Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

EPSTEIN-BARR VIRUS TRANSFORMATION OF B LYMPHOCYTE SUBPOPULATIONS

Epstein-Barr virus is a herpes virus distinguished by its remarkable specificity for the B lymphocyte of humans and certain other primates. Although the transformation process is very efficient, is has become clear that only a fraction of B lymphocytes is susceptible. Therefore the question may be raised if transformation is related to B cell stage of activation. B cells were purified from peripheral blood mononuclear cells by the removal of monocytes using elutriation and sheep red blood cell rosetting to remove T cells. Retesting B cells were purified using discontinuous Percoll gradients. Activation of resting cells for 24 hours with anti-mu or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G(,1) phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr as shown by an increase in thymidine incorporation. By this criterion, SAC activated cells entered S phase on day 2 and anti-mu treated cells on day 3. Control (G(,0)) cells and cells activated for varying lengths of time (G(,1), G(,1) plus S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a precursor frequency of 1% to 2%. With continued activation, however, precursor frequency decreased as a function of the duration of activation. The decrease in frequency of transformable cells correlated with the entry of the population into S phase. The transformation frequency in the SAC-treated population was reduced twenty-fold on day 4, whereas in the anti-mu treated population it was reduced ten-fold. Treating cells with BCGF in conjunction with low concentrations of anti-mu decreased the transformation frequency to levels lower than anti-mu alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation and that with in vitro activation into the cell cycle B cells become progressively insensitive to EBV. ^

EFFECTS OF THE IMMUNOMODULATING AGENT GLUCAN ON MURINE HEMOPOIESIS

BDF(,1) mice received a single intravenous injection of glucan, a potent immunomodulating agent, and at various times thereafter the proliferation of pluripotent (CFU-S), committed granulocyte-macrophage (GM-CFC) and committed B-lymphocyte (BL-CFC) hemopoietic stem cells was measured in the bo

PHENOTYPIC CHARACTERIZATION OF SPONTANEOUS AND IN VITRO-MAINTAINED AKR LYMPHOMAS

T-cell lymphomas from AKR mice were studied to determine their potential as a model of T-cell differentiation. Homogeneous tumor cell lines have been used as model to study normal lymphocyte subpopulations, including differentiation lineages, functional properties, and the inducibility to maturation. The underlying concept is that each lymphoid tumor represents a monoclonal neoplastic proliferation of a discrete lymphoid subpopulation arrested at a particular differentiation stage.^ Individual tumors were analyzed to determine the extent of intertumor heterogeneity, and to determine whether lymphomas represented different thymocyte subsets, by determining the cell-surface antigenic phenotype, PNA-binding capacity, and terminal deoxynucleotidyl transferase (TdT) activity. Splenic and thymic tumor cells were compared to determine if the particular lymphoid microenvironment influenced T-cell marker expression. Several of the lymphomas were passaged in syngeneic hosts to verify the original tumor phenotype and to assess the stability of the cell surface and TdT phenotype after transplantation.^ Lymphomas were adapted to in vitro culture to determine whether the T-cell phenotype was maintained in the absence of the host microenvironment. Clonal progeny were analyzed and compared with each other and with parent cell lines to determine the extent of intratumor heterogeneity in this lymphoma system. Parent and cloned cell lines were passaged in vivo to determine whether alterations in surface phenotype occurred after transplantation.^ Our investigation has verified that most spontaneous AKR lymphomas phenotypically resemble known T-cell subsets, including both immature and mature thymic subpopulations. The in vitro lines, however, expressed a highly unstable phenotype in culture that included loss of Ly-1 and Ly-2 antigen expression. After transplantation in vivo, the in vitro lines exhibited alterations in phenotype, including re-expression of Ly antigen on some lymphomas. The inducibility of T-cell antigen markers on tumor cell lines passaged in vivo suggests that the in vitro lines may serve as a possible model system to study the molecular events involved in gene expression in the T-cell system. ^

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

TH and XFKBP12 interact and regulate proliferation and differentiation in neural ectoderm through the TOR signaling pathway

During development, embryos must carefully integrate the processes of cell proliferation and differentiation. TH has been identified in Xenopus laevis as a gene product that functions in regulating differentiation of the neural ectoderm through its effect on cell proliferation. However, the mechanism and molecular pathway through which TH functions are not known. We identified the Xenopus FK506 binding protein homolog (XFKBP12) as a protein that interacted with TH in a yeast two-hybrid screen with TH as the bait. The direct and specific interaction between TH and XFKBP12 was supported by several tests including CO-IP, drug competence assay and mutagenesis analysis. To investigate the function of XFKBP12 during embryogenesis, we created an XFKBP12 loss of function embryo using antisense morpholino oligonucleotides (MO). XFKBP12 MO injected embryos displayed similar phenotypes as TH depleted embryos. We also demonstrated that both TH and XFKBP12 functioned through the TOR signaling pathway which is a target for cancer therapies. The interaction between TH and XFKBP 12 was required to regulate the proliferation of neural cells. Therefore, our study indicates that TH represents the endogenous ligand of XFKBP12 and together they coordinate neural cell proliferation and differentiation through the conserved rapamycin sensitive TOR pathway. Thus, understanding how this pathway functions in development will not only provide us important insights into the relationship between proliferation and differentiation, but help design rational cancer therapies targeting this pathway. ^

Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

SFRP4 regulation of Wnt7a signaling and cellular proliferation in the endometrium

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication and thus mediate hormone action. Characterization of specific Wnt signaling components in the endometrium was performed using cellular localization studies and evaluating hormone effects in a rat model. Wnt7a was expressed in the luminal epithelium, whereas the extracellular Wnt modulator, SFRP4, was localized to the endometrial stroma. SFRP4 expression is significantly decreased in endometrial carcinoma and aberrant Wnt7a signaling has been shown to cause uterine defects and contribute to the onset of disease. The specific Fzds and SFRPs that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of Wnt7a and SFRP4 in the endometrium has not been addressed. A survey of all Wnt signaling proteins expressed in the endometrium was conducted and Fzd5 and Fzd10 were identified as two receptors capable of transducing the Wnt7a signal. Biologically active recombinant Wnt7a and SFRP4 proteins were purified for quantitative biochemical studies. In Ishikawa cells, Wnt7a binding to Fzd5 activated β-catenin/canonical Wnt signaling and increased cellular proliferation. Wnt7a signaling mediated by Fzd10 induced a non-canonical/JNK-responsive pathway. SFRP4 suppressed Wnt7a action in both an autocrine and paracrine manner. Treatment with SFRP4 protein and overexpression of SFRP4 inhibited endometrial cancer cell growth and induced apoptosis in vitro. A split-eGFP complementation assay was developed to visually detect Wnt7a-Fzd interactions and subsequent pathway activation in cells. By employing a unique ELISA-based protein-protein binding technique, it was demonstrated that Wnt7a binds to SFRP4 and Fzd5 with equal nanomolar affinity. The development of these novel biological tools could lead to a better understanding of Wnt-protein interactions and the identification of new modulators of Wnt signaling. This study supports a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent upon the Fzd repertoire of the cell and can be regulated by SFRP4. The potential tumor suppressor function of SFRP4 suggests it may serve as a therapeutic target for endometrial carcinoma. ^