Compact Colonies Research Articles

Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells

The first steps of invasion and metastasis include the dissociation of adherens junctions and the induction of migratory phenotype, through a program that resembles epithelial-mesenchymal transition (EMT). The L1 cell adhesion molecule, which is normally found primarily in the brain, was recently shown to be expressed in different types of cancer and to have tumor-promoting activity. We now find that L1 mediates EMT-like events in MCF7 breast carcinoma cells. MCF7 predominantly expresses the nonneuronal isoform of L1, as do 16 of 17 other cell lines derived from different types of cancer. L1 protein expression in MCF7 cells, which form E-cadherin-containing adherens junctions, is inversely related to cell density. Analysis of MCF7 cells with overexpression or knockdown of nonneuronal L1 isoform revealed that L1 expression leads to the disruption of adherens junctions and increases beta-catenin transcriptional activity. As a result, L1 expression promotes the scattering of epithelial cells from compact colonies. Expression of the full-length L1 protein, but not of its soluble extracellular moiety, increases the motility of the MCF7 epithelial monolayer in a wound-healing assay, in which L1 expression is preferentially observed and required in cells leading the movement of the monolayer. Based on these results, we propose a model for the role of L1 as a trigger of EMT-like events in transformed epithelial cells.

The Mll-AF9 Knock-In Fusion Gene Results in Reprogramming and Enhanced Self-Renewal of Hematopoietic Progenitors, Especially in the HSC and CLP Populations.

The relative importance of the major hematopoietic progenitor/stem cell populations in MLL fusion gene leukemias remains largely undefined. Several studies have demonstrated that retrovirally transformed populations will produce leukemia in mice. However, because the levels of gene expression are known to alter cellular effects and MLL fusion gene expression in previous studies is generally supraphysiologic, uncontrolled and variable, comparison of individual population is difficult. In this study, we used knock-in mice with the fusion gene inserted in the genomic DNA by homologous recombination as an alternative model, permitting study of cells in which the fusion gene is under the control of the endogenous promoter and thus expressed at physiological levels in all progenitor/stem cells. Such knock-in cells also have the advantage that there is haploinsufficiency of the Mll gene expression which may be important in cellular dysfunction. Mll-AF9 knock-in mice develop myeloproliferation as early as 6 days of age and develop myeloproliferative disease-like (MPD-like) myeloid leukemia after six months latency. We postulated that Mll-AF9 induced deregulation would result in progenitor cells with quantifiably different biologic properties. In this study, we evaluated marrow progenitor/stem cells from young mice before leukemia development. Wild type or Mll-AF9 marrow cells from 3–6 young mice (8 weeks old) were sorted into HSC, CLP, CMP and GMP populations using standard protocols and grown in methylcellulose medium containing IL-3, IL-6, SCF, and GM-CSF. After 7 days' culture, 3.6 fold more myeloid colonies were found in GMP and 2.5 fold more in CMP groups from Mll-AF9 compared to WT mice. After the third plating generation (21 days in culture), a very different pattern emerged. As expected, only rare colonies were found from WT mice whereas Mll-AF9 HSC and CLP groups resulted in significantly more myeloid colonies (60.1±5.5 and 67.5±10.5, respectively) than the CMP and GMP groups (27.2±3.8 and 20.9±4.1, respectively). Dense compact (Type I) colonies composed of immature myeloid cell were frequent in the Mll-AF9 HSC and CLP groups. Two-dimensional Sca-1 and c-kit plots showed that Mll-AF9 resulted in increases from a mean of 6.4% to 12.9% in HSCs and 12.4 to 27.3% in CLPs. Remarkably, Mll-AF9 CLP day 7 cultures had notably high number of CD11b+Gr1+ cells (mean of 57%). Mll-AF9 CLPs cultured under myeloid conditions also showed mixed lineage characteristics: expression of myeloid genes (GM-CSF receptor, c-fms, and lysozyme) and early B lymphoid genes (IL-7 receptor, TdT but not PAX5) and D-J rearrangement. In summary, our results show that Mll-AF9, when expressed at physiologic levels, reprograms progenitor/stem cells from young mice months before development of leukemia. CLPs are remarkable because in contrast to lymphoid restricted wild type CLPs, Mll-AF9 CLPs are mixed lineage in phenotype. Mll-AF9 resulted in increased self-renewal in all four HSC, CLP, CMP, and GMP populations compared with WT populations. However, importantly, in Mll-AF9 mice, self-renewal was most enhanced in cells with more intrinsic self-renewal potential and/or more evidence of reprogramming (HSCs, CLPs) than in committed myeloid progenitors. A predicted consequence of these differences is that cells with the highest Mll-AF9 induced self-renewal (HSCs, CLPs) will be more efficient than transformed CMPs and GMPs in inducing leukemia.

