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Kinetics of pinocytosis of FITC-Dextran (mw 70 000) was analysed in mouse L-cells using flow cytometry with a fluorescence-activated cell sorter (FACS). In each experiment information on 2 X 10(4) individual, living cells was obtained. The population of L-cells thus analysed was shown to be rather homogeneous with respect to cell size, and the peak of the FITC-fluorescence curve--a histogram of the accumulated signals from L-cells exposed to FITC-Dextran--was shown to be representative of the average size of the L-cells. It was found that the cellular accumulation of FITC-Dextran was proportional to the tracer. However, the rate of accumulation decreased with increasing time of incubation (up to 3 h). When cells were pulse-labeled for 10 min at 37 degrees C, rinsed carefully with ice-cold PBS, and reincubated at 37 degrees C for various periods of time, about 50% of the initial cellular FITC-fluorescence disappeared within approximately 15 min of reincubation, whereafter no measurable decrease in cellular fluorescence was seen. In addition, a rapid increase of intact FITC-Dextran molecules in the reincubation medium occurred within the first 15 to 30 min of reincubation, whereafter no further increase took place. Thus, a large portion of previously endocytosed FITC-Dextran becomes rapidly exocytosed, while the rest becomes sequestered within a compartment from where little or no exocytosis occurs. The deviation from linearity in accumulation related to time, and the distinct biphasic kinetics of exocytosis are taken to support a generalized two-compartment model for endocytosis and membrane recycling, the two compartments being endocytic vacuoles (endosomes) and (secondary) lysosomes, respectively.

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.

The relationship between growth and cytodifferentiation was studied in cultured human mammary myoepithelial cells under serum-free culture conditions. Myoepithelial-cell differentiation was monitored by quantifying cells showing immunoreactivity to the muscle isoform of actin; to the membrane glycoprotein common acute lymphoblastic leukemia antigen (CALLA); and to type IV collagen. Growth was quantified either by measuring the actual increase in cell number, or in a more-sensitive assay using immunoreactivity to the cell-proliferation-associated nuclear antigen Ki-67 as a measurement of the number of cells leaving the G0-phase of the cell cycle. The results showed that: (a) Primary cultures of myoepithelial cells on DME-F12 supplemented with cholera toxin (CT) alone resulted in the formation of quiescent cell islets (in the G0-phase of the cell cycle) showing phenotypic traits preserved from the in vivo situation (actin- and CALLA-positive cells with little or no type-IV-collagen immunoreactivity). (b) After addition of epidermal growth factor (EGF), with an ED50 of 1-10 ng/ml, in the presence of CT, the cells entered the G1-phase of the cell cycle, without further increase in cell number. At the same ED50 of EGF, the frequency of CALLA-positive cells decreased, while the number of cells immunoreactive for type IV collagen increased with a maximal effect of EGF seen after 7-11 days. During the same period, the cells remained fully differentiated with respect to actin immunoreactivity. (c) Further addition of insulin (I) to the medium in the presence of EGF and CT resulted in the cells entering an exponential growth phase associated with simultaneous decrease in actin immunoreactivity with a maximal effect of I after 11 days of exposure. The dose-response curve to I was virtually identical for stimulating cell proliferation and for reducing the frequency of actin-immunoreactive cells (ED50 in the range of 30 ng/ml), suggesting that the two processes were controlled by the same initial I-receptor interaction. (d) Some reduction in the number of actin-positive cells was exerted by I-EGF-CT independently of the mitogenic response, but this reduction was further augmented if the cells were allowed to proliferate. (e) Time-course studies of quiescent (G0-phase) cells stimulated to exponential growth revealed that entrance of cells into the G1-phase of the cell cycle preceded the loss of muscle actin filaments. (f) Exponentially growing actin-negative epithelial cells did not resume a myoepithelial phenotype in density-arrested postconfluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used. All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).

A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types.

