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Based on analyses in Arabidopsis thaliana ecotype C24, the AtSUC1 protein was previously characterised as a male gametophyte-specific H + ⁄sucrose symporter. Later, expression analyses in ecotype Columbia-0 (Col-0) identified AtSUC1 expression also in trichomes (not detected in trichome-less C24 plants) and roots, suggesting ecotype-specific differences in AtSUC1 expression. Here, we present data on additional ecotype-specific differences in AtSUC1 expression in other tissues. Using different AtSUC1 promoter‐reporter gene lines, we performed comparative analyses of AtSUC1 expression in floral tissues of C24 and Col-0 plants, and using an AtSUC1-specific antiserum, we performed immunohistochemical analyses on tissue sections from C24, Col-0, Landsberg erecta (Ler) and Wassilewskaija (Ws) ecotypes. We show that AtSUC1 expression occurs in the funicular epidermis of C24, Ler and Ws, but not in Col-0. In contrast, we observed high levels of AtSUC1 protein in pollen grains of Col-0, lower levels in pollen of C24 and Ler, and no AtSUC1 protein in Ws pollen. Moreover, our reporter gene analyses identified a previously undetected expression of AtSUC1 in the female gametophyte, and revealed that AtSUC1 expression in the funicular epidermis is absent from unpollinated siliques and is induced upon successful pollination. The impact of these findings on the potential physiological role of AtSUC1 is discussed.

Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-β-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Abscisic acid (ABA) and sugars regulate many aspects of plant growth and development, and we are only just beginning to understand the complex interactions between ABA and sugar signaling networks. Here, we show that ABA-dependent transcription factors bind to the promoter of the Arabidopsis thaliana AtSUC1 (At1g71880) sucrose transporter gene in vitro. We present the characterization of a cis-regulatory element by truncation of the AtSUC1 promoter and by electrophoretic mobility shift assays that is identical to a previously characterized ABA-responsive element (ABRE). In yeast 1-hybrid analyses we identified ABI5 (AtbZIP39; At2g36270) and AREB3 (AtbZIP66; At3g56850) as potential interactors. Analyses of plants expressing the beta-glucuronidase reporter gene under the control of ABI5 or AREB3 promoter sequences demonstrated that both transcription factor genes are co-expressed with AtSUC1 in pollen and seedlings, the primary sites of AtSUC1 action. Mutational analyses of the identified cis-regulatory element verified its importance for AtSUC1 expression in young seedlings. In abi5-4 seedlings, we observed an increase of sucrose-dependent anthocyanin accumulation and AtSUC1 mRNA levels. This suggests that ABI5 prevents an overshoot of sucrose-induced AtSUC1 expression and confirmed a novel cross-link between sugar and ABA signaling.

This paper presents a detailed analysis of the PmSUC1 gene from Plantago major, of its promoter activity in Arabidopsis, and of the tissue specific localization of the encoded protein in Plantago. PmSUC1 promoter activity was detected in the innermost layer of the inner integument (the endothel) of Arabidopsis plants expressing the gene of the green fluorescent protein (GFP) under the control of the PmSUC1 promoter. This promoter activity was confirmed with a PmSUC1-specific antiserum that identified the PmSUC1 protein in the endothel of Plantago and of Arabidopsis plants expressing the PmSUC1 gene under the control of its own promoter. PmSUC1 promoter activity and PmSUC1 protein were also detected in pollen grains during maturation inside the anthers and in pollen tubes during and after germination. These results demonstrate that PmSUC1 is involved in sucrose partitioning to the young embryo and to the developing pollen and growing pollen tube. In the innermost cell layer of the inner integument, a tissue that delivers nutrients to the endosperm and the embryo, PmSUC1 may catalyze the release of sucrose into the apoplast.

Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival. Figure Figure

Nontumoral accessory cells such as marrow stromal cells (MSC) or nurselike cells (NLC), which constitute the leukemia microenvironment, constitutively secrete the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). CXCL12 transduces signals via its receptor CXCR4, which is expressed at high levels by Chronic Lymphocytic Leukemia (CLL) B cells. Via the CXCL12-CXCR4 axis, CLL cells migrate and adhere to stromal cells. Adhesion to stromal cells protects CLL cells from spontaneous and drug-induced apoptosis in a contact-dependent fashion. Signaling pathways regulating these processes in CLL B cells are largely unknown. Here, we examined the importance of phosphatidyl-inositide 3-kinases (PI3-K) for migration and viability of CLL B cells using non-specific and isoform-specific PI3-K inhibitors. The importance of PI3-K for migration of CLL cells to CXCL12 was determined by transwell chemotaxis and pseudoemperipolesis (PEP) assays. Inhibition of PI3-K resulted in a significant reduction of CLL cell migration in chemotaxis and PEP assays. In comparison to untreated CLL cells, Ly 294002 inhibited chemotaxis to 65 ± 4.6% of untreated controls. Using a panel of isoform-specific PI 3-K inhibitors (PI-103, PIK-90, IC87114, TGX-115, ZK-75), we observed inhibition of chemotaxis by the multi-targeted compounds PI-103 (51.4 ± 0.2%) and PIK-90 (57.5 ± 8.9%), whereas p110beta and delta inhibition had no effect. We conclude from this part of the study that PI3-kinases play an important role for CXCR4 signaling in CLL B cells, mediating migratory responses and protection from apoptosis. Experiments with inhibitors of PI3-K with higher target selectivity suggest a dominant role for the class I PI3-K p110alpha for migration in response to CXCL12. Because adhesion to stroma mediates protection from chemotherapeutic drugs, we tested PI3-K inhibitors alone and in combination with fludarabine in CLL-stroma co-cultures. Pre-treatment of CLL cells with the PI3-K inhibitors Ly 294002, PI-103, and PIK-90 resulted in a significant decrease in viability of CLL cells co-cultured with and without stroma. Moreover, PI3-K isoform specific inhibitors enhance the cytotoxicity of Fludarabine and partially reverse the protective effect of stromal cells on fludarabine-induced apoptosis. Collectively, this study establishes that PI3-Ks play an important role in CXCR4 signaling for CLL cell migration and adhesion to stromal cells. New, isoform-specific PI3-K inhibitors enhance the cytotoxicity of fludarabine in suspension cultures and in co-cultures with stromal cells. Therefore, the therapeutic potential of PI3-K inhibitors alone or in combination with fludarabine should further be investigated. Download : Download high-res image (82KB) Download : Download full-size image Figure.