Activation of a nuclear sphingomyelinase in radiation-induced apoptosis.

The subcellular origin of ceramide signaling in ionizing radiation-triggered apoptosis was investigated using two previously described subclones of the autonomous erythro-myeloblastic cell line TF-1, radio-resistant and -sensitive TF-1-34 and TF-1-33, respectively. We show in nuclei-free lysates and cytoplasts that both cell lines failed to generate ceramide in response to ionizing radiation. Moreover, whereas cytoplasts did respond to anti-Fas stimulation through phosphatidylserine externalization, no effect was observed with ionizing radiation. Only in highly purified nuclei preparations did we observe ceramide generation, neutral sphingomyelinase activation, and apoptotic features (PARP cleavage, nuclear fragmentation, DNA laddering) in TF-1-33, but not in TF-1-34 cells. These observations suggest that nuclear sphingomyelinase and ceramide formation may contribute to ionizing radiation-triggered apoptosis.

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.

In situ assay of acid sphingomyelinase and ceramidase based on LDL-mediated lysosomal targeting of ceramide-labeled sphingomyelin.

The activity of lysosomal sphingolipid hydrolases is usually estimated in vitro from complex assays on cell lysates under artificial conditions including the presence of detergents and substrate analogs. However, the measure of their effective activity in situ (i.e., in living cells) is necessary to understand the normal intracellular sphingolipid turnover. Moreover, their determination in cells from patients with genetic enzyme deficiencies represents a key parameter of the pathophysiology of sphingolipid storage disorders. In this report, we have developed a procedure for estimating the effective activity of lysosomal sphingomyelinase and ceramidase in situ. This procedure is based on the selective targeting to lysosomes of a natural substrate under physiological conditions of substrate influx. Epstein-Barr virus-transformed human lymphoid cells and human skin fibroblasts were incubated with purified human low density lipoproteins (LDL) containing [3H]ceramide-labeled sphingomyelin. Data demonstrate that this substrate is internalized through the apolipoprotein B/E receptor pathway and targeted to lysosomes. Lysosomal localization of the incorporated substrate was evidenced by ultrastructural autoradiography and subcellular fractionation as well as by metabolic studies in mutant cells. Short-term pulse-chase experiments with LDL-associated [3H]ceramide-labeled sphingomyelin allowed us to determine the effective activity of lysosomal sphingomyelinase and ceramidase in normal cells. Initial velocities of sphingomyelin and ceramide degradation were, respectively, estimated at 0.66 and 1.14 nmol.h-1.mg cell protein-1 in lymphoid cells, and 5.4 and 3 nmol.h-1.mg cell protein-1 in skin fibroblasts. The advantages and applications of these in situ studies are discussed.

Increased content of annexin II (p36) and p11 in human placenta brush-border membrane vesicles during syncytiotrophoblast maturation and differentiation.

Annexins are a group of proteins abundant in placental membranes where they may play diverse functional roles. Annexins are expressed in high levels in mature placenta but little is known about their presence at very early stages of gestation and later. We used the model of brush-border membrane vesicles (BBMV) at different stages of gestation to assess precise localization of some of these proteins in syncytiotrophoblast apical membrane and to determine their appearance along the maturation process of placenta. Here we describe annexins type I, II, IV, V and VI which are present all along gestation in BBMV. Annexin II (p36) is present with the S100 like calcium-binding protein p11 in BBMV, where they can constitute heterotetrameric forms of annexin II linked to cytoskeleton structures. No variation of annexins I, IV and VI content was observed in BBMV along pregnancy. Annexin V undergoes significant decrease after 12th week, which could be related to local anticoagulant activity. Levels of annexin II and p11 increased progressively during gestation suggesting that heterotetrameric forms of annexin II play a role in the differentiation process of placenta and in function of the mature microvilli.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle.

It has been claimed repeatedly that gamma-tubulin is exclusively localized at the spindle poles in mitotic animal cells, where it plays a role in microtubule nucleation. In addition to this localization, we have observed a gamma-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xenopus and Drosophila) using five polyclonal antibodies raised against unique gamma-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, gamma-tubulin was detected in the mitotic spindle from late prometaphase to telophase. In contrast, in other cell types, it was detected in metaphase only. In all cases we failed to detect gamma-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the gamma-tubulin localized on the surface of spindle microtubules with a preferential distribution along kinetochore microtubules. In HeLa cells, the amount of antigenic gamma-tubulin was fairly constant in the spindle poles during mitosis from prometaphase to telophase. In contrast, gamma-tubulin appeared in the mitotic spindles in prometaphase. The amount of gamma-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluorescence was localized at the spindle poles, while 75-85% of the fluorescence was distributed over the rest of the spindle. These results suggest that the localization and timing of gamma-tubulin during the cell cycle is highly regulated and that is physiological role could be more complex and diverse than initially assumed.

