Production of laminin and fibronectin by Schwannoma cells: cell-protein interactions in vitro and protein localization in peripheral nerve in vivo.

We studied a rat Schwannoma cell line (RN22F) to determine if it produced the basement membrane glycoproteins laminin and fibronectin, and how it interacted with these proteins in vitro. We used antisera to laminin and fibronectin for immunoprecipitation experiments and immunocytochemical localization at the electron microscope level. Polyacrylamide gels of antilaminin immunoprecipitates of conditioned medium and solubilized Schwannoma cells contained bands of reduced Mr 200,000 and 150,000. Antilaminin immunoprecipitates of conditioned medium contained nonreduced bands of 850,000 daltons and 150,000, and immunoprecipitates of solubilized cells contained nonreduced bands of 850,000, 400,000, 200,000, and 150,000 daltons. Antifibronectin immunoprecipitates of conditioned medium contained a reduced band of 220,000 daltons, and nonreduced bands of 440,000 and 220,000 daltons. Radio-labeled protein was not detected in antifibronectin immunoprecipitates of solubilized cells. By immunocytochemistry, laminin was found along the cell surface in a continuous band, whereas fibronectin was only sparsely distributed along the cell surface. In cell adhesion assays, Schwannoma cells bound preferentially to laminin-coated substrates as compared to fibronectin or noncoated substrates. A number of Schwannoma cells displayed a curved and elongated morphology on laminin substrates, as compared to a uniformly spread morphology on fibronectin, and a round, nonspread morphology on noncoated substrates. Immunofluorescent staining showed laminin in the endoneurium and perineurium and fibronectin predominantly in the perineurium of mouse sciatic nerve in vivo. The production of laminin and fibronectin by Schwann cells may be important in the development and myelination of peripheral nerves, and the proper regeneration of axons following nerve injury.

Migration by haptotaxis of a Schwann cell tumor line to the basement membrane glycoprotein laminin.

Laminin is a large (greater than 850-kdalton) glycoprotein that is localized within basement membranes. Recent work has indicated that this protein is present within the endoneurium of mouse sciatic nerve. Furthermore, it has been shown that a rat Schwannoma cell line, RN22F, produced laminin and that laminin promoted the attachment of these cells to bacterial plastic. This report presents evidence that RN22F cells migrate in vitro to laminin in a concentration-dependent fashion. Laminin was extracted from the mouse EHS tumor and purified by molecular sieve and heparin-agarose affinity chromatography. The migration of Schwannoma cells to laminin, as assessed in a microwell modified Boyden chamber, was inhibited in a dose-dependent manner by affinity-purified antilaminin antibody. Zigmond-Hirsch checkerboard analysis experiments indicated that laminin stimulated both random and directed movement of RN22F cells. Additionally, reversal of the laminin gradient in the chambers also stimulated RN22F migration in a concentration-dependent manner, suggesting that directed migration of RN22F cells was due to a substratum-bound laminin (haptotaxis) as opposed to cell movement in response to fluid-phase laminin (chemotaxis). Binding studies using [3H]laminin demonstrated that laminin bound to the filter surface under the assay conditions used, and support the contention that cells are migrating to substrate-bound material. Furthermore, RN22F cells were shown to migrate on filters coated with laminin in the absence of additional fluid-phase laminin. The magnitude of this response could be altered by changing the relative density of bound laminin. In contrast, fibronectin promoted only marginal migration of RN22F cells. Collectively, these observations indicate that haptotaxis may be a mechanism by which laminin may guide cells during development and raise the possibility that it may be involved in peripheral nervous system myelination.

Metastasis inhibition of different tumor types by purified laminin fragments and a heparin-binding fragment of fibronectin.

Tumor cell metastasis is a complex process that depends in part on tumor cell adhesion to components of basement membranes and the extracellular matrix. Previous studies have indicated that the experimental metastasis of murine melanoma cells can be inhibited by ex vivo pretreatment of cells with purified adhesion-promoting fragments of laminin or the synthetic peptide arginyl-glycyl-aspartyl-serine (RGDS) prior to tail vein injection. This study extended the earlier reports to demonstrate that adhesion-promoting fragments of laminin and fibronectin can inhibit the metastasis of a tumor of different histologic origin, such as murine fibrosarcoma cells. Furthermore, ex vivo pretreatment of cells with a purified 33-kDa heparin-binding fragment of fibronectin, which promotes tumor cell adhesion by an RGDS-independent mechanism, was effective at inhibiting experimental melanoma and fibrosarcoma pulmonary metastases. The survival rate of animals receiving tumor cells pretreated with this fragment was significantly enhanced relative to control groups. As with previous studies, the mechanism of inhibition appeared to involve an increased clearance rate of tumor cells from the pulmonary microcirculation. These results suggest a role for cell surface proteoglycans in the adhesion and metastasis of certain malignant neoplasms. Furthermore, this study emphasizes the complexity of tumor metastasis and suggests that multiple strategies may be developed to inhibit hematogenous metastasis formation.

