Binding and cytotoxicity of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and erythrocytes

The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of 125I-lectins to these cells gave biphasic lines except for HeLa cells at 0 degree C. The association constants of lectins for the three cell types at 37 degrees C were lower than those at 0 degree C. The numbers of total binding sites were estimated to be 7 to 16 X 10(7) per HeLa cell, 3 to 4 X 10(7) per Sarcoma 180 ascites tumor cell and 0.4 to 1 X 10(6) per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37 degrees C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1 M lactose, whereas no lectin was irreversibly bound at 0 degree C. In the case of erythrocytes, no lectin became irreversibly bound even at 37 degrees C. The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.     

Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Stem-like and non-stem human pancreatic cancer cells distinguished by morphology and metastatic behavior

We report here that XPA1 human pancreatic cancer cells are dimorphic. After injection in the spleen, XPA1 cells isolated from the primary tumor in the spleen were predominantly round; while cells isolated from the resulting liver metastasis and ascites were comprised of both round- and spindle-shaped cell types. Cancer cells previously grown in the spleen and re-implanted in the spleen developed large primary tumors in the spleen only. Cancer cells isolated from liver metastasis and re-transplanted to the spleen resulted in a primary tumor in the spleen and liver metastasis. Cancer cells derived from ascites and re-transplanted to the spleen developed primary tumors in the spleen and distant metastasis in the liver, lung, and diaphragm in addition to ascites formation. Spindle and round cells were differentially labeled with fluorescent proteins of different colors. After co-injection of the two cell types in the spleen, cells were isolated from the primary tumors, liver metastasis, and ascites and analyzed by color-coded fluorescence microscopy and fluorescence-activated cell sorting (FACS). No significant differences between the percentages of spindle-shaped and round cancer cells in the primary tumor and the liver metastasis were observed. However, spindle-shaped cancer cells were enriched in the ascites. One hundred percent of the spindle-shaped and round cancer cells expressed CD44, suggesting that morphology and metastatic behavior rather than CD44 expression can distinguish the stem-like cells of the XPA1 pancreatic cancer cell line. The spindle-shaped cancer cells had the greater capability for distant metastasis and ascites formation, suggesting they are stem-like cells, which can be readily targeted for therapy.     

Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cells

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA(1), but not LPA(2), with small interference RNA, suggesting a key role of LPA played in the malignant ascites-induced migration. LPA induced activation of ERK through pertussis toxin-sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA-induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA-induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium.     

Isolation and characterization of mistletoe extracts (Viscum album L.). II. Effect of agglutinating and cytotoxic fractions on mouse ascites tumor cells

Lectin from mistletoe (Viscum album L.) was studied for its relations with the toxins from Viscum album, ascites tumor cells of mouse, and human immunoglobulins. Using affinity chromatography on glutaraldehyde-crosslinked IgG (human) from viscum crude extract, a fraction was isolated which exhibited full agglutination capacity and high toxicity. The supernatant showed no agglutination capacity but a strong toxic effect on mouse ascites tumor cells. This toxic effect could not be influenced by further additions of insolubilized IgG. Chromatography on DEAE cellulose also gave agglutinating fractions with toxic effects and a non-agglutinating toxic portion. Column chromatography on Sephadex G 75 allowed separation of toxic from agglutinating components. The molecular weight of the toxin remaining after lectin removal was above 10,000. Lectin was found to bind more readily to mouse ascites tumor cells than to erythrocytes.     

Proteinase activity and agglutination of leukemia cells.

The proteinase activity was assayed in the leukemia cells L 1210 and in the ascites fluid with [3H] acetylated haemoglobin as a substrate. The proteinase activity at pH 4.1 increased in cells and in the ascites fluid with age of the tumor. The proteinase activity at pH 7.8 was low, but the enzyme activity in the cell homogenate increased between 5th and 7th day of the tumor growth and it was also present in the ascites fluid. It was observed that the leukemia cells aggregate in vivo and in vitro at pH values of the ascites fluid above pH 7.0. It was suggested, that the aggregation of leukemia cells is due to the tumor cell proteinase activity released to the ascites fluid.     

Amphotericin B (Fungizone®) enhancement of nitrogen mustard uptake by human tumor cells

In HT29 human colon carcinoma cells, amphotericin B at doses above 120μg/ml increased nitrogen mustard uptake, and this was due to an increase in the apparent V_max without a change in the apparent K_m. Longer incubations (24 to 48 hr) of ascites fluid human ovarian carcinoma cells or SKMES-1 human epidermoid carcinoma cells with amphotericin B 4μg/ml enhanced the uptake of nitrogen mustard to a greater degree than that observed when cells were incubated for only 30 min. Therefore, amphotericin B can enhance nitrogen mustard by human tumor cell lines and by fresh human tumor cells.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Deficiency of methionine synthesis enzyme activity in ascites tumor cells

Betaine-homocysteine- and S-adenosylmethionine-homocysteine-methyltransferases which catalyze synthesis of methionine from homocysteine are absent in tumor cells such as mouse Ehrlich ascites tumor cells and rat hepatoma AH-109A ascites cells.     

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.     

A comparative study on the removal of cellular lipids from Landschütz ascites cells by human plasma apolipoproteins.