Noggin Overexpression Enhances Leukemic Progenitors Self-Renewal in AML by Abrogating the BMP Pathway Activation.

Noggin, encoded by Nog gene, is a secreted protein that inhibits the bone morphogenetic proteins (BMPs). BMPs regulate the proliferation and differentiation of many tissues, including bone, kidney and hematopoiesis, mainly through phosporylation of SMAD1, 5 or 8 . By cDNA-RDA, we found overexpression of Nog in OCI-AML2 and OCI-AML5 cell lines compared to normal bone marrow (NBM). To confirm this overexpression, we performed real-time RT-PCR on 6 NBM, 2 NBM CD34+ cells, 6 AML cell lines (OCI-AML1-5 and NB4) and 53 AML patients' (pts) samples. NBM had very low Nog/GAPDH (Median: 1.63 X 10−5, range: 1.55 X 10−5-1.75 X 10−5). Similar levels were found in CD34+ cells. Five cells lines (OCI-AML-1, -2, -4, -5 and NB4) showed high Nog/GAPDH (range: 7.5 X 10−4 - 4.3 X 10−3), whereas OCI-AML3 expessed very low level (8.6 X 10−7). High Nog expression (>4-fold NBM level) was, also, found in 51/53 pts (96%), with a median 45.8-fold NBM level. Protein study revealed a positive correlation between Noggin and Nog RNA levels. In contrast, there was a negative correlation between Noggin and phosphorylated SMAD1, 5, 8, suggesting that Noggin plays a key role in the BMP pathway regulation in hematopoietic tissues. To analyze the effect of Noggin on leukemic behavior, we assessed the effect of Noggin on the clonogenicity of OCI-AML3. OCI/AML-3 cultured with Noggin showed enhanced plating efficiency (56±7 vs 33±6, p=0.005), increased colony size and more compact colonies. To determine whether this higher efficiency is due to enhanced leukemic progenitors self-renewal, cells were grown in suspension culture in the presence of Noggin, washed and plated in methylcellulose medium without Noggin on days 1–3. Cells treated with Noggin showed increasing colony-forming potential with time, suggesting an enhancement of leukemic progenitors self-renewal. To further characterize the effect of Noggin on OCI/AML-3 cells we performed cell-cycle and apoptosis assays. Noggin significantly increased the percentage of cells in S/G2/M phase as early as 3h after treatment with a maximum effect by 24h (38% vs 27%). Noggin, also, decreased the percentage of cells undergoing apoptosis (2.8% vs 9.1%), an effect that became noticeable within 24h. Moreover, Noggin protected the leukemic cells against serum-deprivation and daunorubicin-induced apoptosis. As Noggin is a negative regulator of the BMP pathway we assessed the effect of Noggin on the expression of genes known to be affected by BMP, including DKK1, Wnt1, Wnt3a, Wnt5b, P21, P27, Bcl2, Bax, MSX1, MSX2, Id1, Id2, Id3, DLX1 and DLX2. While complete inhibition of SMAD 1,5,8 phosphorylation could be detected within 1 h of the addition of Noggin, gene expression changes were noticed beginning after 4h. Significant decreased expression at the RNA level was noted for DKK1, Wnt1, Wnt3a, Wnt5b, MSX1, Id1, Id3 and DLX2. Although substantial downregulation of the Wnt-pathway-related genes was seen, there was no change in this pathway overall activity as assessed by B-catenin protein and Wnt-pathway target-genes RNA levels. In summary, AML cells express and respond to the BMP inhibitor Noggin. BMP pathway inhibition increases the leukemic progenitors growth and self- renewal. The reduced expression of MSX1 is of interesting importance as MSX1 promotes p53 apoptotic function. The loss of MSX1 may thus reduce the cells sensitivity to apoptotic stimuli, such as daunorubicin.

The Mll-AF9 Fusion Gene Results in Overexpression of Homeobox Genes and Deregulation of Granulocyte-Monocyte Progenitors.