Pinocytosis was studied in cultured human skin fibroblasts in order to obtain more information about uptake in these cells of non-specific ligands with different net surface charge. The fibroblasts were obtained from a normal person and a patient homozygous for familial hypercholesterolemia. No ultrastructural differences were observed between the two cell types. The ratio between coated and smooth pits at the cell surface was about 1:6. Freeze-fracture revealed that whereas smooth pits were devoid of intramembrane particles, the coated pits occupying 1-5% of the total cell surface area showed numerous intramembrane particles. Cationized ferritin (pI = 8.5) bound to the cell surface and to coated pits of both types of fibroblasts at 4 degrees C. Either type internalized CF at 37 degrees C. Vacuoles (lysosomal elements) containing CF were seen already after 5 min at 37 degrees C, but were much numerous after 30 min of incubation at 37 degrees C, and appeared with the same frequency in both cell types. Smooth (uncoated) pits at the cell surface remained unlabeled. In contrast to CF, native ferritin (pI = 4.6) was internalized to a very limited extent in both cell types, even after 3.5 h of incubation at 37 degrees C. These results indicate that both normal and FH fibroblasts internalize CF exclusively by coated pits, and adsorptive uptake mechanism that apparently is nonspecific but clearly cation-selective.

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

The present study describes a cytochemical approach to demonstrate human breast carcinoma cells in cryosections and in primary monolayer cultures from surgical biopsies. The material consisted of biopsies from 52 carcinomas and 29 benign lesions. Cryosections and cultures were incubated to demonstrate NADPH-neotetrazolium reductase in an atmosphere of 99.5% oxygen. Incubation time and section thickness were adjusted to accomplish the same level of reaction in cells of cryosections and corresponding cultures. Positive reaction was thus confined to epithelial elements and to the wall of some smaller blood vessels. More than one-half of the carcinoma cells showed moderate to strong reaction in cryosections from 29 of 52 carcinomas whereas no reaction was seen in ductules of normal appearance adjacent to these carcinoma cells. Positive reaction was seen in epithelial cell islets in primary cultures of 16 of the 40 carcinomas cultured. In cryosections from fibroadenomas and fibrocystic disease specimens only apocrine metaplasia consistently showed positive reactions compared with less than 10% of other ductular profiles in a given cryosection. This pattern of reaction was reflected in the derived primary cultures in which positive reaction was found in epithelial cell islets in only one of 19 biopsies cultured. The presence of human milk fat globule membrane antigen was used to demonstrate the epithelial nature of the cell islets seen in cultures of biopsies from both benign lesions and carcinomas. NADPH-neotetrazolium reductase positive islets from carcinoma biopsies were frequently aneuploid whereas most negative islets from carcinoma biopsies were diploid as were all islets from benign tissues.

Binding and endocytosis of the ligands transferrin, epidermal growth factor (EGF), and ricin were measured in a number of different cell lines after treatment of cells with compounds that react with SH-groups and under conditions where the cytosolic pH was lowered. N-ethylmalemide and diamide irreversibly inhibited endocytosis of all ligands tested, whereas low pH in the cytosol strongly inhibited endocytosis of transferrin and EGF. Data obtained by electron microscopy indicated that the formation of coated vesicles from coated pits is inhibited in acidified cells. Entry of ricin was much less affected, and ricin endocytosed under these conditions was able to intoxicate the cells. At low pH in the cytosol there was a calcium-dependent increase in the number of transferrin receptors at the cell surface. The increase was even larger in the presence of the calcium ionophore A23187, whereas it was completely blocked by the calmodulin antagonists trifluoperazine and W7. The results show that endocytosis from coated pits can be inhibited in a reversible way by acidification of the cytosol and they suggest that a second pathway of endocytosis exists, possibly involving formation of vesicles from uncoated areas of the membrane.

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

Effects of increased biosynthesis of the membrane-bound ATP synthase of Escherichia coli K-12 were analysed at the physiological and morphological level. Overproduction of the enzyme complex was achieved by molecular cloning of the structural genes into plasmid pBR322. A series of plasmids resulting in 2-fold, 4- to 5-fold and 10- to 12-fold overproduction, respectively, was constructed. The ATP synthase was calculated to represent 3%, 6-7% and 18-23%, respectively, of total protein in cells with these plasmids. In wild-type cells ATP synthase represents 1.5-2% of total protein equivalent to approximately 3000 enzyme complexes per average cell. While 2- or 4- to 5-fold wild-type levels of the ATP synthase had only minor effects it was found that 10- to 12-fold overproduction resulted in pronounced inhibition of cell division and growth and in formation of membrane cisterns and vesicles within the cells. Inclusion bodies, probably representing deposits of excess ATP synthase, were also observed in these cells.

