Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates.

Results from ultrastructural immunocytochemistry on glutaraldehyde-fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules.

The molecular organization of myosin in stress fibers of cultured cells.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.

Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.

Effect of microtubule inhibitors on malignant invasion in vitro.

The malignant C3H/3T3 mouse cells MO4 invaded embryonic chick heart fragments in an organotypic coculture system on semisolid medium, which mimicked malignant invasion. In this system, at a dose of 1 microgram/ml, the microtubule inhibitors colchicine, demecolcine, vincristine sulfate, vinblastine sulfate, or methyl[5-(2-thienylcarbonyl)-1H-benzimidazol-1-yl]-carbamate (Nocodazole) totally inhibited malignant invasion. At the same dose the drugs were also mitostatic, which was apparent from C-mitoses and from the absence of postmetaphase figures. At a mitostatic dose of 10 microgram/ml, 5-fluorouracil (FUra), cytosine arabinoside, or bleomycin did not interfere with malignant invasion. Combined treatment of the cocultures with the antimetabolite FUra (10 microgram/ml) plus the microtubule inhibitor Nocodazole (1 microgram/ml) completely inhibited invasion. These cocultures also showed the effective inhibition of mitosis by FUra, because Nocodazole-induced C-mitoses were absent. The reversibility of the anti-invasive effect of 4-day treatment with Nocodazole (1 microgram/ml) was demonstrated in shaker cocultures with the use of fluid medium. Our in vitro experiments indicated that cytoplasmic microtubules were involved in malignant invasion and that cell division and invasion constituted separate characteristics of malignant cells.

Characterization of spontaneous, chemicak, and viral transformants of a C3H/3T3-type mouse cell line by transplantation into young chick blastoderms.

Cells from a C3H/3T3-type cell line were transplanted into defects of the lower layer of stage-4 chick blastoderms before and after "spontaneous," chemical, and viral transformation. To check the validity of inhibition of lower-layer defect closure as a criterion of malignancy, we compared the behavior of the lower layer toward these cells with their in vitro growth pattern, their capacity to invade embryonic chick skin explants that were organotypicaly cultured, and their tumorigenicity in syngeneic mice. No false-positive results were observed with either test. The study of the in vitro growth pattern gave false-negative results during the early phase of spontaneous transformation, whereas these cells, exhibiting an untransformed growth pattern, were shown to be malignant by the other tests. We concluded that the inhibition of lower-layer defect closure is a reliable, sensitive and rapid test for the detection of malignancy in tissue-cultured cells from any source.

A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells.

A new culture model, which facilitated both mass screening of potential anticancer drugs acting on microtubules and quantitative experiments with known "antitubulins," was found to have the following advantages: use of mammalian cells (either transformed or not), simplicity of the techniques (phase-contrast microscopy or simple microscopy after Giemsa staining), and ease with which it lent itself to quantification. The model was based on the uniform multimicronucleation response induced by antitubulins in MO cells. The specificity (towards antitubulins) of this response was ascertained by the use of many substances, including most of the known antitubulins and a number of nonrelated cytostatic or cytotoxic compounds. The uniformity of the response was established with the use of time-lapse observation of large numbers of cells and quantitative approaches. The results obtained in this model with the standard antitubulins (colchicine, vinblastine, vincristine) showed similar effects. The major difference between colchicine and the Vinca alkaloids was that colchicine was less reversible, which might be an indication of stronger intracellular binding. The Vinca alkaloids acted synergistically with colchicine when threshold subactive doses were combined, although it is known that they bind at a different site on tubulins. A number of substances that have been claimed or were suspected to interfere with microtubules were tested. The results showed that the following substances were indeed active with MO cells: colchicine, vinblastine, vincristine, podophyllotoxin, rotenone, griseofulvin, mercaptoethanol, benomyl, methyl benzimidazol-2-yl carbamate, and R 17934. Compounds that were inactive on these mammalian cells in culture included isopropyl carbanilate and melatonin, both of which have been shown to be active in other systems.

The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro.

Ultrastructural investigations on mammalian cells cultured in vitro show that R 17934, a new synthetic anticancer drug, interferes with the structure and function of microtubules, both in interphase and mitotic cells. The activity of this compound in a wide range of experimental tumor systems can thus be explained partly as a direct antimitotic effect and partly as the disintegration of the normal subcellular organization of the nondividing cells. Preliminary investigations in experimental animals show that malignant cells are more susceptible to the antimicrotubular effect of R 17934 than are the nonmalignant cells of the host.

Effects of tubulozole on the microtubule system of cells in culture and in vivo.

