Ectopic TLX1 expression accelerates malignancies in mice deficient in DNA-PK.

The noncluster homeobox gene HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHµ-TLX1(Tg) mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHµ-TLX1(Tg) mice with mice deficient in the DNA repair enzyme DNA-PK (Prkdc(Scid/Scid) mice). IgHµ-TLX1(Tg)Prkdc(Scid/Scid) mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control Prkdc(Scid/Scid) mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant IgHµ-TLX1(Tg)Prkdc(Scid/Scid) thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.

eNOS overexpressing bone marrow cells are safe and effective in a porcine model of myocardial regeneration following acute myocardial infarction.

AIM: Cell therapy has been shown to be effective in improving LV function postmyocardial infarction (MI). We hypothesized that eNOS-transfected bone marrow cells (BMCs) are safe in a swine model of myocardial infarction (MI). We also hypothesized that endothelial nitric oxide synthase (eNOS) transfection would enhance cell function, as assessed by myocardial functional recovery post-MI. METHODS: Fifteen female Yorkshire pigs underwent bone marrow aspiration and creation of MI. Bone marrow cells were cultured for 7 days, and each pig received either autologous BMCs transiently transfected with eNOS plasmid (eNOS-BMC, n = 5), nontransfected BMCs (nt-BMC, n = 4), or phosphate-buffered saline (PBS) control (n = 6). Cardiac MRI was performed at baseline (1 week post-MI) and 6 weeks post-MI. RESULTS: There was no difference in safety outcomes between groups. Absolute left ventricular ejection fraction (LVEF) at 6 weeks showed a trend toward improvement in both cell therapy groups compared with baseline but worsened in the PBS control group. The absolute improvement in LVEF was significantly greater in both cell therapy groups compared with PBS control. Infarct mass was significantly lower in the eNOS-BMC group between baseline and 6 weeks, but the absolute change in infarct mass was not different between groups. Finally, there was a trend toward reduced LV mass in the eNOS-BMC group. CONCLUSIONS: Bone marrow cell delivery, with and without eNOS overexpression, is safe and leads to improvement in LVEF when administered in the coronary circulation 7 days following acute MI in swine. Transfection of healthy BMCs with eNOS resulted in some improvement in left ventricular remodeling. Further study is warranted in a preclinical model that approximates the impact of cardiovascular risk factors on BMC function.

Resveratrol has anti-leukemic activity associated with decreased O-GlcNAcylated proteins.

CLL cells are characterized by high levels of proteins that are post-translationally modified by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties, but it is not clear whether O-GlcNAc is a relevant therapeutic target. The neutraceutical resveratrol is cytotoxic to chronic lymphocytic leukemia cells in vitro. In this study, we found that resveratrol has therapeutic activity as a single agent in vivo in both human chronic lymphocytic leukemia patients and mice with erythroleukemia. Blood and splenic O-GlcNAc levels reflected the changes in tumor burden. Resveratrol directly lowered O-GlcNAc levels in leukemia cells through proteasomal activation, but increasing O-GlcNAc levels in vitro did not prevent cell death. These findings suggest that resveratrol has potential as a novel treatment for some forms of chronic and acute leukemia, and the measurement of O-GlcNAc levels could be a surrogate marker for therapeutic responses.

The miR-17-92 cluster expands multipotent hematopoietic progenitors whereas imbalanced expression of its individual oncogenic miRNAs promotes leukemia in mice.

The miR-17-92 cluster and its 6 encoded miRNAs are frequently amplified and aberrantly expressed in various malignancies. This study demonstrates that retroviral-mediated miR-17-92 overexpression promotes expansion of multipotent hematopoietic progenitors in mice. Cell lines derived from these miR-17-92-overexpressing mice are capable of myeloid and lymphoid lineage differentiation, and recapitulate the normal lymphoid phenotype when transplanted to nonobese diabetic/severe combined immunodeficiency mice. However, overexpression of individual miRNAs from this locus, miR-19a or miR-92a, results in B-cell hyperplasia and erythroleukemia, respectively. Coexpression of another member of this cluster miR-17, with miR-92a, abrogates miR-92a-induced erythroleukemogenesis. Accordingly, we identified several novel miR-92a and miR-17 target genes regulating erythroid survival and proliferation, including p53. Expression of this critical target results in marked growth inhibition of miR-92a erythroleukemic cells. In both murine and human leukemias, p53 inactivation contributed to the selective overexpression of oncogenic miR-92a and miR-19a, and down-regulation of tumor-suppressive miR-17. This miR-17-92 expression signature was also detected in p53- B-cell chronic lymphocytic leukemia patients displaying an aggressive clinical phenotype. These results revealed that imbalanced miR-17-92 expression, also mediated by p53, directly transforms the hematopoietic compartment. Thus examination of such miRNA expression signatures should aid in the diagnosis and treatment of cancers displaying miR-17-92 gene amplification.

Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses.