Our understanding of the biology of MLL fusion gene leukemias is limited by the lack of knowledge of the effects of the different MLL fusion genes on expression of specific homoebox genes and the specific cell compartment(s) that are subsequently deregulated. In this study we investigated whether cellular deregulation was present in committed myeloid precursors and/or the multi-potent hematopoietic stem cells derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll fusion gene under the control of the endogenous promoter that is present in every hematopoietic stem/progenitor cell. The Mll-AF9 knock-in mice display expansion of the myeloid compartment as early as 6 weeks of age (young adult) and develop myeloid leukemia at approximately 6 months. We purified hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitors (GMPs) from wild type and Mll-AF9 young adult bone marrow. We depleted lineage positive cells using a magnetic separation system and purified the respective populations using fluorescence activated cell sorting with specific panels of antibodies (HSC=Li−/Thy1.1lo/IL-7R−/C-kit+/Sca-1+; GMP=Lin−/IL-7R−/Sca-1+/C-kit+/CD34+/CD16/32hi). We cultured these cells in methylcellulose supplemented with GM-CSF, IL-3, SCF and IL-6, conditions that promote the growth of myeloid colonies. We assessed growth deregulation by increased colony numbers at the end of 7 days of culture and by the predominance of dense, compact colony morphology, the latter comprised of immature myeloid cells. Culture of HSCs from Mll-AF9 and wild type mice yielded an identical number of colonies (1102 and 1315 colonies per 104 cells respectively, average). In contrast, GMPs from Mll-AF9 mice yielded almost four times the number of colonies compared to wild type GMPs (3331 and 920 colonies per 104 cells respectively, average). Additionally, Mll-AF9 GMPs formed a higher number of dense, compact colonies compared to Mll-AF9 HSCs (1314 and 352 colonies per 104 cells respectively, average). Neither HSCs nor GMPs from wild type mice formed dense, compact colonies. These results indicate a greater deregulation of GMPs compared to HSCs in Mll-AF9 mice. MLL fusion gene leukemias are characterized by over-expression of specific homeobox genes, and we have previously shown that Mll-AF9 bone marrow cells display increased expression of 5′ Hox-a genes and of the Hox co-factor Meis1 compared to wild type counterparts. We hypothesized that these genes are over-expressed in Mll-AF9 GMPs compared to wild type GMPs. Real time quantitative RT-PCR showed that expression levels of Hoxa7, Hoxa9 and Meis1 were increased in Mll-AF9 GMPs compared to wild type (2.7 ± 0.8, 11.7 ± 7.8 and 19 ± 11.3 fold respectively, mean ± SEM). Overall, these data support the hypothesis that the Mll-AF9 gene is “instructive” at the molecular level at least in part via specific homeobox gene over-expression, resulting in deregulation and expansion of specific progenitor/stem cells such as the GMP population. This expanded GMP population then becomes a target for secondary mutations and later development of leukemia. Future studies focused on understanding the biology of this compartment in Mll-AF9 mice will help in our understanding of the pathogenesis of leukemia and aid in the development of newer, more effective therapies.

Candida albicans strains deficient in CHS7, a key regulator of chitin synthase III, exhibit morphogenetic alterations and attenuated virulence

Chitin is a structural polysaccharide present in most fungal cell walls, whose synthesis depends on a family of enzymic activities named chitin synthases (CSs). The specific role of each of them, as well as of their regulatory proteins, in cell morphogenesis and virulence is not well understood. Here, it is shown that most chitin synthesis in Candida albicans, one of the fungi most commonly isolated from opportunistic mycoses and infections, depends on CHS7. Thus, C. albicans chs7Delta null mutants showed reduced levels of chitin and CS activity, and were resistant to Calcofluor. Despite the sequence similarity and functional relationship with ScChs7p, CaChs7p was unable to restore CSIII activity in a Saccharomyces cerevisiae chs7Delta null mutant, because it was unable to direct ScChs3p export from the endoplasmic reticulum. C. albicans chs7Delta null mutants did not show any defect in growth rate, but yeast cells displayed minor morphogenetic defects affecting septum formation, and showed an increased tendency to form filaments. CaChs7p was not required for germ-tube emission, and null mutant strains underwent the dimorphic transition correctly. However, colony morphology appeared distinctively affected. chs7Delta hyphae were very curved and had irregular lateral walls, resulting in very compact colonies that seemed unable to spread out radially on the surface, unlike the wild-type. This growth pattern may be associated with the reduced virulence and high clearance rate observed when the chs7Delta strain was used in a murine model of infection. Therefore, CaChs7p is required for normal hyphal morphogenesis, suggesting that in C. albicans CSIII plays an important role in maintaining cell wall integrity, being essential when invading surrounding tissues.