Light and electron microscopic investigations on mammalian cells in vitro and in vivo showed that tubulozole-C (R 46 846), the cis-isomer of tubulozole, a new synthetic anticancer drug, interfered with the structure and function of microtubules in both interphase and mitotic cells. The activity of this compound in experimental tumor systems can thus be explained partly by a direct antimitotic effect and partly by the disintegration of the normal subcellular organization of the nondividing cells. At concentrations which affect the microtubule system, tubulozole-C arrested directional migration of transformed cells and malignant invasion in a three-dimensional organ culture system. Investigations in vivo show that malignant L1210 leukemia cells are more susceptible to the antimicrotubular effect of tubulozole-C than are the normal leukocytes of the host. The trans-isomer of tubulozole (tubulozole-T, R 48 265), which has no antitumor activity in vivo, did not affect the microtubule system of cells in vitro or their capacity for directional migration or for malignant invasion.

Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients.

OBJECTIVE: To compare two antiretroviral regiments, loviride plus lamivudine (3TC) plus zidovudine (ZDV) (triple combination) and loviride plus ZDV (double combination) in terms of pharmacokinetic interactions, tolerability, safety, and immunological and virological efficacy. STUDY DESIGN: An open, case-controlled, pharmacokinetic and 24-week continuous treatment pilot study. PATIENTS: Twenty p24 antigen-positive patients, 10 per treatment group, were matched according to p24 antigenaemia less or more than 100 pg, CD4 count less or more than 150 x 10-(6)/l, and gender. Eight out of 10 cases and seven out of 10 controls had received previous antiretroviral therapy. RESULTS: No clinically relevant pharmacokinetic interactions were observed. Both treatment combinations were well tolerated. Median absolute and percentage CD4 count increases above baseline were more pronounced in the triple combination arm than in the double combination arm. Median p24-antigen and plasma viraemia level decreases below baseline were more pronounced in the triple combination arm. The M(184)I/V mutation was detected in all plasma samples of triple combination patients examined at week 12. Mutations conferring resistance to loviride and ZDV were found in a significant subset of patients in both treatment arms. CONCLUSIONS: Both combination regimens have an excellent safety/tolerability profile, but a higher level of in vivo efficacy is achieved by the triple combination, despite genotypic changes conferring resistance to one or all of these agents. The conclusions drawn are limited by small population size and the heterogenous pretreatment history. However, they support the validity of and strongly encourage a rationally designed multidrug combination approach to HIV therapy.

Polyploid cells in ageing hamster fibroblasts in vitro: possible implication of the centrosome.

Fibroblasts from hamster embryos cultivated in vitro present the typical ageing process of other fibroblastic lines, but they also suddenly give rise to giant non dividing cells which could be considered to represent terminally differentiated cells [36]. We investigated the latter mechanism, first by showing that microtubules in these cells depolymerized from the centrosome and not from the cell periphery as in other cells; secondly we analysed the structure of the centrosome on serial sections and found a diminished pericentriolar material; finally time lapse sequence studies of cell division confirmed that this process sometimes aborts giving rise to these giant polyploid cells. As a consequence, what first appeared as a differentiation process is in fact the result of an environmental deterioration which probably reaches a critical level thus creating a catastrophic consequence for the cell.

Laminin binding and internalization by human and murine mammary gland cell lines in vitro.

We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice.

Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33-59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.

Purine nucleoside phosphorylase, a possible histochemical marker for T-cells in man.

A procedure is described for the histochemical detection of purine nucleoside phosphorylase (PNP) activity in circulating lymphocytes of man. The number of PNP-positive cells, as evaluated on smears of Ficoll--Hypaque purified cells, correlated well with the number of E-rosette-forming cells of the same blood samples of healthy and diseased people with normal or abnormal numbers of E-rosettes. In healthy people, the number of PNP-positive cells was within the range of 70-80% of the total lymphocyte population, whilst the corresponding E-rosette-forming cells were scored between 60-75%. Patients with unusually low or high E-rosettes had equally low or high numbers of PNP-reactive cells. More substantial evidence for the presence of PNP activity in T-cells and not in B cells was gathered from experiments in which PNP activity and surface membrane immunoglobulins (SMIg) were simultaneously demonstrated on the same preparation. These results showed, on the one hand, that the bulk of lymphocytes that are reactive for PNP do not reveal SMIg and, on the other hand, that most Ig-bearing cells were unreactive for PNP.

The mechanism of action of the new antimycotic ketoconazole.

