Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

An investigation of phenolic glycoside and condensed tannin homeostasis in Populus by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, PTSUT4, in planta

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

Enhanced T cell transmigration across the murine liver sinusoidal endothelium is mediated by transcytosis and surface presentation of chemokines

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration. CONCLUSION: Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Modeling Framework for the Establishment of the Apical-Basal Embryonic Axis in Plants

The apical-basal axis of the early plant embryo determines the body plan of the adult organism. To establish a polarized embryonic axis, plants evolved a unique mechanism that involves directional, cell-to-cell transport of the growth regulator auxin. Auxin transport relies on PIN auxin transporters 1], whose polar subcellular localization determines the flow directionality. PIN-mediated auxin transport mediates the spatial and temporal activity of the auxin response machinery 2-7] that contributes to embryo patterning processes, including establishment of the apical (shoot) and basal (root) embryo poles 8]. However, little is known of upstream mechanisms guiding the (re)polarization of auxin fluxes during embryogenesis 9]. Here, we developed a model of plant embryogenesis that correctly generates emergent cell polarities and auxin-mediated sequential initiation of apical-basal axis of plant embryo. The model relies on two precisely localized auxin sources and a feedback between auxin and the polar, subcellular PIN transporter localization. Simulations reproduced PIN polarity and auxin distribution, as well as previously unknown polarization events during early embryogenesis. The spectrum of validated model predictions suggests that our model corresponds to a minimal mechanistic framework for initiation and orientation of the apical-basal axis to guide both embryonic and postembryonic plant development.

ROLE OF GSH METABOLISM IN MEDIATING STROMAL-LEUKEMIA INTERACTION AND PROMOTING CELL SURVIVAL AND DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. My dissertation works proved a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. I showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

THE ROLE OF ADRENAL AND THYROID HORMONES IN GASTRIC DEVELOPMENT (THYROXINE, PARIETAL CELL, CORTICOSTERONE)

The role of adrenal and thyroid hormones on the development of chief and parietal cells was studied in the rat. Administration of corticosterone or thyroxine in the first and second postnatal weeks resulted in the precocious appearance of pepsinogen in the oxyntic gland mucosa and an increase in basal acid output. When pups were adrenalectomized or made hypothyroid, both pepsinogen and basal acid secretion were lowed. Corticosterone injection increased pepsinogen content and acid secretion to levels higher than those of control in hypothyroid and adrenalectomized rats while thyroxine had no such effect in adrenalectomized rats. Morphologically, chief cells responded to corticosterone or thyroxine with increases in both zymogen granules and RER. Chief cells, however, contained less zymogen granules and RER in adrenalectomized and hypothyroid rats. Corticosterone was effective in restoring the normal morphological appearance of chief cells in the hypothyroid rats while thyroxine had no effect in the adrenalectomized rats. In response to corticosterone or thyroxine, parietal cells in normal animals appeared to contain more mitochondria, tubulovesicles and intracellular canaliculi than those of control. Unlike chief cells, parietal cells retained normal ultrastructure in the absence of adrenal and thyroid hormones. These data indicate that (1) corticosterone is necessary for the functional and morphological development of chief cells; (2) the morphological development of parietal cells does not appear to depend upon corticosterone, (3) the effect of thyroxine on the development of chief and parietal cells is due to corticosterone. ^

Suprasellar chordoid neoplasm with expression of thyroid transcription factor 1: evidence that chordoid glioma of the third ventricle and pituicytoma may form part of a spectrum of lineage-related tumors of the basal forebrain.

Chordoid glioma of the third ventricle is a rare neuroepithelial tumor characterized by a unique histomorphology and exclusive association with the suprasellar/third ventricular compartment. Variously interpreted as either astrocytic- or ependymal-like, and speculatively ascribed to the lamina terminalis/subcommissural organ, its histogenesis remains, nevertheless, unsettled. Here, we report on a suprasellar chordoid glioma occurring in a 52-year-old man. Although displaying otherwise typical morphological features, the tumor was notable for expression of thyroid transcription factor 1, a marker of tumors of pituicytic origin in the context of the sellar region. We furthermore found overlapping immunoprofiles of this example of chordoid glioma and pituicytic tumors (pituicytoma and spindle cell oncocytoma), respectively. Specifically, phosphorylated ribosomal protein S6, a marker of mTOR pathway activation, was expressed in both groups. Based on these findings, we suggest that chordoid glioma and pituicytic tumors may form part of a spectrum of lineage-related neoplasms of the basal forebrain.

p73 regulates basal and starvation-induced liver metabolism in vivo

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

Use of cardiac glycosides for treatment of cancer: Determinants of cancer cell sensitivity

Cardiac glycoside compounds have traditionally been used to treat congestive heart failure. Recently, reports have suggested that cardiac glycosides may also be useful for treatment of malignant disease. Our research with oleandrin, a cardiac glycoside component of Nerium oleander, has shown it to be a potent inducer of human but not murine tumor cell apoptosis. Determinants of tumor sensitivity to cardiac glycosides were therefore studied in order to understand the species selective cytotoxic effects as well as explore differential sensitivity amongst a variety of human tumor cell lines. ^ An initial model system involved a comparison of human (BRO) to murine (B16) melanoma cells. Human BRO cells were found to express both the sensitive α3 as well as the less sensitive α1 isoform subunits of Na+,K +-ATPase while mouse B16 cells expressed only the α1 isoform. Drug uptake and inhibition of Na+,K+-ATPase activity were also different between BRO and B16 cells. Partially purified human Na+,K+-ATPase enzyme was inhibited by cardiac glycosides at a concentration that was 1000-fold less than that required to inhibit mouse B16 enzyme to the same extent. In addition, uptake of oleandrin and ouabain was 3–4 fold greater in human than murine cells. These data indicate that differential expression of Na+,K+-ATPase isoform composition in BRO and B16 cells as well as drug uptake and total enzyme activity may all be important determinants of tumor cell sensitivity to cardiac glycosides. ^ In a second model system, two in vitro cell culture model systems were investigated. The first consisted of HFU251 (low expression of Na+,K+-ATPase) and U251 (high Na+ ,K+-ATPase expression) cell lines. Also investigated were human BRO cells that had undergone stable transfection with the α1 subunit resulting in an increase in total Na+,K+-ATPase expression. Data derived from these model systems have indicated that increased expression of Na+,K+-ATPase is associated with an increased resistance to cardiac glycosides. Over-expression of Na +,K+-ATPase in tumor cells resulted in an increase of total Na+,K+-ATPase activity and, in turn, a decreased inhibition of Na+,K+-ATPase activity by cardiac glycosides. However, of interest was the observation that increased enzyme expression was also associated with an elevated basal level of glutathione (GSH) within cells. Both increased Na+,K+-ATPase activity and elevated GSH content appear to contribute to a delayed as well as diminished release of cytochrome c and caspase activation. In addition, we have noted an increased colony forming ability in cells with a high level of Na+,K+-ATPase expression. This suggests that Na+,K+-ATPase is actively involved in tumor cell growth and survival. ^