Patterns of ganglioside expression in B cell neoplasms

Twenty seven B cell neoplasms were examined by high performance thin layer chromatography (HPTLC) and immune thin layer chromatography (ITLC) to determine ganglioside expression. Patterns of expression in the cells were compared with conventional morphology, genotype, and glycoprotein immunophenotype. Patterns of ganglioside expression were found for each of the tumor types analyzed (5 acute lymphoblastic lymphomas (ALL), 5 Burkitt's Lymphomas (BL), 4 chronic lymphocytic leukemias (CLL), and 3 diffuse poorly differentiated lymphomas (DPDL), 7 diffuse histiocytic lymphomas (DHL), and 3 multiple myelomas (MM). GM3 was the predominant ganglioside found in all B cell neoplasms except multiple myeloma where GM2 was equivalent to GM3. GM1 was detected by ITLC in all B cell tumors, but significant amounts were found by HPTLC only in ALL, CLL, and DHL. Small amounts of GD3 and GD2 were found in several B cell neoplasms. Significant amounts of other gangliosides were not found. The expression of GM2 on the MM cell lines, a cell type derived from outside of the nervous system, is unusual. This high level of expression was also seen in metabolic labeling studies. GM2 was readily detectable in the SKMM1 human multiple myeloma cell line by flow cytometry and served as a target for human complement-mediated cytotoxicity. Although the functions of gangliosides are largely known, the patterns of gangliosides found for this system of human B cell malignancies may serve to provide targets for specific immunotherapy and clues to their functions.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Modulation of cell surface components of human breast cancer cells by retinoic acid: enhanced HLA-DR expression

A finite volume method in a boundary-fitted coordinate system together with a zonal grid method is employed to compute the flow fields and shear stresses in a two-dimensional aortic bifurcation. Eddy is found distal to plaques during pulsatile flow, whereas permanent eddies are observed only during steady flow. The computed flow fields are consistent with those visualized experimentally by other authors. It is also found that although the time averaged shear rates in a pulsatile flow are similar to those of a steady flow with mean Reynolds number in most regions, they are different in recirculation zones. This result implies that care should be taken if a steady flow shear rate were to be used in modeling shear-dependent physiological processes. The non-Newtonian viscosity has only a minor effect on the flows.     

Function and specificity of human natural killer cell receptors

In humans, natural killer lymphocytes express HLA class I-specific inhibitory receptors belonging to at least two different molecular families. The first is represented by members of the Ig superfamily that are involved in the recognition of different groups of HLA class I alleles, and the second is represented by a molecular complex formed by CD94 and NKG2A that displays a broad specificity for various class I molecules including the 'non-classical' HLA-G molecules. In addition to the inhibitory receptors, a series of activating receptors has been identified. Some display the same specificities as the corresponding inhibiting receptors and can be viewed as HLA class I-specific activating receptors. Another group of activating receptors appear to be involved in the cytolytic activity against HLA-'negative' target cells. These receptors are clearly non-MHC specific and, under physiological conditions, their function is suppressed by the HLA class I-specific inhibitory receptors.     

TNF-alpha induction of A1 expression in human cancer cells

A1, a member of the Bcl-2 gene family, was originally identified as a hemopoietic-specific early response gene. Later it was found that A1 was overexpressed in human stomach cancer tissues and was induced by tumor necrosis factor-alpha (TNF-alpha) in human vascular endothelial cells. However, its expression in human cancer cells has not been well characterized. In the present study, we examined the expression of A1, as well as the antioxidant manganous superoxide dismutase (MnSOD), in four human thyroid carcinoma cell lines, two human pancreatic carcinoma cell lines, and two human prostate carcinoma cell lines. A1 mRNA was expressed in all four thyroid carcinoma cell lines. TNF-alpha induced A1 in a time- and dose-dependent manner. In contrast, A1 mRNA was not detectable in the pancreatic and prostate carcinoma cell lines in the presence or absence of TNF-alpha. However, TNF-alpha induced manganous superoxide dismutase (MnSOD) mRNA in all the cell lines tested. Furthermore, an agonist antibody to the p55 TNF-alpha receptor induced A1, but the agonist antibody against p75 TNF-alpha receptor did not have this effect. The results indicate that A1 is expressed in human thyroid carcinoma cells and TNF-alpha induces A1 through the p55 TNF-alpha receptor-mediated pathway.     

Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance

Sections of formalin-fixed, paraffin-embedded tissues from 210 human breast cancers were immunohistochemically examined using the mAb against human tenascin (TN) RCB1. Immunoreactive TN was detected in the breast cancer stroma in 77 (36.7%) cases, whereas the remaining 133 (63.3%) were negative. Of the 77, 12 (5.7%) cases also showed positive staining in the carcinoma cell cytoplasm. The positive cells were often observed in the margin of the cancer nests at the site adjacent to the stroma. According to the staining pattern of TN, the breast cancer cases were classified into the three groups of cancer cell TN(+)/stromal TN(+), cancer cell(-)/stromal TN(+), and cancer cell(-)/stromal TN(-). Analysis of the relationship of these TN patterns with various clinicopathological characteristics of the tumors and the patient outcome revealed that, in comparison to the cancer cell(-)/stromal TN(-) group, the cancer cell TN(+)/stromal TN(+) group exhibited increased frequency of lymph node metastasis and exceptionally poor outcome, and the cancer cell(-)/stromal TN(+) group also showed more frequent metastasis and poorer outcome. Most of the cancer cell TN(+)/stromal TN(+) cases were c-erbB-2 positive and estrogen receptor negative. Furthermore, in situ hybridization of freshly obtained breast cancer tissues demonstrated that both cancer cells and stromal cells express TN mRNA. These results indicate that the TN in breast cancer is produced by cancer epithelial cells as well as by stromal mesenchymal cells, and that cancer cell TN might be involved in cancer spreading, resulting in unfavorable patient prognosis.     

Increased expression of human histocompatibility leukocyte antigen-G in colorectal cancer cells

Human histocompatibility leukocyte antigen (HLA)-G is a nonclassical major histocompatibility complex class I molecule. HLA-G is known to provide tolerance from recognition by natural killer cells. We studied HLA-G expression in 39 human colorectal cancers and 23 extra-neoplastic colon tissue samples by RT-PCR. The expression of HLA-G mRNA was significantly more frequent in colorectal cancer (34 of 39 cases) than in the extraneoplastic tissue (10 of 23 specimens; chi2 test, p = 0.0003). HLA-G expression was also confirmed on the cancer cells immunohistochemically. These results suggested that HLA-G on colorectal cancer cells may be correlated with escape from immunological surveillance during colon cancer development.     

Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells

It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections.     

Stromal cell expression of components of matrix-degrading protease systems in human cancer

Components of matrix-degrading protease systems are in human cancer often expressed by tumour-infiltrating stromal cells. The cellular pattern of expression of these molecules appears to be unique for each type of cancer. In several cases there are similarities with patterns observed in nonmalignant remodelling processes in the same tissue. These findings indicate that the stromal cells actively participate in the process of cancer invasion. The implications of this new paradigm for cancer biology and cancer treatment are discussed.     

The relative efficacy of four methods of human milk expression

In view of the current trend toward increased breast-feeding, both of normal term infants as well as sick or premature infants, a successful means for milk expression in order to establish and maintain lactation is of major importance to the mother. The present study was designed to evaluate four methods of milk expression, measuring the amount as well s the fat content of milk expressed by each method during a 10-min period. The four methods included the Egnell electric pump, the Loyd B pump, the Evenflo system, and manual expression. The electric pump enabled mothers to express significantly more milk with adequate fat content during the expression period than any of the other methods tested. No significant differences were found between the other three methods. The Egnell or similar electric pump may be a preferred method for milk expression for some mothers, particularly those anticipating a prolonged need for pumping.     

