Antiproliferative effects of free and liposome-encapsulated retinoic acid in a squamous carcinoma model: monolayer cells and multicellular tumor spheroids

Summary Antiproliferative effects of free retinoic acid (RA) and liposome-encapsulated RA (RAlp) were compared in a squamous carcinoma system using both monolayer cells and multicellular tumor spheroids (MTS), an in-vivo-like model with three-dimensional histological structure. Initial studies examined the effect of lipid composition on the efficiency of RA encapsulation and on the subsequent toxicity of RAlp to red blood cells. In 5-day growth assays for monolayer cells, RA and RAlp (1 M-0.1 nM) produced similar growth inhibition. In 6-day growth assays for MTS, RAlp was shown to have increased effectiveness. Liposomal uptake by the squamous carcinoma cells was examined by culturing monolayers and MTS with fluorescence-tagged liposomes and examining them under fluorescence microscopy between days 1 and 6. Phagocytosed liposomes were present, but their low levels suggested that other mechanisms of drug delivery such as adsorption, fusion or direct lipid transfer probably occurred for RAlp. Histological examination of MTS showed that RA and RAlp produced similar alterations. In this squamous carcinoma system, liposomes are effective in delivering retinoic acid and in producing biological effects in monolayer cells and within the three-dimensional structure of MTS.Abbreviations MTS multicellular tumor spheroids - SCC squamous cell carcinoma - RA free -all-trans-retinoic acid - RAlp lipo-some-encapsulated RA - PtdCho dimyristoylglycerophosphocholine - PtdGro dimyristoylglycerophosphoglycerol This work was supported in part by M. D. Anderson Annual Campaign Funds and by United States Public Health Service grants CA38751 and CA57166 from the National Cancer InstitutePresent address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York; NY 10021, USA     

Role of H-ras in the malignant progression of rat tracheal epithelial cells

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutantras gene than did the injected cells, while a subclone that lacked theras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing aras mutation resulted in an increase in the fraction ofras-mutated cells, which suggests that, in this line,ras activation may confer a selective advantagein vitro as well. However, this was not seen in anotherras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.Abbreviations RTE rat tracheal epithelial - RFLP restriction-fragment-length polymorphism - DMBA 7,12-dimethylbenz[a]anthracene Supported by NIH grants CA 52925, CA 13343, and ES 00260 and ACS Award IRG-14-33.     

A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines

In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated. A murine NIH/3T3-derived cell line, designated 2–12, contains an inducible Ha-ras oncogene, which is regulated by theEscherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl--d-thiogalactoside induction. To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2–12 cells. Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features. Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels. Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2–12 transformed cells. Directly injecting the ribozyme DNA into tumors induced by transformed 2–12 cells in BALB/c mice also caused tumor regression. The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis. These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.Abbreviations IPTG isopropyl -d-thiogalactoside     

Apoptosis of human BEL-7402 hepatocellular carcinoma cells released by antisense H-ras DNA-in vitro and in vivo studies

Recent findings suggest that over-expression of activated H-ras inhibited apoptotic cell death by blocking the activity of apoptotic endonuclease(s). This study was designed using antisense H-ras oligodeoxynucleotides (ODN) to evaluate whether alterations of H-ras expression in BEL-7402 human hepatocellular carcinoma cells could influence the induction of apoptosis in vitro and in vivo. We found that, in vitro, continuous suppression of H-ras expression could decrease the proliferation of BEL-7402 cells and inhibit H-ras-induced entry into S phase. In situ end labeling showed that a large number of cells underwent apoptotic cell death after treatment with antisense H-ras ODN (P<0.01), and gel electrophoresis of DNA extracted from these cells demonstrated a typical DNA ladder, characteristic of apoptosis. In vivo study indicated that pretreatment with antisense H-ras significantly retarded tumor growth in comparison with the untreated controls or tumors treated with non-specific ODN (P<0.01,P<0.01). In situ end-labeling revealed that pronounced apoptotic nuclei were also present in the tissue treated with antisense H-ras ODN (P<0.01). Immunocyto-histochemical study showed that expression of p21^H-ras was significantly decreased after treatment with antisense H-ras. These results indicate that suppression of H-ras over-expression by antisense ODN could effectively inhibit tumor growth and revive the apoptotic pathway by releasing the activity of apoptotic endonuclease(s). The data also suggest the need for further studies to elucidate molecular events involved in antisense H-ras-released apoptosis and evaluate its therapeutic implications.Abbreviations ODN oligodeoxynucleotides - HCC hepatocellular carcinoma - PBS phosphate-buffered saline     

