MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Sphingolipids and the sphingosine kinase inhibitor, SKI II, induce BCL-2-independent apoptosis in human prostatic adenocarcinoma cells.

BACKGROUND: Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance. METHODS: We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II. RESULTS: In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment. CONCLUSIONS: Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC.

Hyperthermia engages the intrinsic apoptotic pathway by enhancing upstream caspase activation to overcome apoptotic resistance in MCF-7 breast adenocarcinoma cells.

Febrile hyperthermia enhanced TNF-stimulated apoptosis of MCF-7 cells and overcame resistance in a TNF-resistant, MCF-7 variant (3E9), increasing their TNF-sensitivity by 10- and 100-fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF-7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase-8 activation, as the specific inhibitor, Z-IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase-8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF-dependent loss of mitochondrial membrane potential and activation of capspase-9 that was initiated and dependent on upstream caspases. MCF-7 and 3E9 cells were equally sensitive to exogenous C(6)-ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro-palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF-7 cells and overcomes TNF-resistance by sustained activation of caspase-8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase-dependent apoptosis by febrile hyperthermia.

Cytotoxic effects of sphingolipids as single or multi-modality agents on human melanoma and soft tissue sarcoma in vitro.

We evaluated the cytotoxic effects of a cell-permeable ceramide (Cer), N-hexanoyl-D-sphingosine (C6-Cer) and of two related sphingoid bases, sphingosine (So) and dihydrosphingosine (sphinganine; Sa) on human melanoma cell lines and on soft tissue sarcoma lines recently established from fresh surgical biopsy specimens. These cell lines ranged from high susceptibility (939 melanoma) to strong resistance (A2058 melanoma and all three sarcomas) to tumour necrosis factor (TNF), an inducer of elevated intracellular Cer levels. However, all the cell lines demonstrated a dose-dependent susceptibility to C6-Cer with protracted cytotoxic kinetics, with the C8161 melanoma being the most sensitive and A2058 the least. Protein kinase C (PKC) antagonizes Cer-dependent apoptosis, and chelerythrine chloride, So and Sa, which inhibit PKC, caused extremely rapid cytotoxicity of melanoma cell lines, irrespective of their relative sensitivity to C6-Cer. So-mediated cytotoxicity was extensive even after only 90 min of treatment, within the time frame of limb perfusion. So and Sa only slightly potentiated the cytotoxic responses to TNF, C6-Cer or melphalan. Sphingolipid-driven intracellular pathways may offer opportunities for therapy of these tumours.

Multi-chemothermoimmunotherapy for human colon adenocarcinoma in vitro.

Effective adjunctive therapies for colorectal carcinoma are clearly needed. We evaluated the cytotoxic responses in vitro of human colon carcinoma cell lines to combined modalities: 5-fluorouracil/leucovorin (5-FU/LV), carboplatin (CP), tumor necrosis factor (TNF) and hyperthermia (HTX). Cytotoxicity was evaluated in a cell proliferation assay using crystal violet staining. 5-FU/LV was administered 2-3 days before TNF and CP, followed 1 h later by HTX. These cell lines were relatively resistant to HTX alone (42 degrees C for 2 h), but were heterogeneous in their responses to various doses of the other single agents. This heterogeneity was also evident for combined modalities: the HCT-15 cell line exhibited significant supra-additivity for selected doses of CP, TNF and 5-FU/LV, which was further enhanced by hyperthermia. In contrast, the HT-29 cell line did not demonstrate a strong pattern for supra-additivity, whereas the DLD-1 cell line had an intermediate response. Thus, our results suggest one approach to develop effective and dose-sparing multimodality therapeutic regimens for colon adenocarcinoma.

Formulation and antitumor efficacy of liposomal-caprylated-TNF-SAM2.