Tumour necrosis factor α confers an invasive, transformed phenotype on mammary epithelial cells

Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor alpha (TNF-alpha) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-alpha abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-alpha induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-alpha-induced 3D scattering. TNF-alpha also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the alpha2 integrin subunit. Addition of a blocking antibody to beta1-integrin or of rhodocetin (a specific alpha2beta1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and alpha2beta1 integrin in the invasive response of 31EG4-2A4 cells to TNF-alpha. We propose that the biological activities described in this study contribute to the ability of TNF-alpha to promote tumour progression and cancer-cell dissemination.

MEK kinase 1 is essential for Bcr–Abl-induced STAT3 and self-renewal activity in embryonic stem cells

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate signal transducers and activators of transcription 3 (STAT3) and to promote self-renewal in embryonic stem (ES) cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction. To investigate the role of MEKK1 in Bcr-Abl-induced transformation of stem cells, p210 Bcr-Abl was stably transfected into wild-type (WT(p210)) and MEKK1-/- (MEKK1-/-(p210)) ES cells. Bcr-Abl enhanced MEKK1 expression in ES transfectants, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT(p210) cells showed constitutive STAT3 activation and formed rounded, compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1-/-(p210) cells, by contrast, showed less STAT3 activity than WT(p210) cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays a key role in Bcr-Abl-induced STAT3 activation and in ES cells' capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.

194 ESTABLISHMENT OF GOAT EMBRYONIC STEM CELL-LIKE LINES DERIVED FROM IN VIVO-PRODUCED BLASTOCYST-STAGE EMBRYOS

Pluripotent embryonic stem cells (ESC) derived from the inner cell mass (ICM) of mammalian blastocysts provide AN unlimited number of cells that can be used in gene targeting and be of great value to agriculture and medicine. Embryonic stem cells with capacity for germ line transmission have been verified only in mouse despite many efforts to derive ESC from other mammalian species. The methods for the derivation, propagation and differentiation of ESC from domestic animals have not been fully established. The objective of this study was the generation and initial characterization of goat embryonic stem cells (GESC) derived from in vivo-produced blastocyst-stage embryos. Goat compact morulae and blastocysts were collected from superovulated adult Saanen crossbreed donors 7 days after insemination with a fertile male. Embryos were collected by uterine flushing using a 12Fr. Foley catheter by means of a laparoscopically assisted mid-ventral laparotomy under general anesthesia. Twenty eight in vivo-derived blastosyst-stage embryos were cultured on a goat fetal fibroblast feeder layer (inactivated by Mitomycin C) in a medium of DMEM containing 0.1 mm �-mercaptoethanol, 0.1 mm MEM nonessential amino acids, 200 mm l-glutamine, and 10% FCS. Following three days in culture 25 of 28 embryos hatched. Ten embryos attached to the feeder layer, nine degenerated, and nine embryos were floating in the medium and expanding in size. After 5-7 days in culture four of the tehn attached embryos appeared with a prominent an ICM outgrowth and 3 of the nine floating embryos formed structures resembling ICM disc surrounded by trophectoderm cells. The ICM and the embryonic discs were isolated mechanically and cultured on goat feeder cells in DMEM medium containing 10 ng/mL horse leukemia inhibitory factor (hLIF) and 10% FCS. The ICM and embryonic disc outgrew into colonies on Day 4 post-culture. Compact colonies of cells from these outgrowths were isolated mechanically and passed onto fresh goat feeder cells every 4-5 days with the addition of 10 ng/mL hLIF to the culture media. Established colonies at passage 6 were tested for immunoreactivity against alkaline phosphatase (AP) and Oct-1 using standard protocols. The result was the estabilshment of embryonic stem cell-like colonies (57%) from both ICM and embryonic discs cultured on goat fetal fibroblast feeder cells. Colonies forming from these outgrowths (50%) of either ICM or embryonic disc stained positive for both AP and Oct-4. Embryoid bodies formed from colonies of either ICM or embryonic disc in suspension (DMEM containing 10% FCS with no hLIF). Two cell lines (one from ICM and one from embryonic disc) have been maintained so far through passage 8 and have been cryopreserved. These GESC-like lines will be used in further characterization and ultimately in transgenic animal production studies.