Reversal of multidrug resistance by B859-35, a metabolite of B859-35, niguldipine,verapamil and nitrendipine

Summary It has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-l-piperadinyl)-propyl]-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 M B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 M (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. In KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (=100%). If 1 M B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 M B859-35 was a reduction in proliferation to 38%, that of 0.1 M verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.Abbreviations B859-35 (4R)-3-[3-(4,4-diphenyl-1-piperidinyl)-propyl]-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylatehydrochloride - B859-35 metabolite, (4R)-3-[3,(4,4-diphenyl-1-piperidinyl)-propyl]-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)-pyridime-3,5-dicarboxylatehydrochloride - MOPS 3-N-morpholinopropanesulfonic acid - MDRI human multidrugresistance gene - WR Walker cells resistant to alkylating agents and also multidrug-resistant     

Effects of okadaic acid on cell growth,anchorage-independent growth,and co-cultures of normal (KMS-6), immortalized (KMST-6), and neoplastically transformed (KMST-6T and KMST-6/RAS) human fibroblasts

The effects of okadaic acid (OA) on normal human (KMS-6), its immortalized (KMST-6) and neoplastically transformed (KMST-6T and KMST-6/RAS) cells were investigated as a model of two-stage carcinogenesis. The presence of OA inhibited cell growth of the normal and immortalized cells but not that of the neoplastic KMST-6T cells. In contrast, cell growth of the other neoplastic KMST-6/RAS cells transformed with the Ha-ras oncogene was inhibited by OA. OA enhanced colony formation of KMST-6T cells in soft agar, but it suppressed that of KMST-6/RAS cells. Co-cultures of KMST-6T cells with normal KMS-6 cells showed an increase in focus formation of KMST-6T cells in the presence of OA, whereas focus formation of KMST-6/RAS cells decreased. These results indicate that OA has growth-promoting effects on certain types of transformed human cells.Abbreviation OA okadaic acid This work was supported by a grant-in-aid from the Ministry of Education, Science, Culture and Sports of Japan     

Auranofin modulates human neutrophil superoxide production and protein phosphorylation

Summary The effect of auranofin (AF) was examined on human neutrophil superoxide production and protein phosphorylation stimulated by phorbol esters. Low concentrations of auranofin (0.5 M) enhanced while higher concentrations (0.5–10 M) inhibited superoxide release stimulated by a suboptimal concentration (0.005 M) of phorbol myristate acetate (PMA). The enhancing but not the inhibitory effect of AF was lost if a maximal stimulating dose (0.05 M) of PMA was used. In contrast AF had a biphasic effect on protein phosphorylation regardless of the stimulating concentration of PMA. Comparison of the dose-response curves for these effects of AF suggest that although changes in protein phosphorylation may be partly responsible for altered activity of the NADPH oxidase responsible for superoxide production, it is unlikely that they are mediated by a direct effect of AF on protein kinase C. Also, measurement of ³H-PDBu-binding to neutrophils showed that these actions of AF could not be attributed to altered binding of phorbol esters to their cellular receptor (protein kinase C).     