The tumor necrosis factor (TNF) mutant TNF-SAM2 has previously been shown to have a therapeutic profile superior to parental TNF. To initially evaluate the characteristics of liposomal formulations of TNF-SAM2, it was modified with the N-hydroxysuccinimide ester of caprylic acid to increase its hydrophobic binding to multilamellar and small unilamellar vesicles (MLVs and SUVs). Native PAGE and fluorescamine analysis of acetylated parental TNF and TNF-SAM2 indicated that these proteins both displayed trimeric structures based on crosslinking/SDS-PAGE analysis and behaved similarly with respect to reactivity of their amino functions. Limited N-terminal sequencing analysis of partially acetylated (approx 3 acetyl groups per trimer) TNF-SAM2 indicated that the N-terminal Val was not modified; this was also concluded based on HPLC/mass spectrometric (LC-MS) analysis of Glu C digests. LC-MS analysis of tryptic digests of the acetylated TNF-SAM2 indicated that Lys-98 was unreactive. Molecular ions corresponding to acetylated Lys-containing peptides for all five other Lys residues could be detected; none appeared hyperreactive, but Lys-11 appeared hyporeactive. MLVs composed of DMPC/DMPG (7:3) and SUVs composed of DPPC/DSPC (1:1) displayed high capacity for binding to caprylated TNF-SAM2. These formulations of caprylated TNF-SAM2 displayed tumor necrotizing and growth-inhibitory activity in a syngeneic tumor model, and may be candidates for clinical development.

Clonogenic survival studies of human colon tumor cell lines in vitro: combined hyperthermia, 5-fluorouracil/leucovorin, carboplatin and tumor necrosis factor.

The responses of DLD-1 and HCT-15 human colon adenocarcinoma cells to hyperthermia, 5-fluorouracil (5-FU)/leucovorin, carboplatin and tumor necrosis factor-alpha, singly and in multiple combinations, were evaluated in clonogenic assays. The combination of hyperthermia with the lower dose combination resulted in a survival fraction of about 0.005 to 0.001 for both cell types, whereas estimated additive interactions alone would have resulted in a survival fraction of about 0.5 (DLD-1) or 0.05 (HCT-15). A survival fraction of 0.00001 or greater was observed when the higher dose levels were combined with hyperthermia, whereas additive interactions alone would have achieved a decrease of only 0.001 or 0.0001 in the surviving fraction. The combination of the three other modalities at either dose level under conditions of hyperthermia or normothermia achieved statistically significant apparently supra-additive losses of clonogenicity in HCT-15 cells; similar results were obtained with the lower dose level in DLD-1 cells. Our results suggest that human colon tumor cells are markedly sensitive to this combination of modalities when used at clinically achievable dose levels.

Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro.

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.

Human Kupffer cells are cytotoxic against human colon adenocarcinoma.

Colorectal liver metastases are a common clinical problem and require more effective therapy. Kupffer cells (KC) perform many important homeostatic functions within the liver and may also possess the ability to mediate tumor cytotoxicity. We investigated the ability of human KC to mediate cytotoxicity against human colon adenocarcinoma targets (HT 29) in vitro. Unstimulated human KC were cytotoxic against the HT 29 targets at all effector/target ratios tested. This cytotoxicity was increased significantly (p less than 0.05) when the KC were stimulated with interferon-gamma and lipopolysaccharide. Human KC produced tumor necrosis factor (TNF), and KC stimulation significantly (p less than 0.05) increased secretion of this monokine. The addition of anti-TNF antibody to the KC-HT 29 cocultures completely neutralized all of the available TNF yet cytotoxicity was not affected, suggesting the participation of a membrane-bound form of TNF or other mechanisms.

L-arginine independent macrophage tumor cytotoxicity.

We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.

Tumoricidal effector mechanisms of murine BCG-activated macrophages. I. Parameters of production and initial characterization of a cytolytic factor serologically related to necrosin.