201 CHARACTERIZATION OF CANINE EMBRYONIC GERM CELLS: AN EXPERIMENTAL MODEL FOR CELL THERAPY

Dogs represent excellent models to test different approaches before use for human therapy. Studies with animal models have suggested that the transplant of stem cells may have success in the treatment of degenerative diseases such as Parkinson's disease, diabetes, Duchenne muscular dystrophy (DMD), and acquired lesions. Embryonic stem cells are pluripotent and therefore have the potential to form all tissues. Our research aims to contribute to the treatment of DMD through the isolation and identification of embryonic germ cells and the development of the methodology of cellular differentiation for future transplantation into dystrophic dogs. Mongrel female dogs were ovariehysterectomized between 25 and 30 days of pregnancy. For recovery of embryos, the excised uterine horns were flushed with heparinized PBS. Samples collected from somites near the mesonephros area of four embryos recovered at 22 to 24 days of pregnancy and designated as A1 through A4 were dissociated and placed in culture. Isolated embryonic cells were allowed to plate onto monolayers of canine fibroblast cells. Flow cytometry was used to identify CD34+ markers. Isolated compact colonies of embryonic germ cells were seen growing around tissue fragments at 7 days of culture and remained in the undifferentiated stage until approximately 21 days in culture. At 14 days of explant, cell colonies were analyzed by flow cytometry. Cells from the A2 embryos contained the highest number of CD34+ cells, whereas no cells from A4 embryos showed specificity for the marker. A small proportion (2.12%) of cells from embryos A1 and A3 showed specificity for the CD34 marker. A quantity of A2 embryo cells that had maintained stem cell characteristics were frozen for future studies. Our results suggest that although spontaneous differentiation occurred, a small population of cells maintain the characteristics of stem cells. We are currently trying to improve the methodology to maintain cells undifferentiated for longer periods and to better control the specific differentiation process.

Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.

Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.

Cyp33 Immortalizes Mouse Bone Marrow Progenitor Cells.

MLL is a gene involved in translocations associated with leukemia. It encodes a protein with PHD fingers and SET domains that are conserved in the related Drosophila protein trithorax. Cyp33, a nuclear cyclophilin with an RNA binding domain, interacts with the MLL PHD finger domain. Overexpression of Cyp33 represses the Hoxc8 gene in human leukemia cells in an MLL dependent manner. This close relationship between Cyp33 and the genes involved in hematopoiesis and leukemogenesis inspires us to further clarify the function of Cyp33 in primary hematopoietic cells. Murine bone marrow progenitor cells (BMPC) were retrovirally transduced to express Cyp33 and their replicative potential was then investigated by repeatedly plating on methylcellulose medium supplemented with GM-CSF. Upon over-expression of Cyp33, p27 is down-regulated and the progenitor cells keep proliferating after the fourth plating while the control cells (infected with empty retroviral vector only) differentiate and stop forming colonies after the third plating. Furthermore, after the third plating, Cyp33 over-expressing cells exhibit primitive cell morphology after Wright staining, while the control cells show mature macrophage/granulocyte characteristics. We also generated a Cyp33-BMPC line by growing the Cyp33 over-expressing cells in liquid culture containing GM-CSF, after the fourth plating. Regardless of the presence of GM-CSF, the Cyp33-BMPC cell line cycles and grows exponentially maintaining stem cell surface markers: Sca-1+, c-Kit+, CD34+ and Lin− (B220−, CD3e−, Mac-1−, Gr-1−). Morphologically, Cyp33-BMPCs are like immature progenitor cells. In addition, these cells can again form compact colonies on the same methylcellulose media as described before. These data suggest that Cyp33 may immortalize mouse bone marrow progenitor cells and arrest them at the primitive cell stage.

BCR-ABL Activates STAT3 Via MEK Kinase 1 in Embryonic Stem Cells.