Bile acid sequestration by the solid phase of stools in cystic fibrosis patients

The distribution of bile acids in the stool of seven cystic fibrosis (CF) patients with severe or mild steatorrhea was examined and compared with that of three controls. Results indicated significantly lower endogenous bile acid concentrations in the stool water phase, obtained by centrifugation, in the CF patients (12.0±3.5%), compared with the controls (25.5±8.1%). In vitroincorporation of labeled cholic acid (CA) and deoxycholic acid (DCA) demonstrated a stronger binding of both to the paniculate matter of stools in the CF group. Using equilibrium dialysis, the calculated concentrations of unbound CA and DCA in the CF group measured 0.78 and 0.3 mol/g homogenate, respectively, and in the control patients 1.76 and 1.39 mol/g homogenate, respectively. Partial release of bile acids from CF stool pellets was achieved by the addition of trypsin and elastase, as well as by alkalinization. It is suggested that in patients with CF, stool bile acids are bound to the undigested protein fraction, which makes them unavailable for colonic resorption.     

Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins

Summary Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of ³H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 M were not effective competitors of the binding of either ³H-retinoic acid or ³H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.     

Vasoactive effects of eicosapentaenoic acid on isolated vascular smooth muscle

Summary Dietary intake of unsaturated fatty, acid of eicosapentaenoic acid (EPA) is thought to reduce the size and incidence of myocardial infarction. These beneficial effects are postulated to be due to chronic antithrombotic properties of EPA itself. We studied the possible direct effects of EPA on vascular smooth muscle as well as the ability of EPA to modify the vasoactivity of constrictor mediators in rabbit and cat aortic rings and isolated cat coronary arterics. EPA concentration-dependently (30 to 300 M) relaxed rabbit and cat aortic rings having an intact endothelium, while EPA did not show any significant vasodilator effects on rings without an endothelium. This EPA-induced vasorelaxation was not altered by the cyclooxygenase inhibitor ibuprofen, but was totally abolished by the guanylate cyclase inhibitor methylene blue, indicating an endothelium-dependent smooth muscle relaxation mechanism. In isolated perfused cat coronary arteries, EPA (3 to 300 M) exerted a dilator effect which was endothelium-independent and not affected by ibuprofen. The response was attenuated by propyl gallate, a lipoxygenase inhibitor. EPA also inhibited leukotriene (LT) C₄, (50 nM) and LTD₄ (50 nM)-induced vasoconstriction of isolated cat coronary arteries ranging from a blockade of 10% to 15% (P<0.05) at 3 M of EPA to a blockade of 89% to 93% (P<0.01) at 300 M. In contrast, the thromboxane analog, CTA₂, induced coronary constriction was not significantly altered by EPA. Thus, EPA produces endothelium-dependent relaxation in rabbit and cat aorta and endothelium-independent vasodilation in cat coronary arteries (i.e., intact vessels or helical strips). Moreover, EPA exerts acute anti-leukotriene actions in coronary arteries. In the case of long-term dietary intake of EPA, these actions may contribute to the protective action of EPA in myocardial ischemia.Supported in part by NIH Grant No. HL-25575 from the National Heart Lung and Blood Institute of the NIH.     

Synergistic interactions between differentiation-inducing agents in inhibiting the proliferation of HL-60 human myeloid leukaemia cells in clonogenic micro assays

