Bone marrow mesenchymal stromal cells from acute myelogenous leukemia patients demonstrate adipogenic differentiation propensity with implications for leukemia cell support.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

Effects of Neddylation and mTOR Inhibition in Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Recently, the neddylation (NEDD8) and the mammalian target of rapamycin (mTOR) signaling pathways have emerged as promising pharmaceutical targets for AML therapy. However, the interaction of these two pathways remains unclear. Here we evaluated the effects of pevonedistat, an inhibitor of the NEDD8 activating enzyme (NAE), and sapanisertib (TAK-228), an inhibitor of mTORC1 and mTORC2 as single agents or in combination on AML cell lines. We found that inhibition of neddylation with pevonedistat partially inhibited mTOR signaling transduction and vice versa, inhibition of mTOR signaling with sapanisertib partially inhibited neddylation in AML cell lines. Pevonedistat alone was able to induce cytotoxicity in most AML cell lines as well as in primary AML, whereas sapanisertib alone decreased cell metabolic activity, reduced cell size and arrested cells in G0 phase with only minimal induction of cell death. In addition, pevonedistat was able to induce cell differentiation, arrest cells in G2/M cell cycle phases, and induce DNA re-replication and damage. However, co-treatment with sapanisertib suppressed pevonedistat induced apoptosis, differentiation, S/G2/M arrest, and DNA damage. Taken together, our data demonstrate that pevonedistat and sapanisertib exhibit distinct anti-tumor effects on AML cells, i.e. cytotoxic and cytostatic effects, respectively; however, sapanisertib can attenuate pevonedistat-induced cellular responses in AML cells. Understanding mTOR and neddylation pathway interaction could provide therapeutic strategies for treatment of AML and other malignancies.

Phenotypic, genotypic, and functional characterization of normal and acute myeloid leukemia-derived marrow endothelial cells.

In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to cell proliferation and survival. Endothelial cells from multiple vascular beds are able to prevent spontaneous or therapy-induced apoptosis in acute myelogenous leukemia (AML) blasts. Marrow-derived endothelial cells from leukemia patients have not been well-characterized, and in this work, endothelial cells were purified from marrow aspirates from normal subjects or from newly diagnosed AML patients to compare these cells phenotypically and functionally. By reverse transcription polymerase chain reaction, these cells express CD31, Tie-2, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS), supporting endothelial origin. They take up acetyl low-density lipoprotein and are able to form tubular structures. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and leukemia colony-forming unit outgrowth. RNA-sequencing analysis revealed 130 genes significantly up- or downregulated in AML-derived endothelial cells as compared with those derived from normal marrow. The genes differentially expressed (p < 0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos and genes involved in chemotaxis such as CXCL16. AML-derived endothelial cells are similar in phenotype and function to their normal marrow-derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes, which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment.

Peroxisome proliferator-activated receptor gamma ligands inhibit transforming growth factor-beta-induced, hyaluronan-dependent, T cell adhesion to orbital fibroblasts.

Thyroid eye disease is characterized by the infiltration of leukocytes and accumulation of hyaluronan (HA) in orbital tissue. Inflamed orbital tissue expands in size due to excessive HA and to the formation of scar tissue (fibrosis) and/or adipose accumulation. Transforming growth factor ß (TGF-ß) acts as a key inducer of fibrosis by enhancing extracellular matrix production. Treatment of primary human orbital fibroblasts with TGF-ß led to significant increases in both HA synthesis and secretion. TGF-ß also strongly induced hyaluronan synthase 1 (HAS1) and HAS2 mRNA levels, which increased 50- and 6-fold, respectively. Remarkably, the addition of the peroxisome proliferator-activated receptor (PPARγ) ligands pioglitazone (Pio) or rosiglitazone (Rosi) to TGF-ß-treated orbital fibroblasts attenuated HA synthesis and reduced HAS1 and HAS2 mRNA levels. The attenuation of TGF-ß function by Pio and Rosi was independent of PPARγ activity. Furthermore, Pio and Rosi treatment inhibited TGF-ß-induced T cell adhesion to orbital fibroblasts. Our findings demonstrate that TGF-ß plays an important role in HA synthesis and in the inflammatory response by enhancing or facilitating inflammatory cell infiltration and adhesion to orbital tissue. Pio and Rosi exhibit anti-fibrotic and anti-inflammatory activity and may be useful in treating thyroid eye disease.

