Functional role of alpha4beta1 and alpha5beta1 integrin fibronectin receptors expressed on adriamycin-resistant MCF-7 human mammary carcinoma cells.

Cytofluorimetric and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that adriamycin-resistant (ADRR), but not sensitive (WT), MCF-7 human mammary carcinoma cell lines express alpha4beta1 and alpha5beta1 integrins. ADR(R) cells adhere to fibronectin (FN), and only alpha5beta1 is involved in cell adhesion to this glycoprotein, while alpha4beta1 mediates cell binding to the cellular counter-receptor VCAM-1. Proliferation assays showed that FN, but not VCAM-1, delivers a mitogenic signal to quiescent ADR(R) MCF-7 cells. The activating signal is mediated by alpha5beta1, since cell proliferation is inhibited in the presence of RGD peptide or specific antibody. Cell cycle analysis demonstrated that cell/FN interaction induces the re-entry of ADR(R) MCF-7 into S phase, and prevents them from undergoing serum deprivation-induced apoptosis. Our data suggest that the presence of alpha5beta1 on the resistant cells enables them to draw advantage from FN for both cell growth and survival.

Interleukin 1-beta-induced protein kinase C-zeta activation is mimicked by exogenous phospholipase D.

Interleukin 1-beta (IL1-beta) is a pleiotropic cytokine that stimulates a number of signal transduction pathways in cells, leading to different cellular responses. In this study we investigated the signal transduction pathways activated by IL1-beta in two different human cell lines: RD/TE671, a rhabdomyosarcoma, and EJ, a bladder-derived carcinoma. We showed that this cytokine induced the activation of protein kinase C-zeta (PKC-zeta) and the accumulation of a putative physiological PKC-zeta activator, phosphatidic acid [Limatola, Schaap, Moolenaar and van Blitterswijk (1994) Biochem. J. 304, 1001-1008]. Exogenously supplied phospholipase D, which generated cellular phosphatidic acid, was able to mimic the cytokine effect, supporting the hypothesis that this lipid second messenger might contribute to cytokine-induced PKC-zeta activation. In addition, we show that IL1-beta stimulation of BOSC23 cells, transiently overexpressing PKC-zeta, induced an increase in PKC-zeta autophosphorylation. These results give the first direct evidence that IL1-beta can activate this atypical PKC isoform and suggest that this enzyme might be involved in mediating some of the biological effects induced by IL1-beta.

Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues.

Several chemokine receptors have been cloned and shown to belong to a superfamily of seven transmembrane, G protein-coupled receptors. We report here the molecular cloning of TER1, a novel human chemokine receptor-like gene. The amino acid sequence deduced from the TER1 cDNA shows 43, 40, 40, and 39% identity to CCR4, CCR5, CCR1, and CCR2B beta chemokine receptors, respectively. By the use of fluorescent in situ hybridization, we have mapped the TER1 gene to chromosome 3p21, clustered with other chemokine receptor genes. By Northern blot analysis, TER1 mRNA is found to be expressed in the thymus, spleen, and at barely detectable levels in peripheral blood lymphocytes. Moreover, TER1 message in abundant in the NK cell line NK3.3 and in the T cell line MOLT-4. The restricted TER1 expression in cells and tissues of the lymphoid lineage suggests that this receptor may play a role in regulating immune functions.

beta 1-Integrin expression and function in human bladder cancer cells: modulation by TNF alpha.

Cytofluorimetric and biochemical analysis in two different grade human bladder cancer cell lines showed that G3 EJ cells exhibited higher levels of alpha5beta1 and alpha6beta1 heterodimers, and the G2 RT112 cell line higher levels of alpha2beta1. Alpha6/beta4 receptor was detected only in RT112 cells. Adhesion assays with extracellular matrix proteins indicated that both cells bound to fibronectin, laminin and collagen 1, the adhesive properties being related to the integrin profile. Inhibition tests revealed that alpha5beta1 mediated adhesion to fibronectin, alpha3beta1 and alpha6beta1 to laminin, and that alpha2beta1 was the main mediator of adhesion to collagen I in both cell lines. In EJ but not in RT112 cells, tumor necrosis factor-alpha induced the upregulation of alpha2, which mediated increased adhesion to collagen I. The different effects of TNFalpha on the two cell lines were not attributable to differences in tumor necrosis factor responsiveness, as both cells expressed comparable levels of tumor necrosis factor receptor-1 and the tumor necrosis factor-inducible intercellular adhesion molecule-1.