Summary All-trans-retinoic acid, hexamethylene bisacetamide and 5-azacytidine are inducers of granulocytic differentiation of HL-60 human myeloid leukaemic cells, which eventually leads to inhibition of cell proliferation. The effect of graded concentrations of all-trans-retinoic acid (RA) (1 nM-1 M), hexamethylene bisacetamide (HMBA) (0.5–4 mM) and/or 5-azacytidine (5azaC) (1 nM-1 mM), alone and in combination with each other on colony formation and growth of HL-60 cells was studied in agar capillary clonogenic micro assays in order to identify new potential therapeutic regimens for elderly patients with acute myeloid leukaemia. ED₉₀ concentrations, inducing 90% inhibition of colony formation for RA, HMBA and 5azaC, were 128 nM, 2.7 mM and 40 M, respectively. The drug interactions between these differentiating agents were analysed by Berenbaum's general algebraic solution. The combinations: RA + HMBA, 5azaC + HMBA and RA + 5azaC were significantly synergistic in inhibiting HL-60 colony formation. Their interaction indices were 0.62, 0.83, and 0.97, respectively, at a specific effect level of 15%. The addition of 1 mM HMBA to 100 nM 5azaC- and 1 nM RA-treated cultures significantly increased the colony-formation inhibition from only 2.6% and 7.0% to 46.4%, and 43.1 %, respectively. Also, HMBA showed marked synergism with RA and 5azaC in inhibiting colony growth. The interaction indices (I) of HMBA + RA and HMBA + 5azaC were 0.013 and 0.009, respectively, at the same specific level of 15%. Moreover, the triple combination of RA + HMBA + 5azaC showed synergism in inhibiting both the colony formation (I=0.7) and colony growth (I=0.4) at the same specific level of 15%. Since RA, HMBA and 5azaC were effective when administered alone in phase I clinical trials of myeloid leukaemic patients, their synergistic combinations could provide shorter and less toxic courses of treatment in elderly myeloid leukaemic patients.I is < 1, =1 or > 1 in synergistic, additive or antagonistic interactions, respectively.Abbreviations AML acute myeloid leukaemia - 5-azaC 5-azacytidine - HMBA hexamethylene bisacetamide - RA all-trans-retinoic acid     

Absence of H-ras point mutation at codon 12 inN-methyl-N-nitrosourea-induced hepatocellular neoplasms in the rat

Summary In order to assess the possibility that activatedras-associated hepatic carcinomas might be much rarer in rats than mice because of the more frequent or rapid occurrence of powerful carcinogenic event(s) other thanras point mutations in the former animals, precancerous lesions and hepatocellular carcinomas induced by a weak hepatocarcinogenN-methyl-N-nitrosourea (MNU) in the rat liver were analyzed for the presence or absence ofras point mutations. MNU was chosen because it is well known that MNU-induced rat mammary carcinomas contain activated H-ras at very high frequency. Male Fisher rats were treated with a single dose of MNU after partial hepatectomy, and then administered dietary phenobarbital or repeated s.c. injections of carbon tetrachloride as promoting procedures. Analyses by oligonucleotide hybridization, MnlI-restriction-fragment-length polymorphism and NIH3T3 cell transfection assays revealed neither H-ras point mutations nor transforming ability of the DNA from 36 MNU-induced rat hepatic neoplasms. The results were in agreement with previous results for rat hepatocellular carcinomas induced by other potent liver carcinogens and did not support our hypothesis that the frequency of findingras activation might be dependent on the strength of the carcinogen.Abbreviations MNU N-methyl-N-nitrosourea - HCC hepatocellular carcinoma     

In vitro studies on drug interaction of ifosfamide and ACNU in primary and metastatic human brain tumours