Response of four human ovarian carcinoma cell lines to all-trans retinoic acid: relationship with induction of differentiation and retinoic acid receptor expression.

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR3 and OVCCR1 were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV1 cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RAR alpha was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RAR beta was not detected in any of the cell lines, while RAR gamma (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV1 cells.

Rhazinilam mimics the cellular effects of taxol by different mechanisms of action.

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0 degrees C and at 37 degrees C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 microM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level.

gamma-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells.

Animal cells undergoing cytokinesis form an inter-cellular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of gamma-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mammalian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corresponding peptides, by Xenopus gamma-tubulin, but was not modified by purified alpha beta-tubulin heterodimer. An identical staining was obtained with affinity-purified antibodies against the carboxyl-terminal amino acid sequence of human gamma-tubulin. No gamma-tubulin could be detected in the interzone during anaphase and early telophase. Material containing gamma-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles constituting the midbody for one hour after metaphase. Micro-injection of gamma-tubulin antibodies into anaphase cells prevented the subsequent formation of the microtubule bundles between the two daughter cells. In contrast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.

Cell cycle-dependent assembly and disassembly of cytoplasmic microtubules in the plasmodium of the myxomycete Physarum polycephalum.

The giant syncytium of Physarum plasmodia possesses a complex cytoplasmic microtubule network except during the occurrence of the intranuclear mitosis. In early prophase stages, intranuclear spindles assemble concomitantly as the cytoplasmic microtubule network disassembles. No cytoplasmic microtubules are present in metaphase. They begin to reassemble in telophase. The complex cytoplasmic microtubule network reappears in early reconstruction stages. The assembly of cytoplasmic microtubules occurs on cytoplasmic foci, both in telophase stage and during rewarming after cold microtubule disassembly. These foci, independent of the nuclei, correspond to the foci observed in the cytoplasm during interphase, both by immunofluorescence and electron microscopy. As cytoplasmic and intranuclear microtubule-organizing centers are spatially distinct, plasmodial syncytia offer the possibility to study the effects of cell regulatory pathways on two types of microtubule-organizing centers that differ in their nucleating activity during the cell cycle.

Physarum plasmodia do contain cytoplasmic microtubules!

It has been claimed that the plasmodium of the myxomycete Physarum polycephalum constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.

Genetic analysis of the relationships between the amoebal extranuclear spindle-organizing centre and the plasmodial intranuclear spindle-organizing centre of Physarum during conjugation.

Physarum amoebae possess an extranuclear spindle-organizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.

Differential in vitro action of S-12363, a new vinblastine derivative, and of its epimer on microtubule proteins.

The action of two epimers of a new vinblastine derivative that differ in their in vivo antitumor activity and their cytotoxicity was studied in vitro in brain microtubule proteins. These two compounds, called S-12363 and S-12362, could not be distinguished from one another or from other active vinca alkaloids by their ability to prevent microtubule assembly. However, they differed strongly both from one another and from vincristine and vinblastine in their ability to induce the formation of tubulin paracrystals and in the stability of the paracrystals following temperature shifts from 0 degree to 37 degrees C and vice versa. The most potent drug, S-12363, induced considerable tubulin aggregation, which was even more pronounced than that observed in the presence of vincristine. Previous results have shown that S-12363, in contrast to vincristine, induces no neurotoxic effects. This observation is in disagreement with a direct relationship between tubulin aggregation and neurotoxicity.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum.

The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. The mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cytoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.

Acetylcholine metabolism by cultured neurons from rat nodose ganglia: regulation by a macromolecule from muscle-conditioned medium.