Effects of temperature and duration of storage on granulocyte adhesion, spreading, and ultrastructure.

The effects of storage duration and temperature on granulocyte adhesion, spreading, and ultrastructure were examined. Normal adherence and spreading of granulocytes detected by either phase contrast or scanning electron microscopy was maintained for up to 48 hours when granulocytes were stored at 20 to 24 degrees C. By contrast, storage of granulocytes for 24 hours at 1 to 6 degrees C. led to a substantial decrease in granulocyte adherence and spreading. When granulocytes were stored for 24 hours at 1 to 6 degrees C. and then shifted to 20 to 24 degrees C. for a second 24 hours, granulocytes failed to regain the normal adherence and spreading. The cytoskeleton has been implicated in cell morphology, movement, chemotaxis, and spreading of cells. Transmission electron microscopy of thin sections of whole cells or cytoskeleton preparations revealed a well-organized system of microtubules and microfilaments in granulocytes stored for up to 48 hours at 20 to 24 degrees C. In granulocytes stored at 0 to 6 degrees C. that have the decreased adhesion and spreading, there is a paucity of microtubules, and the microfilaments have a disorganized criss-crossed appearance when compared with normal cells. It appears that storage of granulocytes at reduced temperatures is associated with decreased adhesion and spreading, and a concomitant alteration in the cytoskeleton may be responsible for this.

Alternative model for the internal structure of laminin.

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.

Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain.

The first long tract to form in the brain of a vertebrate embryo is the ventral longitudinal pathway. In order to investigate what chemical cues may guide nerve growth cones along this pathway, affinity-purified antibodies to laminin and collagen type IV were used to stain sections of mouse embryos from Embryonic Days 8 through 17. A monoclonal anti-neurofilament antibody was used to show the development of the ventral longitudinal pathway in relationship to immunoreactivity for laminin and collagen type IV. At Day 8 fluorescent immunoreactivity for laminin is bright in the external limiting membrane of the neural tube, but the neuroepithelium does not show bright laminin or neurofilament immunoreactivity. At E9 the ventral longitudinal pathway is forming and punctate immunoreactivity for laminin is present on the surfaces of neuroepithelial cells in the marginal zone, through which axons of the ventral pathway extend. Punctate immunofluorescence for laminin remains concentrated in the marginal zone on Days E10 through E14, but on E16 punctate immunofluorescence was much reduced, although immunoreactivity for laminin remained bright in the maturing pial and arachnoid membranes and on blood vessels in the brain. Immunoreactivity for collagen type IV was strong in the external limiting membrane and on blood vessels, but never showed concentrated punctate immunofluorescence in the marginal zone. These results indicate that laminin may be available on cell surfaces and in extracellular spaces as an adhesive ligand for growth cones during the formation of the ventral longitudinal pathway.

Transient expression of laminin in the optic nerve of the developing rat.

The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin-negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.

Peptide fragments of laminin and fibronectin promote migration (haptotaxis and chemotaxis) of metastatic cells.

The migration of tumour cells through basement membranes and extracellular matrices is an integral component of tumour invasion and metastasis. Laminin (LMN) and fibronectin (FN) at 1-100 micrograms/ml promote the directed migration of metastatic murine melanoma cells 40-70-fold greater than controls in modified Boyden chambers. Antibodies abrogated the migration of cells in response to the respective protein. Preincubation of melanoma cells with plasma FN had no effect on subsequent migration to LMN or FN. The migration of these cells was largely related to substratum-attached molecules and increasing adhesion gradients of cells; this has been termed haptotaxis. Peptide fragments of both FN and LMN were isolated by affinity chromatography with monoclonal antibodies, heparin or other constituents. FN has two unique domains, 80-125 K and 66 K, which promote the adhesion of tumour cells, whereas only one appeared to be responsible for promoting migration. Peptides of LMN, isolated with heparin and monoclonal antibody, define a cell migration-promoting activity within the 200 K chains of LMN. Serum spreading factor and epinectin, the latter an adhesion molecule derived from squamous epithelial tumour cells, are also capable of promoting the migration of malignant cells. Thus, directed migration of metastatic tumour cells may be promoted with peptide fragments of adhesion molecules and blocked with the respective antibody.