BACKGROUND: In utero hematopoietic cell transplantation offers a means of early intervention for the treatment of diseases before birth. Delivery of cells to the yolk sac is a minimally invasive approach that results in low levels of chimerism. However, there is little information on the optimal doses, timing of delivery, and migration of transplanted cells from the yolk sac into the fetus. METHODS: Varying cell doses of mesenchymal stromal cells or bone marrow mononuclear cells labeled with fluorescent supraparamagnetic iron oxide nanoparticles and a fluorescent intracellular dye, 5- and 6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine, were transplanted under ultrasound guidance to the yolk sacs of day 25 or day 35 canine fetuses. Ex vivo whole body fluorescence imaging and microscopy of tissue sections were correlated with the presence of iron oxide in injected and control fetuses. RESULTS: Day 25 and day 35 recipients showed similar survival rates after injection of cells into yolk sacs, although increased fetal morality was associated with cell doses greater than 10 cells/kg to day 25 fetuses. The fluorescence and iron oxide signals were predominantly localized to the abdominal regions, with no fluorescence visible in yolk sacs. Microscopy of tissues revealed colocalization of fluorophore with iron oxide in donor cells detected in the fetal livers and bone marrow of recipients 7 and 17 days after receiving mesenchymal stromal cells or bone marrow mononuclear cells. CONCLUSIONS: These studies demonstrated that cells injected into the yolk sacs of early gestation canine fetuses migrate to recipient hematopoietic tissues. Thus, yolk sac injection offers a safe and effective approach for engraftment of cells to fetal hematopoietic tissues.

Phosphorylation of HOX11/TLX1 on Threonine-247 during mitosis modulates expression of cyclin B1.

BACKGROUND: The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11. RESULTS: Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis. CONCLUSIONS: Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint.

Long-term tracking of bone marrow progenitor cells following intracoronary injection post-myocardial infarction in swine using MRI.

Magnetic resonance imaging (MRI) can track progenitor cells following direct intramyocardial injection. However, in the vast majority of post-myocardial infarction (MI) clinical trials, cells are delivered by the intracoronary (IC) route, which results in far greater dispersion within the myocardium. Therefore, we assessed whether the more diffuse distribution of cells following IC delivery could be imaged longitudinally with MRI. In 11 pigs (7 active, 4 controls), MI was induced by 90-min balloon occlusion of the left anterior descending coronary artery. Seven (0) days [median (interquartile range)] following MI, bone marrow progenitor cells (BMCs) were colabeled with an iron-fluorophore and a cell viability marker and delivered to the left anterior descending coronary artery distal to an inflated over-the-wire percutaneous transluminal coronary angioplasty balloon. T2*-weighted images were used to assess the location of the magnetically labeled cells over a 6-wk period post-MI. Immediately following cell delivery, hypointensity characteristic of the magnetic label was observed in the infarct border rather than within the infarct itself. At 6 wk, the cell signal hypointensity persisted, albeit with significantly decreased intensity. BMC delivery resulted in significant improvement in infarct volume and ejection fraction (EF): infarct volume in cell-treated animals decreased from 7.1 +/- 1.5 to 4.9 +/- 1.0 ml (P < 0.01); infarct volume in controls was virtually unchanged at 4.64 +/- 2.1 to 4.39 +/- 2.1 ml (P = 0.7). EF in cell-treated animals went from 30.4 +/- 5.2% preinjection to 34.5 +/- 2.5% 6 wk postinjection (P = 0.013); EF in control animals went from 34.3 +/- 4.7 to 31.9 +/- 6.8% (P = 0.5). Immunohistochemical analysis revealed intracellular colocalization of the iron fluorophore and cell viability dye with the labeled cells continuing to express the same surface markers as at baseline. MRI can track the persistence and distribution of magnetically labeled BMCs over a 6-wk period following IC delivery. Signal hypointensity declines with time, particularly in the first week following delivery. These cells maintain their original phenotype during this time course. Delivery of these cells appears safe and results in improvement in infarct size and left ventricular ejection fraction.

Derivation and characterization of canine embryonic stem cell lines with in vitro and in vivo differentiation potential.

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation-stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, whereas they expressed very low levels of SSEA-1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC-based therapies in large animal models of human disease.

Gene expression profiling in the inductive human hematopoietic microenvironment.

Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC milieu.

In utero injection of alpha-L-iduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of multiple tissues and neonatal gene expression.

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Human hematopoietic progenitors engraft in fetal canine recipients and expand with neonatal injection of fibroblasts expressing human hematopoietic cytokines.

OBJECTIVE: The development of large-animal models for human hematopoiesis will facilitate the study of human hematopoietic stem cells and their progenitors in vivo. In previous studies, human hematopoietic progenitors engrafted in fetal dogs and contributed to hematopoiesis for one year. Despite initially high levels of human cells, the proportion declined to less than 0.1% at 6 months, possibly due to inability of the canine hematopoietic microenvironment to support ongoing human hematopoiesis. In the current experiments we examined the potential of co-transplanting fibroblasts expressing human hematopoietic cytokines with the hematopoietic graft to increase the contribution of human progenitors to chimeric hematopoiesis. METHODS: Mid-gestation canine fetuses were injected with 1-3 x 10(7) human cord blood cells and 1 x 10(7) murine fibroblasts engineered to express human cytokines. Neonatal pups were boosted with additional injections of cytokine-expressing fibroblasts. Human cell engraftment was monitored by PCR amplification of human-specific DNA sequences from recipient hematopoietic tissues. RESULTS: Human hematopoietic cells were detected in 13/15 fetal recipients for at least 7 months. At time points up to 30 weeks of age, human DNA was detected in stimulated lymphocyte cultures, approximately 0.1% of blood leukocytes and 1.5% (85/5757) of myeloid colonies. Eight months postinfusion, 1.7% of colony-forming units (CFUs) were of human origin. By one year 0.5% or less of myeloid colonies and less than 0.01% of blood leukocytes carried human DNA. Following an infusion of cytokine-expressing fibroblasts at one year, the proportion of human myeloid progenitors rose to 11.5% and remained detectable for 8 months. CONCLUSION: These studies confirm that human hematopoietic progenitors can engraft in fetal pups and contribute to multilineage hematopoiesis. Infusion of cells expressing human cytokines is one approach to stimulate human hematopoietic progenitors in vivo and thus increase their contributions to chimeric hematopoiesis.