Unmodified Cre recombinase crosses the membrane.

Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous 'protein transduction domain' surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPalpha(fl/fl) mice also led to excision of the 'floxed' C/EBPalpha gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.

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.

HOXB4's road map to stem cell expansion.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Stromal cells selectively reduce the growth advantage of human committed CD34+ hematopoietic cells ectopically expressing HOXB4.

Key players in self-renewal of hemopoietic stem cells are homeobox (HOX) transcription factors. In murine cells, overexpression of HOXB4 results in expansion of hematopoietic stem- and committed progenitor cells in vitro without obvious hematopoietic alterations. In vivo, HOXB4 induced HSC expansion continued until stem cell regeneration reached pretransplantation levels. HOXB4 is thus an attractive candidate for amplification of stem cells provided that human HOXB4 overexpressing cells can also be restricted to normal growth in vivo. The stromal microenvironment provides the regulatory mechanisms controlling the balance of stem cell self-renewal and differentiation. Here, we compared the response of HOXB4- and GFP-control vector transduced human CD34(+) cells to stroma encoded signals in vitro. In serum-sustained cocultures MS-5 stroma contact reduced the output of late CD34- HOXB4(+) cells in relation to GFP-controls 9-fold whereas the expansion of early CD34(+)HOXB4(+) cells remained unchanged as compared to liquid cultures. In presence of insulin HOXB4 overexpressing cells do not react to stroma encoded growth-restricting signals. Our results show that ectopic expression of HOXB4 in combination with MS-5 stroma exerts different effects in early and late human cord blood CD34(+) cells resulting in an enhanced proliferation of early CD34(+) cells in absence or presence of MS-5 stroma and an impaired output of late committed CD34(+) cells on MS-5 stroma.

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.

HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues.

Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.

Diverse isoforms of colony-stimulating factor-1 have different effects on the development of stroma-dependent hematopoietic cells.

Maintenance and differentiation of hematopoietic stem and progenitor cells are controlled by complex interactions with the stroma microenvironment. Stroma-cell interactions can be supported by locally expressed membrane-spanning cell-surface (cs) growth factors. CSF-1 is expressed by stroma as a soluble glycoprotein, as proteoglycan, or as a membrane-spanning cs glycoprotein. CSF-1 regulates the survival, proliferation, and differentiation of mononuclear phagocytes. Whereas the biological role of soluble CSF-1 is well characterized, the function of the membrane-spanning cell-surface CSF-1 (csCSF-1) remains unclear. To analyze the biological significance of csCSF-1 in vitro, we used an epithelial cell line to ectopically express the different CSF-1 isoforms. In co-cultures of CSF-1 transduced epithelial cells with primary, early hematopoietic progenitor cells we examined whether interaction between csCSF-1 and its receptor mediates cell proliferation, self-renewal, or differentiation. csCSF-1 induces long-lasting proliferation of stimulated cells and furthermore supports self-renewal. Ectopic secretion of soluble CSF-1 does not permit long-term growth of progenitor cells but induces differentiation of monocytes into macrophages. Previously, we showed that the soluble and cs isoforms of stroma-encoded SCF differently affect the development of hematopoietic cells. Cell-surface SCF (csSCF) promotes self-renewal of stimulated cells whereas soluble SCF causes clonal extinction. These results and those presented here for CSF-1 provide evidence for diverse functions of the isoforms of the ligands SCF and CSF-1 for two tyrosine kinase receptors of the subclass III both regulating hematopoiesis on stroma.

HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

In vivo analysis of retroviral enhancer mutations in hematopoietic cells: SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.

The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct cis elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.

Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range.

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.

Activation of the gene for the PDGF receptor beta1 (PDGFRbeta) in interleukin-3-dependent myeloid cells by retroviral insertional mutagenesis: implications for the transforming potential of PDGFRbeta.

Retroviral insertional mutagenesis has proven to be a powerful tool to identify genetic lesions disrupting normal hematopoiesis. The gene encoding the beta receptor for platelet-derived growth factor (PDGFRbeta) was identified as a target of retroviral mutagenesis in mutants selected for interleukin-3 (IL3)-independent growth. Expression of PDGFRbeta in the parental cells using a retroviral vector increased the frequency of factor-independent growth, confirming the significance of the retroviral integration site. Significantly, however, expression of the receptor did not induce IL3-independent growth in one step. In contrast, TEL-PDGFRbeta, the fusion protein generated by the t(5;12) translocation associated with chronic myelomonocytic leukemia, induced factor-independent growth in all transductants, demonstrating that the TEL-PDGFRbeta fusion protein is a more potent mitogenic signal. Nevertheless PDGFRbeta overexpression is sufficient to give a selective advantage to IL3-dependent cells under adverse conditions, allowing the selection of secondary mutations that impart IL3-independent growth. These results underline the power of insertional mutagenesis to identify subtle but initiating mutations that synergize with other lesions in oncogenic transformation.

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.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P <.03) and in vivo (P =.01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

Genetic protection of repopulating hematopoietic cells with an improved MDR1-retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice.

This study was undertaken to analyze the hematotoxicity of paclitaxel (Taxol) and to test whether transduction of repopulating hematopoietic cells with a retroviral vector (SF1m) expressing the human multidrug resistance 1 gene (MDR1) would permit dose intensification following bone marrow transplantation (BMT). While the regimen chosen (8 x 20 mg/kg i.p. within 12 days) produced a non-lethal, reversible hematotoxicity in mice with steady-state hematopoiesis, only 35.3% (6/17) of control mice survived when treated starting 14 days post BMT. In contrast, 83.3% (15/18) of mice transplanted with SF1m-transduced cells survived, owing to a significant protection against severe acute myelotoxicity (as determined by neutrophil counts, white and red blood cell counts and values for hemoglobin and hematocrit). After recovery from chemotherapy, an increase of myeloid cells that were resistant to colchicine and effluxed the fluorochrome Rhodamine 123 was observed in SF1m-mice, but not in controls. These results reveal that the lethal, dose-limiting hematotoxicity of an intensified post-transplantation chemotherapy with paclitaxel can be prevented by retroviral transfer of the MDR1 gene to a minor proportion of repopulating cells. Our mouse model, mimicking clinically achievable gene transfer rates, thus suggests that bone marrow chemoprotection may widen the therapeutic window and permit an earlier onset of post-transplantation chemotherapy.