The effects of human plasma lipoprotein-proteins on the removal of cellular lipids from Landschütz ascites cells were studied. Cellular lipids were labeled by injecting mice previously injected with ascites with either [3H]cholesterol or [3H]choline. Apoproteins from very low density (apoC-I, C-II, and C-111) and high density (apoA-I and A-II) lipoproteins were used. Each of the apoproteins alone was ineffective in removing cellular [3H]cholesterol. However, when synthetic phosphatidylcholines of known composition were added to each apoprotein and the experiments were repeated using either apoprotein-lipid mixtures or ultracentrifugally isolated complexes, the removal of sterol was considerably enhanced. Complexes of saturated phosphatidylcholines with apoA-II, apoC-I, or apoC-III were the most effective in releasing cellular sterol. Apoprotein-phospholipid complexes were much less effective in removing cellular [3H]phosphatidylcholine than the free apoproteins; apoA-I and apoC-I were the best of the five apoproteins studied. When a comparison was made of the adsorption of iodinated apoproteins to ascites cells, 3 to 4 times more apoA-II and apoC-III were bound than apoA-I. The binding of apoproteins was time and temperature dependent. Approximately 50% of the radioactivity that remained in the washed cells was removed with trypsin. To determine if the counts remaining in the trypsin-treated cells were internalized, identical experiments were performed using human erythrocytes, cells that do not exhibit pinocytosis. Again, approximately 50% of the radioactivity of the iodinated apoproteins was not released by trypsin. Succinylation of apoA-II not only destroys its phospholipid-binding properties but also its adsorption to red cells. These results suggest that the plasma apoproteins differ in their ability to remove cellular lipids and bind to both ascites and red cell membranes, and possibly to specific phospholipids, in such a way that only a part of the apoprotein is degraded with proteases.     

A species difference in platelet aggregation induced by tumour cells

Ehrlich ascites tumour cells (EATC) induced the aggregation of human platelets but not of sheep or rabbit platelets in native platelet-rich plasma. Aggregation was initiated by the interaction of EATC with a component(s) of human plasma, possibly related to the complement system, which led to the release of cellular ADP, a potent platelet aggregating agent. EATC previously incubated with human platelet-poor plasma induced immediate aggregation in platelet-rich plasma from all three species. The species difference in platelet aggregation by EATC is therefore related to the activity or availability of plasma component(s) responsible for release of cellular ADP rather than to intrinsic differences in platelet responsiveness to the tumour cells.     

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.     

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.     

Adhesion defect of ascites cells corrected with membrane-bound attachment molecules

Adhesion is obligatory for cell proliferation in most types of cells. This function becomes defective after malignant transformation. An extreme example is ascites cells which proliferate in suspension. The nature of their defects remains obscure. Here we show that the linking of biotin molecules to the Ehrlich ascites carcinoma cells enables these cells to spread normally on an avidin-coated substrate. The spreading was the result of specific avidin-biotin interaction. The morphology of the spread cells and sensitivity to different inhibitors are similar to those of normal epithelia. Thus it is enough to supply appropriate substrate-adhesive molecules to the ascites cell surface to normalize their adhesion.     

Increase of superoxide dismutase activity in various human leukemia cells.

(1) Superoxide dismutase activity in polymorphonuclear cells from human blood is considerably lower than that in lymphocytes. Macrophages from ascites show the middle level between the other two cells. (2) In myelocytic, monocytic, and lymphocytic leukemia cells, the enzyme activities are increased compared to those in the corresponding normal cells. (3) Gel electrophoresis patterns of all normal cells reveal bands corresponding to the cytosol and mitochondrial bands reported in previous studies. However, the mitochondrial Mn-containing superoxide dismutase activities are diminished or absent in leukemia cells. CN-insensitive superoxide dismutase activity in leukemia cells is not detected under the conditions.     

Flow cytometric method for determining folate receptor expression on ovarian carcinoma cells.

The alpha-folate receptor (alpha-FR) is a folate transporter with restricted expression levels in normal tissues. It is over-expressed in several cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma. It offers a novel therapeutic target for selective imaging and cytotoxic agents. Measurement of the receptor could be a valuable tool in selecting patients more likely to respond to new drugs that target the alpha-FR, and monitoring them while on treatment. While tumor samples are often unavailable, a number of patients who relapse develop ascites, which are often rich in tumor cells. We have therefore developed a triple antibody flow cytometric method to assess alpha-FR expression on tumor cells from ascites. An antibody to BerEP4, an epithelial cell marker expressed on >90% ovarian cancers, labeled with fluorescein, and an alpha-FR antibody labeled with antimouse-phycoerythrin have been used to label tumor cells, with a CD45-phycoerythrin-cyanine5 antibody used to exclude white blood cells from the analysis. The method was optimized using human carcinoma cell lines (JEG-3, IGROV-1, and KB cells). Calibrated beads were used to quantify the number of antibodies bound per cell. The triple antibody protocol successfully measured alpha-FR expression levels in cell lines spiked with blood. Tumor cells were obtained from ascites in 25 patients with relapsed ovarian cancer. In each case sufficient cells were harvested to identify an epithelial cell population to estimate the number of binding sites/cell. All the samples contained a single population of BerEP4, alpha-FR positive cells between 5x10(3) and 5x10(5) antibody binding sites/cell. The method can be used to determine the number of anti-alpha-FR antibodies bound per epithelial cell in ascites from patients with ovarian carcinoma. The results obtained were reproducible and the method could be applied to specimens that had been stored at -80 degrees C.     

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.