Previous studies have shown that peritoneal macrophages from mice chronically infected with Bacillus Calmette-Guérin (BCG) become highly cytotoxic for tumor targets upon further in vitro triggering with a variety of agents. In the current studies, achievement of this activated state was characterized by the production and release of a cytotoxin, herein termed cytolytic factor (CF), which appeared in the fluid phase. Production/release of CF by the macrophage required transcription, translation, glycosylation, and an intact secretory apparatus, as evident from inhibition by treatment with actinomycin-D, cycloheximide, tunicamycin, and monensin, respectively, prior to and during triggering with lipopolysaccharide (LPS). CF obtained by culture of BCG-activated macrophages appeared rapidly in the supernatant after triggering. Using the actinomycin-D-treated L-929 or EMT-6 targets in a microassay, CF secreted by macrophages cultured in low molecular weight serum components was detected as an approximately 150-kD component on Sephacryl S-200. CF demonstrated a spectrum of cytotoxic activity against a number of tumor and normal targets in vitro. Its lytic activity appeared equally effective whether the targets were cultured in medium containing fetal calf serum (FCS) or in Neuman-Tytell medium without serum during the assay. CF was moderately sensitive to treatment with TLCK and TAME; however, its activity in serum and apparent molecular weight distinguish it from a moiety obtained from BCG-activated murine macrophages and previously described. A rabbit heteroantiserum raised against highly purified necrosin, a product of the murine macrophage cell line J774.1, was extremely effective in neutralizing the biological activity of CF.

Tumoricidal effector mechanisms of murine Bacillus Calmette-Guérin-activated macrophages: mediation of cytolysis, mitochondrial respiration inhibition, and release of intracellular iron by distinct mechanisms.

Murine Bacillus Calmette-Guérin-activated macrophages mediate discrete cytotoxic effects in cocultured tumor target cells in vitro. These effects include: the loss of intracellular iron, in part associated with reversible inhibition of the Kreb's cycle enzyme, aconitase; cytostasis, associated with reversible lesions inflicted in the electron transport chain (ETC) of the mitochondria resulting in reversible loss of proliferative capacity; and cytolysis, manifested by eventual gross perturbation of the integrity of the plasma membrane. We demonstrate that these manifestations of cytotoxicity are the result of three independent mechanisms employing apparently distinct macromolecules for their commission. Analysis of target cells that are highly susceptible (L-929), highly resistant (L-1210), or have incomplete resistance (EMT-6) to the cytolytic effects of cocultured activated macrophages indicates that there is no consistent relationship between the release of intracellular 59Fe and 51Cr. Thus, perturbation of intracellular iron pools did not appear to be an obligatory step on the pathway to cytolysis. Further evidence for this dissociation was obtained by employing a specific heteroantiserum reactive with cytolytic molecule(s). This antiserum could block the cytolytic response (51Cr release of cocultured L-929 and EMT-6 targets) but had no effect on the extent of iron release from viable EMT-6 or L-1210 targets. Furthermore, the cytolytic factor itself was incapable of mediating effects on the ETC or in causing release of intracellular iron. Two lines of evidence suggested that effects on the ETC are not linked with loss of intracellular iron. First, the monokine respiration inhibitory factor was incapable of causing release of intracellular iron from target cells in which the mitochondria were strongly suppressed. Second, the kinetics of release of respiration inhibitory factor from endotoxin-triggered Bacillus Calmette-Guérin-activated macrophages indicate a retarded appearance compared to the time at which a factor mediating release of intracellular iron was detectable. Our results strongly suggest that these three distinct cytotoxic reactions are under differential control by the effector cell.

Sensitization of tumor target cells to cytolysis by murine macrophage cytolytic factor by drugs inhibiting DNA, RNA, and protein synthesis.