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.

Immunotoxins, that is conjugates of enzymatically active toxins with antibodies against cell surface antigens, are being used in targeted cell destruction. The directed toxin enters the cytosol of the target cells and inactivates components of the protein synthesis machinery. In this review, Sjur Olsnes, Kirsten Sandvig, Ole W. Petersen and Bo van Deurs discuss the mechanism of action of such conjugates and how they enter the cytosol. Emphasis is placed on points that should be considered in the preparation of efficient conjugates.

Numerous biochemical and drug transport studies have focused on the role of the plasma membrane or membrane-associated processes in anthracycline and vinca alkaloid resistance. Cationized ferritin, which binds electrostatically to anionic sites on the plasma membrane, was used as a membrane marker in a morphometrical ultrastructural study. The nonspecific adsorptive endocytosis was significantly increased in Ehrlich ascites tumor cell lines resistant to daunorubicin (DNR), doxorubicin, vincristine, and vinblastine compared to that in sensitive cells. Thus cells resistant to DNR internalized 2.04% of their plasma membrane per minute, in contrast to 1.17% in sensitive cells. Because the measured cell surface areas in sensitive and resistant cells were unchanged during the experiments, increased membrane traffic (recycling) from intracytoplasmic compartments to the plasma membrane must have occurred in resistant cells. Possible implications of these findings include a resistance-linked phenotype without significance for resistance per se, an increase in membrane repair, and a connection between increased membrane traffic and increased active drug extrusion in resistant cells.

Carcinoma cells typically show little or no polarity as compared to normal, differentiated epithelial cells. We have studied polarity in two established human breast carcinoma cell lines, T47D and MCF-7, by various techniques (electron microscopic enzyme- and immunocytochemistry, freeze-fracture) and show that one of them (MCF-7) is characterized by a high degree of polarity. Thus, in contrast to T47D cells, MCF-7 cells in monolayer culture form apical tight junctions, do not allow a ricin-horseradish peroxidase conjugate, which binds to terminal galactose residues on the apical surface, to stain the basolateral membrane domain, and express a surface antigen (MFGM-A) only in the apical surface membrane domain, as do normal mammary epithelial cells in vivo. This polarization is independent of a basement membrane, since it is maintained when MCF-7 cells, which do not deposit type IV collagen themselves, are grown directly on plastic. Moreover, even though MCF-7 cells express estrogen receptors rather homogeneously, estrogen has no effect on this polarity, neither in vitro nor after transplantation to nude mice. We conclude that polarity is a stable, differentiated feature of MCF-7 cells.

Ultrastructural immunocytochemistry was used to analyze cell surface distribution and intracellular localization of milk fat globule membrane antigen (MFGM-A) in cryosections from human breast carcinomas and benign breast biopsy specimens. The specimens were fixed in formaldehyde and frozen. Cryostat sections were cut at 15 micron, incubated with mouse monoclonal antibody to MFGM-A, and then with a peroxidase-conjugated goat anti-mouse antibody. After glutaraldehyde fixation, the sections were incubated with diaminobenzidine-H2O2 and further processed for electron microscopy. MFGM-A was specific for epithelial cells. MFGM-A staining was strictly confined to the apical surface membrane of normal ductal epithelium, never involving basolateral membranes below the tight junctions. In normal epithelial cells, MFGM-A was readily detected in cisternae of the endoplasmic reticulum (ER), but only to a lesser extent in Golgi complexes and presumptive secretory vesicles. In carcinoma cells, surface staining for MFGM-A was either distributed in a non-polarized manner on the entire cell surface or else was totally absent. In some carcinoma cells without surface-associated MFGM-A, very pronounced intracellular MFGM-A staining was seen in the ER, in the nuclear envelope, and in annulate lamellae. The observations on MFGM-A expression were supported by studies on a cell culture model system.