Hyaluronan synthesis mediates the fibrotic response of keratocytes to transforming growth factor beta.

TGFß induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFß. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFß by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFß. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFß induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFß-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFß induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFß up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFß responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFß.

Mast cell-derived prostaglandin D2 controls hyaluronan synthesis in human orbital fibroblasts via DP1 activation: implications for thyroid eye disease.

Thyroid eye disease (TED) is a debilitating disorder characterized by the accumulation of adipocytes and hyaluronan (HA). Production of HA by fibroblasts leads to remarkable increases in tissue volume and to the anterior displacement of the eyes. Prostaglandin D(2) (PGD(2)), mainly produced by mast cells, promotes orbital fibroblast adipogenesis. The mechanism by which PGD(2) influences orbital fibroblasts and their synthesis of HA is poorly understood. We report here that mast cell-derived PGD(2) is a key factor that promotes HA biosynthesis by orbital fibroblasts. Primary orbital fibroblasts from TED patients were isolated and used to test the effects of PGD(2), prostaglandin J(2), as well as prostaglandin D receptor (DP) agonists and antagonists on HA synthesis. The expression of HA synthase (HAS), hyaluronidase, DP1, and DP2 mRNA levels was assessed by PCR. Small interfering RNAs against HAS1 or HAS2 were used to assess the importance of HAS isoforms on HA production. Treatment of human orbital fibroblasts with PGD(2) and PGJ(2) increased HA synthesis and HAS mRNA. HAS2 was the dominant isoform responsible for HA production by PGD(2). The effect of PGD(2) on HA production was mimicked by the selective DP1 agonist BW245C. The DP1 antagonist MK-0524 completely blocked PGD(2)-induced HA synthesis. Human mast cells (HMC-1) produced PGD(2). Co-culture of HMC-1 cells with orbital fibroblasts induced HA production and inhibition of mast cell-derived PGD(2) prevented HA synthesis. Mast cell-derived PGD(2) increased HA production via activation of DP1. Selectively targeting the production of PGD(2) and/or activation of DP1 may prevent pathological changes associated with TED.

Stem cell therapy restores transparency to defective murine corneas.

Corneal scarring from trauma and inflammation disrupts vision for millions worldwide, but corneal transplantation, the primary therapy for corneal blindness, is unavailable to many affected individuals. In this study, stem cells isolated from adult human corneal stroma were examined for the ability to correct stromal opacity in a murine model by direct injection of cells into the corneal stroma. In wild-type mice, injected human stem cells remained viable for months without fusing with host cells or eliciting an immune T-cell response. Human corneal-specific extracellular matrix, including the proteoglycans lumican and keratocan, accumulated in the treated corneas. Lumican-null mice have corneal opacity similar to that of scar tissue as a result of disruption of stromal collagen organization. After injection with human stromal stem cells, stromal thickness and collagen fibril defects in these mice were restored to that of normal mice. Corneal transparency in the treated mice was indistinguishable from that of wild-type mice. These results support the immune privilege of adult stem cells and the ability of stem cell therapy to regenerate tissue in a manner analogous to organogenesis and clearly different from that of normal wound healing. The results suggest that cell-based therapy can be an effective approach to treatment of human corneal blindness.

A rapid transient increase in hyaluronan synthase-2 mRNA initiates secretion of hyaluronan by corneal keratocytes in response to transforming growth factor beta.