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate STAT3 and to promote self-renewal in ES cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction (Oncogene 22:7774, 2003). To investigate the role of MEKK1 in Bcr-Abl-induced transformation of ES cells, p210 Bcr-Abl was stably transfected into wild type (WT+p210) and MEKK1−/− (MEKK1−/−+p210) ES cells. Bcr-Abl enhanced both MEKK1 expression and activation in ES cells, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT+p210 cells showed constitutive STAT3 activation and formed compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1−/−+p210 cells, by contrast, showed less STAT3 activity than WT+p210 cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays an essential role in Bcr-Abl-induced STAT3 activation and in the capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.

Differentiation of Rhizomucor Species on the Basis of Their Different Sensitivities to Lovastatin

The opportunistic pathogens Rhizomucor pusillus and Rhizomucor miehei may be agents of frequently fatal mycotic diseases. In the present study, the susceptibilities of 27 clinical and environmental isolates of R. miehei and R. pusillus to lovastatin under different culturing conditions were investigated. Most of the R. miehei strains grew at lovastatin concentrations as high as 64 to 128 microg/ml. In contrast, the inhibitory effect of lovastatin on all of the R. pusillus strains was evident at lovastatin concentrations as low as 1 to 2 microg/ml. A simple and reliable method for species-level differentiation, based on the significantly higher sensitivity of R. pusillus to lovastatin than that of R. miehei, was elaborated. According this, on malt extract agar containing 6 mug of lovastatin/ml, R. pusillus is not able to produce colonies, while R. miehei will form compact colonies.

192ISOLATION AND CULTURE OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO FERTILIZED PORCINE BLASTOCYSTS

The establishment of porcine embryonic stem (ES) cell lines should be useful for the production of transgenic pigs and studies of developmental gene regulation. Recent development of techniques for production of embryos in vitro could be a useful source for the isolation of ES cells. Therefore, to establish porcine ES cells, this study was conducted to isolate and culture inner cell mass (ICM) from in vitro-fertilized (IVF) porcine blastocysts. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Oocytes were then fertilized using a modified swim-up method to prevent polyspermy and cultured to the blastocyst stage. Initial culture of ICM was conducted after either culture of whole embryos or isolation of ICM by immunosurgery. Developing IVF embryos were continuously cultured in 50% DMEM and 50% F-10 with 15% fetal bovine serum, 1% non-essential amino acids, 1.7mM L-glutamine, 1% penicillin/streptomycin, 0.1mM α-mercaptoethanol, 1000 unit recombinant human LIF, 40ngmL−1 recombinant human SCF and 20ngmL−1 recombinant human basic FGF on a mytomycin-C-inactivated murine embryonic fibroblast (MEF) feeder layer. Antibodies against porcine cells were produced in rabbit. After removal of zona pellucida, ICMs were isolated by immunosurgery and cultured on feeder cells the same as described above. After IVF, the rates of 2-cell embryos and blastocysts were 70.8% and 20.4%, respectively. Results from the isolation and culture of ICMs of porcine blastocysts are shown in following table. ICM isolated by immunosurgery showed better attachment to feeder cells and ES cell colony formation than cultured whole blastocysts. Morphology of colonies was similar to that of mouse ES cells, showing compact colonies with delineated boundary. Also, these colonies showed alkaline phosphatase activity. Porcine ES-cell like colonies were passed 3 times through physical separation on fresh feeder layers. These results indicated that porcine ES-like cell line can be established from IVF porcine blastocysts. Further characterization of these porcine ES-like cell lines is required. Table 1 Isolation and culture of ICM from porcine blastocyst produced by IVF