Cost recovery and usage tracking of CD-ROM databases with menuing software.

CD-ROM database products are in widespread use and offer potential cost savings over online products. Many libraries would find a cost-benefit analysis useful. Libraries often do not have sufficient funding simply to underwrite all costs related to CD-ROM workstations. Inexpensive menuing software permits usage tracking for data analysis and cost recovery of CD-ROM subscription, start-up, and operating costs. This article will explore the approach taken at the Fox Chase Cancer Center in Philadelphia to recover CD-ROM related expenses through user fees.

Preparation and utilization of fluorescent synthetic peptides.

In this study, several methods for controlled labelling of synthetic peptides by the use of fluorescent compounds (fluorescein isothiocyanate and dimethylaminonaphthalene sulfonyl chloride) were investigated. The first reagent yielded monofluoresceinated, active compounds only when the peptides lacked lysine residues. Monolabelling of peptides in solution with dimethylaminonaphthalenesulphonyl chloride was hindered by the broad reactivity of the reagent, but was achieved by reacting the fluorochrome on protected resin-bound peptides in solid-phase synthesis. The remarkable stability of the linkage allowed the cleavage of the peptide from the resin and deprotection of side-chain functions without hydrolysis of the labelled group. The binding of antipeptide antibodies to the labelled fragments was then estimated using different techniques.

Effects of VM-26 and lonidamine on a B16 melanoma cell line.

The treatment of exponentially-growing B16 melanoma cells with teniposide causes a dose- and time-dependent decrease of cell survival. By means of the nucleoid technique, the formation of double strand breaks was demonstrated in the nuclei of the treated cells, indicating a possible involvement of topoisomerase II. DNA double strand breaks were rapidly but ineffectively repaired. Morphometric and densitometric analyses showed that teniposide treatment causes a considerable increase of nuclear area, nuclear DNA and cell size, associated with a lowering of the mitotic index to less than one hundredth of that of the controls. The cytocidal effect of VM-26 can be potentiated by the addition of a non-lethal dose of lonidamine, whose synergism is particularly evident at low teniposide concentrations.

Use of NEXIS in a medical research library.

The NEXIS online system is used a great deal in business and law libraries, but has been virtually overlooked by medical librarians due to their perception of the system as expensive and difficult to search. The changing health care environment has placed new demands on medical searchers for just the type of legal, business, governmental, and news information found in the full-text files in NEXIS. Through the recent introduction of News Plus software, NEXIS can be searched as never before by those unaccustomed to full-text search techniques. Besides simplifying the search process, the new software has cost-saving features since search building and file selection are accomplished offline, and several activities may be done simultaneously. NEXIS via News Plus should be seriously considered as an addition to the repertoire of online systems accessed by medical librarians.

Polymorphonuclear leukocyte stimulation measured by phage inactivation.

A quantitative assay for determining the polymorphonuclear neutrophil (PMN) stimulation is described. The method is based on the inactivation of lambda vector phages that occurs after a brief exposure to stimulated PMNs. The determination of the number of residual plaque-forming units on the appropriate bacterial host allows a reproducible and sensitive quantitative assay for measuring the stimulation level of the PMN. In comparison with other methods that employ bacteria or eukaryotic cells, this assay provides several advantages and can be used for investigating the biochemical and physiological processes responsible for PMN stimulation.

On the mechanism of action of lonidamine: a study on human erythrocyte membrane.