Acetylcholine metabolism has been studied in primary cultures of neurons dissociated from newborn rat nodose ganglia. Neither nerve growth factor nor muscle-conditioned medium had any detectable effect on the long-term survival of these neurons, which appeared well differentiated upon phase-contrast and electron microscopic examination. [3H]Acetylcholine synthesis and accumulation by 2-3-week old nodose cultures and choline acetyltransferase activity were increased (2.0 +/- 0.15)-fold and (2.0 +/- 0.48)-fold, respectively, upon growth with muscle-conditioned medium, whereas acetylcholinesterase was decreased (1.5 +/- 0.1)-fold (means +/- SEM, n = 5-9). The same effects were observed when comparing nodose cultures grown in the presence of proteins purified from conditioned medium in four fractionation steps. This purified material had no effects on the protein content of the cultures, or on lactate dehydrogenase activity, and thus did not affect the overall growth of the cultures. We demonstrate that this factor copurifies with a factor(s) involved in the regulation of choline acetyltransferase and acetylcholinesterase in neuron cultures from newborn rat superior cervical ganglia [Swerts, Le Van Thai, Vigny and Weber (1983) Devl Biol. 100, 1-11] and from embryonic day 13 rat embryo spinal cord [Giess and Weber (1984) J. Neurosci. 4, 1442-1452]. Although the cholinergic factor from muscle-conditioned medium has not been purified to homogeneity, the data suggest that the same extracellular, macromolecular factor may be involved in the regulation of acetylcholine metabolism in derivatives from the neural crest, the neural tube and the epibranchial placodes.

Localization of human placental opiate binding sites on the syncytial brush border membrane.

Opiate binding sites were measured in different placental membrane fractions which were characterized by marker enzyme analysis and electron microscopic examination. The distribution pattern of opiate binding sites in the different fractions closely parallels that of placental alkaline phosphatase. These results clearly show that opiate binding sites are mainly located on the syncytial brush border membrane. The opiate binding sites found on microvillus membrane fraction have the same pharmacological characteristics as the Kappa opiate binding site previously characterized on placental crude membrane fraction.

Comparative cytotoxic and antitumoral effects of ellipticine derivatives on mouse L 1210 leukemia.

Twelve derivatives of the antitumoral alkaloid ellipticine (E) and ellipticinium were assayed in vitro on cultured L 1210 cells. These drugs possess varying abilities to decrease the cell growth rate in a 1--1000-fold range. Some of them have a highly cytotoxic effect in the 10(-8)--10(-6) M range. Non-specific intracellular damages are produced: multilobation of nuclei, occurrence of numerous lipid granules, diminution of the size and increase in the number of mitochondrial profiles and several modifications of the internal architecture of mitochondria. 2-Methyl-9-hydroxyellipticinium (2-CH3-9-OHE) was submitted to a bioassay; it inactivates the tumorigenic potency of the cells exposed to it, when they are grafted back into mice in the same dose range which reduces in vitro the growth rate of the cells. A fairly good correlation holds between the in vitro and in vivo (antitumor effect) assays, offering a possible prescreening test for a cheaper and rapid evaluation of chemotherapeutic activity of these compounds. The results stress again the importance of the 9-hydroxy substitution in these series for improving the anticancer efficiency. The nature of the biochemical target of E and derivatives is discussed according to our data.

[Demonstration of the action of methyl benzimidazole 2 yl carbamate (MCB) and methyl(5(2 thienyl carbonyl) 1 H benzimidazole 2 yl carbamate) (R17934) on the nucleus of Physarum polycephalum (Myxomycetes)]. / Mise en évidence de l'action du méthyl benzimidazole 2 yl carbamate (MBC) et du méthyl[5(2 thiényl carboyl) 1 H benzimidazole 2 yl carbomate] (R 17934) sur le noyau de Physarum polycephalum (Myxomycètes)

The toxicity of these compounds was determined in the amoebae and plasmodia. An electron microscopic study shows an increase in nuclear size which is in agreement with the increase of the total amount of DNA. Microtubules are present but they are not organized in a normal mitotic apparatus. Some other nuclear abnormalities are described.

Characterization of mitochondrial DNA from the pika (Ochotona rufescens rufescens).

Further to anatomical and physiological studies performed on the pika (Ochotona rufescens rufescens), a new laboratory animal, the main characteristics of the mitochondrial DNA from its liver are defined. The buoyant density of this DNA is 1.695 g/cm3, its length 5.30 mum,, i.e., 3.17 times that of the replicative form of phiX 174. It could have approximately 16 500 base pairs. The DNA from the pika is very similar to that of the rat or the rabbit, although these animals show great physiological differences.