The mechanism(s) with which tumor target cells actively resist the lethal lesion induced by murine macrophage cytolytic factory (CF) has been probed by drug-induced sensitization of these cells. We have examined whether drug-induced sensitization is attributable to the action of these drugs on cellular DNA, RNA, or protein synthetic rates. The murine mammary adenocarcinoma cell line EMT-6 pretreated with a dose range of actinomycin-D (.03-3.0 micrograms/ml) for 4 hr was inhibited from 66 to 98% in DNA synthesis rate, from 81 to 93% in RNA synthesis rate, and from 38 to 52% in protein synthesis rate. The maximum sensitization toward lysis by CF was achieved with a drug dose of 1.0 micrograms/ml. The lack of correlation between the drug-induced effects on sensitization and effects on these metabolic rates was evident from the correlation coefficients for the percentage of maximum sensitization of the target toward CF-induced lysis, and the percent of inhibition of DNA, RNA, and protein synthesis. These were 0.11, -0.11, -0.44, respectively. Similarly, target cells treated with a dose range of cycloheximide (0.3-30 micrograms/ml) were inhibited from 81 to 95% in DNA synthesis rate, 68 to 81% in RNA synthesis rate, and 82 to 93% in protein synthesis rate. However, at none of the drug levels employed was significant sensitization of the target cell to CF-induced lysis observed. This was reflected in the correlation coefficients of -0.55, -0.28, and -0.34 for DNA, RNA, and protein synthetic rates, respectively. The essential role of cellular microtubule-dependent events early in the lytic mechanism was demonstrated by exposing colchicone-treated targets to CF. While colchicone could markedly inhibit lysis if introduced before CF, this inhibitory effect was not detected if the drug were withheld for 4 hr after CF exposure. The importance of the repair mechanism(s) early in the cellular response to lesion formation was demonstrated by altering the schedule for CF and actinomycin D administration to the target. If actinomycin D treatment was withheld from the targets for as little as 3 hr following introduction of CF, the lytic mechanism had already bypassed the drug-sensitive steps, manifest in markedly decreased susceptibility to lysis. Collectively, the data show that the ability of the EMT-6 target cells to resist CF-induced lysis does not depend solely on the ability of the cells to synthesize DNA, RNA, or protein.

[Irregular anti-P1 allo-antibodies and sinensis clonorchiasis]. / Allo-anticorps irréguliers anti-P1 et clonorchiase á Clonorchis sinensis.

A parasitological and immuno-hematological study was undertaken simultaneously in fifty South-East asian refugees at the time of their arrival in France. --in this series the frequency of individuals having a P2 erythrocyte phenotype is 80%. --54 % of these immigrants were found to be carriers of Clonorchis sinensis, a parasite rarely found in Europe. --in 40,7 % of these subjects infested by Clonorchis sinensis, the following properties were disclosed concerning the P1 allo-antibody: slow-P1 red cell agglutination at 22 degrees C, no hemolysis of P1 red cells in vitro, IgM antibody, in weak titers. The immuno-hematological study of the immuno-serums with respect to distomian antigens coupled with adsorption-elution using P1+++ red cells shows a close immunological relationship between the antibody of parasite origin and the anti-P1 allo-antibody.

[Peculiarities of hemostasis in the term newborn infant]. / Particularités de l'hémostase du nouveau-né à terme

Haemostasis in the new-born is a product of various factors which are both qualitative and quantitative. The only factors that compare in levels and quality with those in the adult are factors V, VIII and XIII. They are alterations in the semi-analytical tests for coagulation except in the Stypven time. In contrast with this deficit shown up in haemostasis by global tests coagulation is normal and in truth there is hypercoagulability. Using Laurell's method of immunoelectrophoresis for levels of alpha 2 M high levels of this are shown contrasting with progressively lowered antithrombitic action. These paradoxes no doubt arise from the fact that neonatal haemostasis is analysed using standardised techniques and reactions which were developed for adult haemostasis, from which it is certainly as different as from those of other animals. Neonatal haemostasis is perfectly balanced in normal conditions. Important changes occur however in respiratory distress where there is a drop in factor V and soluble complexes appear, bringing about rough shapes suggestive of the subclinical syndrome of defibrination. This situation will be further developed in a forthcoming article.

[Compared efficiency of mean corpuscular volume (MCV) and serum gamma-glutamyltransferase (gamma-GT) as screening tests for excess-ethanol drinkers (author's transl)]. / Efficacite comparee du volume globulaire moyen (vgm) et de la gamma-glutamyltransferase (gamma-gt) serique comme tests de triage des buveurs excessifs d'alcohol

53 percent of ethanol drinkers had, before detoxication, a gamma-GT higher than the upper limit of the reference interval at the 2.5 percent risk level (36 mU/ml). 44 percent had a mean corpuscular volume (MCV) higher than the limit (99.2 mum3). In alcoholics not previously "weaned" during a rest cure or in a hospital the proportion becomes 67 percent for gamma-GT but remains at 44 percent for MCV. gamma-GT thus seems a better test for the screening of an excessive ethanol intake than MCV, especially when the subject has not been previously weaned.