The incubation of human erythrocytes with increasing levels of the antineoplastic drug Lonidamine clearly indicated a dose-dependent effect on the lipid composition of the plasma membranes. A selective transfer of phosphatidylcholine and cholesterol from membrane to incubation medium and the consequent enrichment in phosphatidylethanolamine of the membrane itself was observed. Moreover, the membranes were found to contain increasing levels of the drug which was incorporated at a constant membrane/medium partition ratio. The changes in composition appeared to be consistent with morphological alterations observed by scanning and freeze-fracture electron microscopy demonstrating changes in cell shape, the presence of numerous intracellular vesicles, and a membrane protein rearrangement. The analysis of intact red cells by nuclear magnetic resonance ruled out the possibility that the alterations described above could be due to an ATP depletion. This further confirmed that cell membranes were the primary target of the Lonidamine action, the previously described energy metabolism impairment being a consequence of a selective damage of cellular membranes, probably originating from the incorporation of the drug into the lipid bilayer.

Effect of hyperthermia on electron transport in Ehrlich ascites tumor mitochondria.

The effect of hyperthermia (1 hr, 41 degrees C) on the functional properties of Ehrlich ascites tumor mitochondria was investigated. Mitochondria isolated from Ehrlich ascites tumor after exposure of whole cells to 41 degrees C for 1 hr still phosphorylate and maintain a normal acceptor control ratio (ACR). The temperature decreases state 4 and ADP-and FCCP-stimulated respiration on various substrates entering at three energy-conserving sites of the respiratory chain. The inhibition of oxygen consumption by NAD- and FAD-linked substrates was 40% for state 4 and 70% for ADP- or FCCP-stimulated respiration. State 4 and FCCP-stimulated respiration of mitochondria on TMPD + ascorbate was affected 38% and 45%, respectively. ATPase activity was unaffected by hyperthermia, indicating that under these experimental conditions, the inhibition of ADP-stimulated respiration does not depend on an effect on either Fo F1-ATPase or adenine translocase, the activity of which is required for ATP entry prior to ATPase activity. Because of the inability to detect a specific site of action of temperature, it is conceivable that hyperthermia might inhibit substrate oxidation by altering some components of the inner mitochondrial membrane, which regulates the kinetic properties of the membrane-associated enzymes.

Cell membrane changes induced by lonidamine in human erythrocytes and T lymphocytes, and Ehrlich ascites tumor cells.

Lonidamine, a derivative of indazol carboxylic acid, has been found to exert a powerful inhibitory effect on oxygen consumption and aerobic glycolysis of neoplastic cells through mechanisms yet to be defined. Recent freeze-fracture studies have shown that Lonidamine alters the distribution of intramembranous particles in the plasma membrane, suggesting that the cell membranes, rather than the energy metabolism, are the drug's primary target. The present study was carried out to further evaluate the effects of Lonidamine on cell membranes, using normal human erythrocytes and T lymphocytes and Ehrlich ascites tumor cells as cell models. These studies indicate that plasma and mitochondrial membranes are the primary site of the drug's action, though other cell membranes seem to be affected as well. Thus, Lonidamine inhibition of energy metabolism in nucleated cells reported in previous studies must be considered as a consequence of the structural damage of the inner and outer mitochondrial membranes, which in turn affects respiration and glycolysis and then cell viability.

Induction of stress proteins by lonidamine in human and murine melanoma cells.

The ability of lonidamine [1-(2,4)-dichlorobenzyl-1H-indazol-3-carboxylic acid], to induce a stress response in human and murine cultured melanoma cells has been demonstrated. In the M14 and M10 human melanoma cell lines, lonidamine enhances the synthesis of a unique set of proteins, characterized by SDS-PAGE by an Mr of about 72 kDa. In the B16 murine melanoma cell line, exposure to lonidamine increases the synthetic rate of two polypeptides of mol mass 86 and 72 kDa, respectively. Lonidamine is a drug which specifically acts on mitochondria. Therefore the observation that it can also promote a stress response indicates that the mitochondria might be one of the primary cellular targets and postulates a causal relationship between an impairment of the energy supply and induction of stress protein synthesis.

Effect of lonidamine on protein synthesis in neoplastic cells.

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Effect of lonidamine on the aerobic glycolysis of normal and phytohemagglutinin-stimulated human peripheral blood lymphocytes.