A major goal in analyzing the structure of tight junctions by freeze-fracture has been to create a generalized model for this membrane specialization conforming to both physiological and morphological data, and providing a basis for understanding variation in tight junction appearance and function. In the present review we have focused on some of the problems involved. Results obtained by double replication, rotary shadowing and rapid freezing are discussed. A model for tight junctions with a general scaffolding of inverted lipid micelles (cylinders) binding the adjacent membranes together, and associated with integral membrane proteins of significance, for instance, in stabilization as well as in regulation of permeability, appears promising.

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.

Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed. Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15-60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested. In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.

A new, nontumorigenic human breast epithelial cell line, HMT-3522, has been established from fibrocystic breast tissue. Cells were explanted and propagated in chemically defined medium including insulin, transferrin, epidermal growth factor, hydrocortisone, estradiol, prolactin, and Na-selenite. The epithelial nature of the cell line was established by immunocytochemical detection of cytokeratins. Moreover, electronmicroscopy revealed monolayers of polarized cells connected by desmosomes and provided with apical microvilli. Milk fat globule membrane antigen, specific for the apical membrane domain of normal, luminal breast epithelial cells, was expressed only in confluent cultures where some cells overlaid others, indicating "stem cell"-like properties. After 25 to 30 passages, the cells are diploid with a few marker chromosomes and loss of chromosomes in the D-group. The cells are nontumorigenic in athymic mice; they lack estrogen receptors, and estradiol does not stimulate growth. The HMT-3522 cell line may represent a useful model for the study of breast cell differentiation and carcinogenesis in vitro.

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).

The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2 h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70% of the HRP- and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland also permeable to peptides synthesized and secreted by the pineal gland.

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.

Fragments and suspensions of human fetal thymus were incubated in the presence of 3H-TdR to permit study of the distribution and morphology of DNA-synthesizing cells. Results of light and EM autoradiography showed that 1. although DNA-synthesizing cells were present in the medulla, the vast majority of these cells were localized in the thymic cortex, 2. cells with the typical EM appearance of small lymphocytes and lymphoid blast cells both synthesized DNA, and 3. cells in S-phase were predominantly 8 to 12 mum in size.

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.

Interpretation of primary monolayer culture of organs and tissues with different epithelial cell types demands well-defined criteria for distinguishing between such cells. Epithelial components in breast carcinomas comprise, in addition to carcinoma epithelial cells (CEP), at least two epithelial cell types organized in mammary ductules of normal appearance as an inner layer of luminal epithelial cells (LEP) and an outer layer of basal or myoepithelial cells (MEP) resting on a basement membrane. In a previous study (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986) we have defined a population of CEP in vivo and in vitro, appearing in about 50% of primary carcinomas, by a cytochemical reaction for reduced nicotinamide adenine dinucleotide phosphate neotetrazolium reductase. Carcinoma derived epithelial cells not showing this cytochemical reaction in culture were believed to originate from either carcinoma cells or from mammary ductules of normal or benign appearance. In the present study we show that carcinoma derived NADPH-NT reductase negative cell islets often exhibit phenotypic traits of apparently normal mammary ductules as defined in vivo. Moreover, it is shown that the reductase positive CEP, apart from the reductase reaction, has preserved several other features in culture distinguishing them from cells of apparently normal origin. Thus, whereas reductase positive CEP often consisted of only one cell type, as revealed by phase contrast microscopy, some reductase negative cell islets showed a distinct two-cell-type composition. One cell type exhibited cobblestone-like appearance and remained in the center of the islets whereas the other was more loosely arranged and rapidly left the central area by migration below the cobblestone-like cells to the periphery of the islets. Cobblestone-like cells and loosely arranged cells were found by immunocytochemistry to express elements of LEP and MEP phenotype, respectively. LEP phenotype was defined in vivo by expression of milk fat globule membrane antigen and cytokeratins, whereas MEP expressed basement membrane-associated type IV collagen. Computerized image analysis revealed mean population doubling times for cells with MEP phenotype of 1 day and for those with LEP phenotype of 2 days. Both cell types showed a diploid DNA pattern as revealed by fluorimetry. Reduced nicotinamide adenine dinucleotide phosphate neotetrazolium reductase positive CEP expressed milk fat globule membrane antigen and cytokeratins, thus resembling the reductase negative LEP.(ABSTRACT TRUNCATED AT 400 WORDS)