed in human progenitors is low, despite transcription levels equivalent to those found in AMLs.

Animals↗

PubMed2004

Tumor cells escape suicide gene therapy by genetic and epigenetic instability.

Transfer and expression of suicide genes is one cornerstone of cancer gene therapy and is also considered as a proactive tool to enhance the safety of somatic transgenesis. Here we addressed whether retrovirus-mediated suicide gene therapy would result in a predictable antitumor efficiency, given that problems related to gene transfer are solved or that the suicide gene is used in a proactive approach. Using retroviral vectors encoding the thymidine kinase gene of herpes simplex virus, we transduced EL-4 lymphoma cells and induced experimental tumors in congeneic C57Bl/6 mice. Systemic administration of ganciclovir (GCV) resulted in remission of transduced clonal and polyclonal tumors in vivo. However, GCV-resistant relapses occurred and were found to be associated with postinsertional alterations of transgene structure or loss of the entire transgene. Complete loss of a retrovirally marked fusion chromosome was confirmed by spectral karyotyping. Transgene silencing occurred in another clone. We conclude that genetic as well as epigenetic instability related to biologic features of the tumor, the insertion site, and the vector represent relevant limitations of retroviral suicide gene therapy. Considering the mechanisms of escape identified here, the proactive use of suicide genes to prevent complications of insertional mutagenesis may still be efficient.

Animals↗

PubMed2004

Recombination at chromosomal sequences involved in leukaemogenic rearrangements is differentially regulated by p53.

Chromosomal translocations and retroviral integration events at breakpoint cluster regions (bcrs) have been associated with leukaemias. To directly compare the effect of different cis-regulatory sequences on recombination, we adapted our SV40 based model system to the analysis of correspondingly selected bcrs from the TAL1, LMO2, retinoic acid receptor alpha (RARalpha) and MLL genes. We show that a 399 bp fragment from the MLL bcr is sufficient to cause a 3-4-fold stimulation of spontaneously occurring DNA exchange and to respond to etoposide by up to 10-fold further elevated frequencies, i.e. to mimic the fragility of the 8.3 kb bcr during chemotherapy. To analyse the regulatory role of p53 in recombination involving leukaemia-related sequences, we stably expressed wtp53 and a transactivation negative mutant. Consistent with the proposed role of p53 as a suppressor of error-prone recombination, both p53 proteins down-regulated recombination with most of the sequences tested, even with the MLL bcr after etoposide treatment. Surprisingly, however, p53 stimulated recombination, in constructs carrying the RARalpha bcr fragment. This is the first study, which provides evidence for a stimulatory role of p53 in homologous recombination. Our data further indicate that inhibition of topoisomerase I can mimic the effects of p53 on stimulating recombination on the RARalpha bcr. Thus, these data also firstly describe a biological role of the biochemical interactions between p53 and topoisomerase I that may have implications for a gain-of-function phenotype of certain p53 mutants in genetic destabilization.

Animals↗

PubMed2004

Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors.

BACKGROUND: Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. METHODS: To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses beta-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of beta-galactosidase. RESULTS: Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or beta-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. CONCLUSIONS: The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells.

Animals↗

PubMed2003

A dominant-negative mutant of C/EBPalpha, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is an essential transcription factor for granulocytic differentiation. C/EBPalpha mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region, resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in AML. To test this hypothesis, we introduced a cDNA encoding an N-terminal mutated C/EBPalpha (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34+ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly, mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBPalpha protein sequences, expression levels, or inefficient targeting of relevant cells. Taken together, our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs.

Animals↗

PubMed2003

An increase in the expression and total activity of endogenous p60(c-Src) in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1).

Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60(c-Src). The kinase activity of p60(c-Src) was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60(c-Src) triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common beta chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60(c-Src) in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.

Base Sequence↗

PubMed2003

10A1-MuLV but not the related amphotropic 4070A MuLV is highly neurovirulent: importance of sequences upstream of the structural Gag coding region.

Recombinants of Moloney murine leukemia virus (MoMuLV) with either an amphotropic (MoAmphoV) or 10A1-tropic host range (Mo10A1V) induce a spongiform neurodegenerative disease in susceptible mice. To test whether MoMuLV -derived sequences are required for induction of neuropathology, mice were inoculated with either the original 10A1 or the amphotropic (4070A) MuLV isolate. Strikingly, wild-type 10A1 was more neurovirulent than Mo10A1V, inducing severe neurological clinical symptoms with a median latency of 99 days in 100% of infected mice. In contrast, no motor disturbances were detected in any of the 4070A-infected mice, although limited central nervous system lesions were observed. A viral determinant conferring high neurovirulence to 10A1 was mapped to a region encompassing the first 676 bases of the viral genome, including the U5 LTR and encoding the amino-terminus of glycosylated Gag (glycoGag). In contrast to studies with the highly neurovirulent CasFr(KP) virus, an inverse correlation between surface expression levels of glycoGag and neurovirulence was not observed; however, this does not rule out a common underlying mechanism regulating virus pathogenicity.

Amino Acid Sequence↗

PubMed2003

Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose.

OBJECTIVE: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. MATERIALS AND METHODS: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. RESULTS: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. CONCLUSION: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.

Animals↗

PubMed2003

Transient expression of PU.1 commits multipotent progenitors to a myeloid fate whereas continued expression favors macrophage over granulocyte differentiation.

OBJECTIVES: The Ets-family transcription factor PU.1 is expressed specifically in the hematopoietic system, in which it is absolutely required for the generation of B lymphocytes and macrophages. In contrast, overexpression of PU.1 blocks terminal differentiation of the erythroid lineage, in which it can act as an oncogene. In this study we used a multipotential progenitor cell line to examine the effects of PU.1 overexpression on myeloerythroid commitment within a single model system. MATERIALS AND METHODS: PU.1 cDNA was introduced transiently and stably into the multipotent, nonleukemic hemopoietic cell line FDCPmix. Transiently transfected cells were isolated by fluorescence-activated cell sorting within 18 hours of transfection. Stable transfectants were selected by antibiotic resistance over a number of weeks. The effects of short- and long-term overexpression of PU.1 on self-renewal, proliferation, and differentiation were investigated. RESULTS: A transient pulse of expression in multipotent progenitor cells eliminated the options of self-renewal and erythroid differentiation, resulting in commitment to the myeloid lineage. However, this transient pulse of expression did not affect the subsequent lineage choice of bipotent granulocyte/macrophage progenitors. In contrast, continuous expression of PU.1 resulted in a strong bias toward macrophage rather than granulocyte differentiation. CONCLUSIONS: These results demonstrate promyeloid effects of PU.1 at two distinct stages of hematopoiesis.

Animals↗