Antigen-presenting function and B7 expression of murine sinusoidal endothelial cells and Kupffer cells

BACKGROUND & AIMS: Inflammatory liver disease as well as rejection of liver allografts are thought to be mediated by resident antigen-presenting cells in the liver. At the same time, in vivo antigen presentation in the liver appears to be a more tolerogenic than systemic antigen challenge. The aim of this study was to show and characterize the antigen-presenting capability of sinusoidal endothelial cells and Kupffer cells. METHODS: Purified murine sinusoidal endothelial cells and Kupffer cells were studied for their ability to serve as accessory cells and antigen-presenting cells by proliferation assays. They were also studied for their expression of interleukin 1 and the B7 costimulatory molecules by Northern blotting, polymerase chain reaction, and flow cytometry. RESULTS: Both cell types expressed interleukin 1 messenger RNA and could serve equally well as accessory and antigen-presenting cells. B7-2 messenger RNA and surface expression on sinusoidal endothelial cells and on Kupffer cells was shown. Antibodies to the B7 molecules inhibited antigen presentation. Addition of interleukin 10 as a regulatory cytokine secreted by Kupffer cells was suppressive. CONCLUSIONS: Sinusoidal endothelial cells carry functional B7-2 molecules and can serve as effective antigen-presenting cells. However, antigen presentation by sinusoidal endothelial cells may be locally down-regulated by interleukin 10.     

Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture

Cell density-dependent inhibition of growth and neural differentiation in the human neuroblastoma cell line SK-N-MC are associated with a ganglioside sialidase-mediated increase of GM1 and lactosylceramide at the cell surface. Because these glycolipids expose galactose residues, we have initiated the study of the potential role of galectins in such cellular events. Using specific antibodies, galectin-1 but not galectin-3 was found to be present at the cell surface. Assessment of carbohydrate-dependent binding revealed a saturable amount of ligand sites approaching 2.6 x 10(6) galectin-1 molecules bound/cell. Presence during cell culture of the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of the GM1-binding cholera toxin B subunit effected a decrease of the presentation of galectin-1 ligands by 30-50%. The assumption that GM1 is a major ligand for galectin-1 was reinforced by the correlation between the number of carbohydrate-dependent 125I-iodinated GM1-neoganglioprotein binding sites and the amount of immunoreactive surface galectin-1, the marked sensitivity of probe binding to the presence of anti-galectin-1 antibody, and the inhibition of cell adhesion to surface-immobilized GM1 by the antibody. The results open the possibility that the carbohydrate-dependent interaction between ganglioside GM1 and galectin-1 may relay sialidase-dependent alterations in this cell system.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.     

Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells

Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.     

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.     

Identification of murine p120 isoforms and heterogeneous expression of p120cas isoforms in human tumor cell lines

Heat stress is a common problem for cattle. General consequences of heat stress include increased body temperatures and reduced feed intakes. As a measure of heat stress, core body temperatures of unshaded feedlot steers (crossbred Bos taurus) were monitored from mid-June to early November in Nebraska using transmitters implanted in the peritoneum of 10 steers (initially 10 mo of age). Steers were fed at 0630 and 1430 using a finishing diet of 1.52 NEg Mcal/kg with 13% protein and 4% roughage per day and housed in two open lots with stocking densities of 15.2 or 19.3 m2/steer. Core body temperatures, ambient temperature, relative humidity, and wind speed were measured at 3-min intervals and mathematically filtered to produce 120 readings/ d. For 94 usable daily records, body temperature means (39.04 +/- .12 degrees C), maxima (39.89 +/- .21 degrees C at 1836 +/- .73 h), minima (38.33 +/- .29 degrees C at 0823 +/- .38 h), and patterns were similar among steers. As daily maximum ambient temperatures increased, minimum body temperatures decreased slightly (.04 degree C per 5 degrees C; P < .01). After daily maximum ambient temperatures reached a threshold of 25.6 degrees C, daily maximum body temperatures increased linearly with maximum ambient temperatures (.42 degree C per 5 degrees C; P < .01). Sharp peaks in body temperature were often seen in the late evening (approximately 2200) after ambient temperature had decreased to well below maximum values. These evening peaks occurred on an average of 25% of the days, had amplitudes ranging from .7 to 3.5 degrees C relative to mean daily temperatures and lasted for 1.5 h. From a practical standpoint, we suggest that producers monitor meteorological forecast of peak ambient temperatures and make special efforts, such as spraying animals, when exceptionally hot weather is predicted.