193CHARACTERIZATION OF PORCINE TROPHECTODERM-DERIVED CELLS IN THE PRESENCE OF HBMP4 IN THE ABSENCE OF A FEEDER LAYER

A porcine trophoblastic cell line could provide a powerful model for understanding trophoblast cell biology as well as placental gene expression and proteomics in vitro. In this experiment, we derived porcine trophoblastic cells from trophectoderm tissue and assessed their growth on three different extracellular matrix substrates and in three different concentrations of human recombinant bone morphogenetic protein 4 (hBMP4). Human BMP4 has been shown to induce differentiation of human embryonic stem cells into trophoblast lineages. Elongated embryos were flushed using DPBS supplemented with 1% fetal calf serum and penicillin-streptomycin (1X) from the hysterectomized uteri of superovulated and bred prepuberal gilts 15 days post-insemination. The embryonic disc was visualized with a dissecting microscope. The trophectoderm tissue was cut 2–3mm away from the embryonic disc with a scalpel and the trophectoderm tissue was manually dissected into cell aggregates. These aggregates were plated on collagen type IV, Matrigel, and human extracellular matrix (laminin, collagen type IV and heparan sulfate proteoglycan derived from human placenta) in culture medium (DMEM with 15% FCS, 0.1mM 2-mercaptoethanol, 4ngmL−1 basic FGF4 and 1X P/S) in the presence or absence of hBMP4 at 0, 10, or 20ngmL−1. Cell outgrowth was observed within 24 hours of culture. After three days of culture, various cell types (based on size and morphology) were present. Among cultures of predominant large cells were colonies of smaller cells with epithelial-type morphology that had a prominent nucleus and a high nuclear-to-cytoplasmic ratio. The epithelial-type cells grew in tight colonies with definite borders and contained cytoplasmic structures resembling lipid-containing vesicles. These colonies initially appeared on all matrices across all hBMP4 concentrations. After seven days in culture the colonies developed distinct differences across groups. Cell growth on collagen was comprised of tight colonies having definite borders among large cells. Colonies on collagen were larger and more pronounced in both the hBMP4-supplemented groups than when cultured without hBMP4. The Matrigel coated plates contained large sheets of epithelial-type cell growth instead of compact colonies. This type of growth characteristic was present in all hBMP4 treatments on Matrigel. In contrast, few cells survived and propagated on human extracellular matrix. Only small colonies having the desired morphology were among the large cells on human extracellular matrix when cultured in medium containing 10ngmL−1 hBMP4. Cells were passaged and only cells growing on Matrigel could be further cultured. These data suggest that both the cell substrate and hBMP4 affect initial trophoblast outgrowths. Further analysis including immunocytochemistry and RT-PCR is currently being performed to better characterize these cells. Epidemiology/Diseases

HOXD3 Mediates the Switchover of Cadherin 4 to β3 Integrin Gene Expression in Human Erythroleukemia HEL Cells

We previously reported that overexpression of the HOXD3 homeobox gene in human erythroleukemia HEL cells gave rise to changes in cell adhesiveness. In the present study, we examined the consequences of over- or underexprcssion of HOXD3 in HEL cells for colony morphology, the ability to adhere to fibronectin, and expression of cell adhesion molecules in order to furtlicr confirm the effects attributed to varied levels of HOXD3 expression. Cells overexpressing HOXD3, which formed compact colonies composed of condensed cells, had a greater affinity for fibronectin and expressed higher levels of β3 integrin mRNA than the control cells transfected with an empty vector. Cells which did not express any HOXD3 mRNA failed to form colonies, had a low affinity for fibronectin, and expressed low levels of β3 integrin mRNA. Instead, the cells underexpressing HOXD3 exhibited higher levels of cadherin 4mRNA than the control and cells overexpressing HOXD3. These results demonstrate that the ability to form colonies and attach to fibronectin in HEL cells was dependent on levels of HOXD3 expression, and that HOXD3 served as a switch from cadherin 4 to β3 integrin gene expression.

Colony patterning and collective hyphal growth of filamentous fungi

Colony morphology of wild and mutant strains of Aspergillus nidulans at various nutrient and agar levels was investigated. Two types of colony patterning were found for these strains. One type produced uniform colonies at all nutrient and agar levels tested, and the other exhibited morphological change into disordered ramified colonies at low nutrient levels. Both types showed highly condensed compact colonies at high nutrient levels on low agar media that was highly diffusive. Disordered colonies were found to develop with low hyphal extension rates at low nutrient levels. To understand basic pattern selection rules, a colony model with three parameters, i.e., the initial nutrient level and the step length of nutrient random walk as the external parameters, and the frequency of nutrient uptake as an internal parameter, was constructed. At low nutrient levels, with decreasing nutrient uptake frequency under diffusive conditions, the model colony exhibited onsets of disordered ramification. Further, in the growth process of A. nidulans, reduction of hyphal extension rate due to a population effect of hyphae was found when hyphae form three-dimensional dense colonies, as compared to the case in which hyphal growth was restricted into two-dimensional space. A hyphal population effect was introduced in the colony model. Thickening of colony periphery due to the population effect became distinctive as the nutrient diffusion effect was raised at high nutrient levels with low hyphal growth rate. It was considered that colony patterning and onset of disorder were strongly governed by the combination of nutrient diffusion and hyphal growth rate.