Ketoconazole is one of the new members of the imidazole series with a broad-spectrum antifungal profile. Although sharing its basic active principles with the other imidazoles, ketoconazole obtains its superior in vivo activity mainly from its good oral absorption and its lower degree of inactivation once absorbed. Its selective toxicity for yeasts and fungi is found to be primarily linked to the inhibition of ergosterol biosynthesis and to interference with other membrane lipids. In vitro growth studies revealed that ketoconazole's activity was more pronounced against the invasive morphogenetic form than against the saprophytic form of Candida albicans, which at least partly explains its prominent in vivo potency. At extremely low concentrations (10 ng/ml-1) ketoconazole prevents the development of the very form that is responsible for the expression of clinical symptoms. In contrast to other imidazoles, ketoconazole's action on the morphogenesis of the organism is not influenced by serum. The synergistic action with host defense cells, as demonstrated in culture systems, is another inherent property of this drug and may have a great impact on the eradication of systemic fungal infections. These effects of ketoconazole have been studied in a variety of fungal organisms with the aid of phase-contrast, scanning, and transmission electron microscopy in order to characterize ketoconazole's profile in comparison to the other imidazole derivatives.

Interaction between the microtubule inhibitor tubulozole and gamma-irradiation in murine tumors in vivo.

The combined effect of the microtubule inhibitor tubulozole and gamma-irradiation has been investigated in vivo in subcutaneous MO4 fibrosarcomas and Lewis Lung carcinomas. A marked interactive effect on tumor growth was observed when 160 mg/kg tubulozole was orally administered before the tumors were treated with 10 Gy radiation. Dose dependency and optimal effect were obtained on tumor growth of MO4 tumor bearing animals when the drug treatment was given 6 hr prior to the irradiation. The optimal pretreatment time coincided with the time at which a peak mitotic index in the tumor tissue was observed. An enhancing effect is also noticed at other doses of radiation in MO4 tumors pretreated 6 hr before with 160 mg/kg tubulozole. The interactive effect is maintained in a clinically relevant dose fractionation schedule whereby 8 fractions of 2 Gy each were pretreated 6 hr before with 80 mg/kg tubulozole. Tubulozole-T, the stereo-isomer of tubulozole, neither exhibits any antimicrotubular action nor exerts an antitumoral effect on its own or in combination with gamma-irradiation. The possible mechanisms of interaction between tubulozole and gamma-irradiation in tumor tissue are discussed.

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

Microtubules and microfilaments in ageing hamster embryo fibroblasts in vitro.

Microtubules and microfilaments were investigated in hamster lung fibroblasts, during their in vitro life-span. These cells show a senescence process characterized by a drastic phenotypic change, resulting in two phenotypes: the type 1 cells, characteristic of young cultures and the type 2 cells appearing progressively with culture passages. Microtubules and microfilaments were observed at the TEM and also visualized by the unlabelled peroxidase-anti-peroxidase method. Moreover, the susceptibility of microtubules to nocodazole was tested in type 1 and 2 cells. We could not provide evidence for a different susceptibility to the drug. However the depolymerization wave occurred centripetally in type 1 cells whilst centrifugally in type 2 cells. These observations are discussed in relationship with the early arrest of division growth of the type 2 differentiated cells.

Unchanged levels of interleukins, neopterin, interferon-gamma and tumor necrosis factor-alpha in cerebrospinal fluid of patients with dementia of the Alzheimer type.

Several histopathological studies suggest that amyloidogenesis in dementia of the Alzheimer type is accompanied by activated glia and glia-derived cytokines, leading to chronic, self-propagating, cytokine-mediated molecular and cellular reactions. As studies regarding inflammatory changes in cerebrospinal fluid of patients with dementia of the Alzheimer type has been inconclusive, we set up a prospective study to assess cerebrospinal fluid levels of interleukin-1beta, interleukin-6, interleukin-10, interleukin-12, soluble interleukin-2 receptor, interferon-gamma, tumor necrosis factor-alpha and neopterin in 20 patients with dementia of the Alzheimer type and 20 age- and sex-matched controls. Comparing both groups, no significant differences in concentrations and specific activities could be revealed. An additional 22 patients were included to enlarge the study population. No statistically significant differences were shown comparing patients (n=42) with the control group (n=20). We conclude that the immune-mediated inflammatory changes found in histopathological studies are not reflected in cerebrospinal fluid of patients with dementia of the Alzheimer type. Probably, cytokine production appears very localized in the central nervous system, not allowing representative detection in cerebrospinal fluid. Further studies assessing cytokine levels in various regions of central nervous system of patients with dementia of the Alzheimer type will be of interest to confirm this hypothesis.