Keratocytes of the corneal stroma produce transparent extracellular matrix devoid of hyaluronan (HA); however, in corneal pathologies and wounds, HA is abundant. We previously showed primary keratocytes cultured under serum-free conditions to secrete matrix similar to that of normal stroma, but serum and transforming growth factor beta (TGFbeta) induced secretion of fibrotic matrix components, including HA. This study found HA secretion by primary bovine keratocytes to increase rapidly in response to TGFbeta, reaching a maximum in 12 h and then decreasing to <5% of the maximum by 48 h. Cell-free biosynthesis of HA by cell extracts also exhibited a transient peak at 12 h after TGFbeta treatment. mRNA for hyaluronan synthase enzymes HAS1 and HAS2 increased >10- and >50-fold, respectively, in 4-6 h, decreasing to near original levels after 24-48 h. Small interfering RNA against HAS2 inhibited the transient increase of HAS2 mRNA and completely blocked HA induction, but small interfering RNA to HAS1 had no effect on HA secretion. HAS2 mRNA was induced by a variety of mitogens, and TGFbeta acted synergistically to induce HAS2 by as much as 150-fold. In addition to HA synthesis, treatment with TGFbeta induced degradation of fluorescein-HA added to culture medium. These results show HA secretion by keratocytes to be initiated by a rapid transient increase in the HAS2 mRNA pool. The very rapid induction of HA expression in keratocytes suggests a functional role of this molecule in the fibrotic response of keratocytes to wound healing.

Protection of hepatocytes from apoptosis by a novel substance from actinomycetes culture medium.

A novel substance, #675, found from an Streptomyces sp. SM675 culture medium, dose-dependently stimulates the proliferation of human functional liver cell 4 (FLC4). When FLC4 cells were incubated under conditions without fetal bovine serum (FBS), typical features of apoptotic cell death such as shrinkage and nuclear condensation appeared; high molecular weight (HMW) DNA fragments were found; and caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were cleaved. When FLC4 cells were incubated with #675 and without FBS, the cells grew healthy, no HMW DNA fragments were found, and caspase-3 and PARP cleavage weakened, suggesting that #675 protects FLC4 cells from apoptosis induced by FBS-deprivation. The quantitative reverse-transcribed polymerase chain reaction did not show differences in PARP or Bcl-2 mRNA expression in FLC4 cells incubated with or without #675, indicating other genes may be involved in this anti-apoptosis effect. These results show that #675 enhances FLC4 proliferation via an apoptosis-inhibition pathway, implying potential pharmacological and clinical applications.

Repeated immunization induces the increase in fucose content on antigen-specific IgG N-linked oligosaccharides.

OBJECTIVE: In order to investigate whether repeated immunization induces changes in IgG glycosylation, we analyzed the composition of oligosaccharides on antigen-specific IgGs obtained from mice that received different amounts of immunization boosts. METHODS: Three groups of mice were immunized with ovalbumin (OVA) and boosted once, twice, or three times, respectively, with an interval of 1 week. The patterns of oligosaccharides present in anti-OVA specific IgGs were analyzed using lectin-enzyme-linked immunosorbent assay (ELISA) and lectin-blot. RESULTS: The repeated injection of OVA induced both the production of specific IgGs and an increase of fucose content in N-linked oligosaccharides of the IgGs. The lowest IgG fucosylation was observed in mice boosted once, whereas the highest fucosylation rate was observed in mice boosted three times. ELISA assay demonstrated that there was a positive relationship between the fucose content and amount of immunization boosts. CONCLUSIONS: IgG fucosylation increases during repeated immunization with ovalbumin. The alteration of IgG fucosylation may have important biological significance.

Pharmacological mechanism in slip-down behavior of mice.

Slip-down from a raised platform was previously found in mice treated with morphine, and this behavior was also recognized in mice treated with a monoamine releaser methamphetamine. Pharmacological examination on the slip-down indicated that the behavior was induced by receptor stimulations by D2 agonist PPHT and 5HT-2 agonist DOI. In mice treated with PPHT, antagonists of D1, alpha2, 5HT-2 and opioid mu and kappa suppressed the behavior. In mice treated with DOI, antagonists of D1, alpha2, 5HT-1A, 5HT-3 and opioid mu and delta suppressed the behavior. These present findings suggest that the slip-down was mainly induced by opioid mu receptor activity regulated with monoamine activities. When the slip-down is considered as an anxious behavior, it may be also suggested that the anxiety induced by 5-HT activities furthermore stimulated the behavior via the other opioid receptor activities.

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats.

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.