Combination chemotherapy is widely employed in clinical oncology; however, there is no generally accepted model to evaluate individual tumour susceptibility to a given drug combination protocol. We therefore investigated the drug interaction of ifosfamide (4-hydroxyperoxy-ifosfamide) and ACNU in a recently developed in vitro model of paired sequential combination chemotherapy in primary and metastatic malignant brain tumours. A long-term standard [6,3-³H]-thymidine-incorporation assay, employing a liquid scintillation counting protocol, was selected to assess the drug sensitivity of human tumours. In vitro drug exposures were derived from correlating in vivo-(systemic and CNS) and in vitro-pharmacokinetic drug parameters. In combination experiments tumour cells were treated sequentially by two drugs in both sequences: drug exposures were calculated for 2 h with a 1-h drug-free interval in between. Cut-off concentrations (maximum in vitro exposure doses) were calculated as 1.74M (for primary CNS tumours: 0.58M) for ifosfamide and 5.4M (for primary CNS tumours: 1.33M) for ACNU. Dose/response relations were derived from isotope incorporation rates after cells had grown for approximately five population doubling times. Combination isoboles were plotted after drug doses had been transformed into equieffective doses, enabling comparison of drug combination effects. In all three glioblastomas (with CNS exposure dose) an additive or supra-additive effect could be observed in either sequence (in one tumour a biphasic additive isobole was found for both sequences). Out of three bronchial carcinomas (small-cell type, brain metastases) in two nonidentical sequences a supra-additive effect was observed in two tumours, with antagonistic effects in the third tumour. In all three malignant melanomas and in one renal carcinoma antagonistic effects were observed, whereas in a second renal carcinoma supra-additive effects were demonstrated for both sequences. We conclude that drug combination chemotherapy effects at the cellular level may be extremely heterogeneous.Presented at the Satellite Symposium Ifosfamide in Tumor Therapy: Questions for the Nineties; 15th International Cancer Congress, Hamburg, August 16–22, 1990     

Alterations in articular chondrocyte growth and proteoglycan synthesis due to prostanoid precursors

Summary The effects of the prostaglandin precursors, dihomo-gamma-linolenic acid and arachidonic acid, on chondrocyte proliferation, proteoglycan synthesis and morphological structure were studied using lapine articular chondrocytes in vitro. Neither substance exerted a cytotoxic effect on chondrocytes. Dihomo-gamma-linolenic acid caused a dose-dependent inhibition of chondrocyte proliferation (8% and 35% reduction at 10 and 100 mol/l respectively) (P<0.01), whereas arachidonic acid failed to cause any significant alteration: 10 mol/l of dihomo-gamma-linolenic acid stimulated proteoglycan synthesis by 14% (P<0.01), whilst 100 mol/l elicited a reduction of 14% (P<0.01); 100 mol/l of arachidonic acid also caused a statistically significant inhibition (31%) (P<0.001) of ³⁵SO₄ incorporation into proteoglycans. The inhibitory effects on proteoglycan synthesis may be mediated by intracellularly synthesized prostaglandins, which are known to exert this effect. This may also help explain the susceptibility of articular cartilage to damage with increasing age, as arachidonic acid is found in increasing concentrations in the superficial layers of articular cartilage.     

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells

Summary Binding studies using recombinant human¹²⁵I-labelled insulin-like growth factor I ([¹²⁵I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines withK _d ranging from 74×10^–12 M to 100×10^–12 M andB _max=36–63 fmol/mg cell protein. [¹²⁵I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC₅₀ values of 1.5 nM, 6.3 nM and 0.7 M respectively. Recombinant human [¹²⁵I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II withK _d=(110–175)×10^–12 M andB _max varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [¹²⁵I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC₅₀=0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 M IGF-I and 0.1 M IGF-II, the EC₅₀ value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC₅₀ value of about 0.7 nM for both peptides. -IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and longterm biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.     

Cell signalling associated with fibrinolytic ligand binding to human colorectal carcinoma cells

Summary Addition of purified plasmin or plasminogen (0.1 M) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP ₃) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca²⁺ channels of the type blocked by 5 M nifedipine, and 100 M EGTA, a Ca²⁺ chelator, did not suppress plasmin's ability to elevate InsP ₃. Binding assays at 4° C with¹²⁵I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.Abbreviations InsP ₃ inositol trisphosphate - Ptd phosphatidyl     

Plasma glutamate levels,lymphocyte reactivity and death rate in patients with bronchial carcinoma

Summary Elevated glutamate concentrations are commonly observed in patients with advanced carcinoma, and glutamate was recently found to inhibit the membrane transport of cystine and to impair the function of macrophages and lymphocytes in vitro. We therefore investigated the possibility that elevated plasma glutamate levels may be quantitatively correlated with reduced lymphocyte reactivity and an impaired host response to the tumor. Here we report the results of a study on patients with bronchial carcinoma, which show that patients with plasma glutamate levels above 120 M have a lower lymphocyte response to mitogens and a substantially higher death rate than those with glutamate levels below 120 M. This correlation does not prove a causal role of glutamate, but it confirms predictions from the in vitro laboratory data.     