Nesting Ecology of the Greater Adjutant Stork in Assam, India

Abstract The nesting ecology of the Greater Adjutant Stork (Leptoptilos dubius), the rarest stork in the world, was studied in the Brahmaputra Valley, Assam, India during 1994-97. We made a study of nine colonies in the valley during the 1994-95 nesting season, followed by a more intensive study in two subsequent nesting seasons (1995-96 and 1996-97) of two colonies at Nagaon, in central Assam. We measured the dimensions of both nest and non-nest trees, nearest distance between nest trees, and nearest house, nearest forest, nearest road, nearest water source and nearest foraging ground from each colony. All of the colonies were near human settlements. The structure of nest trees differed significantly from non-nest trees in both the overall and the intensive studies. Number of nests was highly correlated with tree height, diameter at breast height (DBH) and canopy area. About 90% of nests were built on the top of the canopy. Storks preferred to nest in compact colonies on large, widely branched trees with...

The fine structure of human embryonic stem cells

The fine structure of human embryonic stem (ES) cell colonies was analysed by transmission electron microscopy (TEM) after 35 passages of in-vitro culture. Most cells formed compact, saucer-shaped colonies with epithelioid cells on the periphery and polygonal cells within the colony. Three morphological types of cells were identified based on their fine structure: undifferentiated cells resembling inner cell mass (ICM) cells of blastocysts; protein-synthesizing cells at the onset of cellular differentiation; and compact masses of secretory cells resembling unicellular goblet cells of the intestine. The predominant cell type was the undifferentiated ES cells resembling ICM cells of blastocysts. These cells had large nuclei containing reticulated nucleoli, well-developed rough endoplasmic reticulum (RER), Golgi complexes, elongated tubular mitochondria, lysosomes and typical centrosomes with centrioles associated with microtubules and microfilaments, organizing the cytoskeleton. Some ES cells have very large nuclei and scanty cytoplasms with fewer organelles. The isolated or attached protein-synthesizing cells at the onset of differentiation had extensive RER and large Golgi complexes. The morphologically differentiated cells formed compact colonies and resembled goblet-like cells in microstructure. They had RER and large Golgi complexes associated with secretory vesicles. The epithelioid cells at the periphery were columnar and largely polarized by centrosomes associated with Golgi complexes. Epithelioid cells in all three categories had specialized cell junctions (desmosomes), anchored by tonofilaments, and surface blebs. Isolated cells were seen on the surface, towards the centre of the colony, and their free surfaces had microvilli and larger blebs. Approximately 3–5% of all cells were mitotic, with typical bipolar spindles organized by centrosomes, pivotally located at the poles, and appeared to resemble typical somatic cells.

Colony shape as a genetic trait in the pattern-forming Bacillus mycoides

BackgroundBacillus mycoides Flügge, a Gram-positive, non-motile soil bacterium assigned to Bacillus cereus group, grows on agar as chains of cells linked end to end, forming radial filaments curving clock- or counter-clockwise (SIN or DX morphotypes). The molecular mechanism causing asymmetric curving is not known: our working hypothesis considers regulation of filamentous growth as the prerequisite for these morphotypes.ResultsSIN and DX strains isolated from the environment were classified as B. mycoides by biochemical and molecular biology tests. Growth on agar of different hardness and nutrient concentration did not abolish colony patterns, nor was conversion between SIN and DX morphotypes ever noticed. A number of morphotype mutants, all originating from one SIN strain, were obtained. Some lost turn direction becoming fluffy, others became round and compact. All mutants lost wild type tight aggregation in liquid culture. Growth on agar was followed by microscopy, exploring the process of colony formation and details of cell divisions. A region of the dcw (division cell wall) cluster, including ftsQ, ftsA, ftsZ and murC, was sequenced in DX and SIN strains as a basis for studying cell division. This confirmed the relatedness of DX and SIN strains to the B. cereus group.ConclusionsDX and SIN asymmetric morphotypes stem from a close but not identical genomic context. Asymmetry is established early during growth on agar. Wild type bacilli construct mostly uninterrupted filaments with cells dividing at the free ends: they "walk" longer distances compared to mutants, where enhanced frequency of cell separation produces new growing edges resulting in round compact colonies.