The effect of lonidamine on the aerobic glycolysis and on the ultrastructure of normal and phytohemagglutinin-stimulated human peripheral blood lymphocytes has been investigated. In quiescent lymphocytes lonidamine does not affect aerobic lactate production and does not induce ultrastructural modifications. On the contrary, lymphocytes stimulated with phytohemagglutinin become susceptible to lonidamine inhibition. The rate of lactate production is decreased by 50% and mitochondria appear swollen with rarified matrix and disrupted cristae. The different effect of lonidamine can be ascribed both to the biochemical modifications induced by phytohemagglutinin and to the mechanism of lonidamine itself. Phytohemagglutinin increases the activity of hexokinase and phosphofructokinase and determines a shift of cytoplasmatic hexokinase toward the mitochondria-bound form which is responsible for the increased lactate production. This interpretation is supported by the finding that lonidamine, which specifically inhibits mitochondria-bound hexokinase only when mitochondria are in a condensed state, decreases lactate production to a value similar to that found in unstimulated cells. The inability of lonidamine to affect the aerobic glycolysis of quiescent lymphocytes can be interpreted along the same line. On this basis it is suggested that the inhibition of mitochondria-bound hexokinase might be ascribed to marked changes in membrane conformation that affect the activity of membrane-associated enzymes, rather than to a direct effect of the enzyme itself.

Effects of Lonidamine on murine and human tumor cells in vitro. A morphological and biochemical study.

Lonidamine induces in murine and human tumor cells severe morphological damage of the mitochondria and other cytoplasmic structures both 'in vitro' and 'in vivo'. Biochemical studies have demonstrated that the drug decreases oxygen consumption and lactate production. The sensitivity of human tumor cells is not related to their histotype. Lonidamine's effects on mitochondria, glycolysis, pentose phosphate pathway, and aromatase activity are discussed.

Inhibition of aerobic glycolysis in normal and neoplastic lymphoid cells induced by Lonidamine [1-(2,4-dichlorobenzyl)-I-H-indazol-3-carboxylic acid].

The in vitro inhibitory activity of Lonidamine on the aerobic glycolysis of normal as well as leukemic lymphocytes has been investigated. The extent of the impairment of lactate production induced by Lonidamine on normal thymus (T)- and bone marrow (B)-derived lymphocytes was found to be dependent on their source of origin, i.e. residing or circulating pool. Among leukemia tested 'null' and B cell leukemias appeared the most affected metabolically by the compound. Administration in vivo of the drug to the patient with B cell chronic leukemia resulted in a decrease of lactate production by leukemic cells comparable to that induced in vitro. These metabolic changes were paralleled in normal, as well as in leukemic cells by ultrastructural lesions, mainly confined to the mitochondrial compartment.

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells.

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. The action of the drug on tumor cells was studied by addition of the drugs to cells harvested from Swiss male mice. The results may be summarized as follows: (1) Low concentrations of Gossypol increase the rate of oxygen consumption by uncoupling oxidative phosphorylation. High concentrations result in an inhibition of oxygen consumption with a mechanism that must be regarded as not directly related to the uncoupling activity. (2) Gossypol, at concentrations at which it exerts an uncoupling activity, stimulates mitochondrial ATPase which in turn increases the aerobic and anaerobic rates of lactate production. The decrease of glycolysis at high concentrations of Gossypol does not depend on the inhibition of enzymes of the glycolytic pathway, but must be ascribed to cell death. (3) The association of a low concentration of Gossypol with Lonidamine brings about a further inhibition of oxygen consumption. Moreover, Lonidamine abolishes the stimulation of glycolysis induced by Gossypol and lowers lactate production to values that are quite similar to those found with Lonidamine alone. (4) It may be concluded that the association of Gossypol and Lonidamine results in a very effective decrease of the energy requirements of cancer cells.

IgM participation in tumor rejection by immunized mice.

IgG and IgM participation in tumor rejection was studied in DBA/2 mice immunized against L1210 leukemia and in Swiss mice immunized against Ehrlich adenocarcinoma. In both the systems, IgM globulins seem to be implicated and are present on the cell surface of macrophages, lymphocytes and cancer cells, while IgG globulins are present only on some lymphocytes. IgM are also present in the peritoneal washing fluids (obtained 24 h of the control group. In the former group, protein content is about three times higher than that in the control group, while the relative amount of heavy proteins (18 S) and light proteins (7 S) is quite similar. These observations are discussed, as is the possibility that some complement components as C3 may participate in the reaction.