Nitric oxide-mediated neurotransmission is attenuated in the anococcygeus muscle from diabetic rats

Summary The effect of STZ-induced diabetes of 8-weeks duration was examined on nitric oxide-mediated neurotransmission in the rat anococcygeus muscle. In the presence of noradrenergic blockade and raised tissue tone, relaxant responses to nerve stimulation (0.5–5 Hz, for 10 s), sodium nitroprusside (5 and 10 nmol/l) and nitric oxide (1 and 3 mol/l) were significantly reduced in anococcygeus muscles from diabetic rats compared to responses from control rats (p <0.05). In contrast, relaxations to papaverine (3 and 10 mol/l were not reduced in tissues from diabetic rats. The nitric oxide synthesis inhibitor NOLA (100 mol/l) abolished relaxant responses to nerve stimulation but had no effect on responses to any of the relaxant agents used. Exposure to NOLA at 10 mol/l reduced stimulation-induced relaxations; this reduction was significantly greater in tissues from the diabetic group than from the control group (p <0.05), probably as a consequence of the smaller relaxant responses in muscles from diabetic rats. Contractile responses to nerve stimulation (1–10 Hz, for 10 s), but not noradrenaline (0.03–30 mol/l), were significantly greater in anococcygeus muscles from diabetic rats than from control rats (p <0.05). NOLA (100 mol/l) significantly enhanced stimulation-induced contractions (p <0.05), however the enhancement was significantly less in tissues from diabetic rats (p <0.05). The results suggest that STZ-induced diabetes impairs smooth muscle reactivity to nitric oxide in the rat anococcygeus muscle.Abbreviations STZ Streptozotocin - NOLA N^G-nitro-l-arginine - NANC nonadrenergic noncholinergic - ANOVA analysis of variance     

Modulation of hepatic glucose production by non-esterified fatty acids in Type 2 (non-insulin-dependent) diabetes mellitus

Summary To study the effect of changes in plasma non-esterified fatty acid concentration on suppression of hepatic glucose production by insulin eight Type 2 (non-insulin-dependent) diabetic patients participated in three euglycaemic, hyperinsulinaemic (108pmol · m^2–1 · min^–1) clamp studies combined with indirect calorimetry and infusion of [3-³H]-glucose and [1-¹⁴C]palmitate; (1) a control experiment with infusion of NaCl 154 mmol/l, (2) heparin was infused together with insulin, and (3) an antilipolytic agent, Acipimox, was administered at the beginning of the experiment. Six healthy volunteers participated in the control experiment. Plasma non-esterified fatty acid concentrations during the insulin clamp were in diabetic patients: (1) 151±36 mol/1, (2) 949±178 mol/l, and (3) 65±9 mol/l; in healthy control subjects 93±13 mol/l. Non-esterified fatty acid transport rate, oxidation and non-oxidative metabolism were significantly higher during the heparin than during the Acipimox experiment (p<0.001). Suppression of hepatic glucose production by insulin was impaired in the diabetic compared to control subjects (255±42 vs 51±29 mol/min, p<0.01). Infusion of heparin did not affect the suppression of hepatic glucose production by insulin (231±49 mol/min), whereas Acipimox significantly enhanced the suppression (21±53 mol/min, p<0.001 vs 154 mmol/l NaCl experiment). We conclude that insulin-mediated suppression of hepatic glucose production is not affected by increased non-esterified fatty acid availability. In contrast, decreased non-esterified fatty acid availability enhances the suppression of hepatic glucose production by insulin.