The effect of lovastatin on [3H]thymidine uptake in HTC-4 and LLC-L1 tumor cells.

Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been shown to inhibit the in vitro and in vivo growth of a number of different cell lines. However, the mechanism by which lovastatin exerts its effect is not clear. In this experiment, we investigated the effect of lovastatin on the incorporation of [3H]thymidine by Hepatoma Tissue Culture-4 (HTC-4) and Lewis Lung Carcinoma L-1 (LLC-L1) tumor cells. Tumor cells were grown under standard conditions and treated with four different concentrations of lovastatin. Cell growth was evaluated by daily hemacytometer cell counts. On Day 4, the plates were pulsed with 10 microCi [3H]thymidine. After 24 hr, the plates were harvested and [3H]thymidine incorporation was measured by scintillation counting. Lovastatin inhibited both HTC-4 and LLC-L1 cell growth in a dose-dependent manner. At the highest lovastatin dose, both LLC-L1 and HTC-4 cell growth was slowed to less than 15% of control. Remarkably, however, both cell lines showed a paradoxical, dose related, increase in [3H]thymidine uptake. Cell cycle analysis using flow cytometry was performed on Day 5 in the LLC-L1 cell line. As the lovastatin concentration increased, a lower percentage of cells was found in the G1 phase of the cell cycle and a higher percentage of cells was found in the S and G2 phases. These findings suggest that these tumor cells are undergoing brisk DNA repair or that lovastatin is more effectively blocking cell division than cellular DNA replication.

Guidance of neurite outgrowth by pathways of substratum-adsorbed laminin.

Neurite outgrowth is guided by narrow pathways of bioactive laminin. These pathways are created by ultraviolet light irradiation of laminin-coated coverslips masked with electron microscope grids. Patterned outgrowth of neurites is independent of gross mechanical guidance and guidance caused by substrate limitation. Cells on unirradiated laminin are less readily displaced by shear forces than cells on irradiated laminin. This study suggests that ultraviolet light alters the adhesive properties of laminin and that differential cell-substratum adhesion may guide extending neurites on the purified naturally occurring substance, laminin.

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin.

Dissociated neurons from embryonic chick dorsal root and sympathetic ganglia (peripheral neurons) and from spinal cord and retina (central nervous system neurons) were cultured on plastic substrata treated with purified fibronectin and laminin. Both central and peripheral neurons attached to and extended neurites on laminin. In contrast, only peripheral neurons initiated neurites on fibronectin; central neurons cultured under identical conditions aggregated into clusters and did not extend neurites. Neurite length, number of neurites initiated, and extent of neurite branching on fibronectin- and laminin-treated substrata were evaluated and compared with similar measurements of neuronal response to poly-L-lysine-treated plastic. Poly-L-lysine provides an adhesive surface for neurite elongation, but fibronectin and laminin appear to promote more rapid neurite elongation. Our observations suggest that neuronal interaction with these glycoproteins may involve neuron-specific cell surface components. These responses to laminin and fibronectin in vitro may be related to the presence or absence of these glycoproteins in specific extracellular environments during specific developmental stages.

Effect of lovastatin alone and as an adjuvant chemotherapeutic agent on hepatoma tissue culture-4 cell growth.

BACKGROUND: Cholesterol is essential for cell viability and growth. Interference with the cholesterol biosynthetic pathway with a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (e.g., lovastatin) may preferentially slow malignant cell growth and offer a new approach to cancer chemotherapy. To test this hypothesis, we evaluated the effect of lovastatin alone, and as an adjuvant chemotherapeutic agent, on the growth and function of hepatoma tissue culture-4 (HTC-4) cells. METHODS: HTC-4 cells were treated with lovastatin at concentrations of 1, 3, 5, and 10 microM, with mitomycin-C at concentrations of 10, 25, 50, and 100 nM, or with combinations of the two drugs. Cell growth was evaluated by daily cell counts and substrate adhesion to fibronectin. RESULTS: Lovastatin alone slowed HTC-4 cell growth at concentrations as low as 1 microM (p < 0.01). Mitomycin-C alone slowed HTC-4 cell growth at concentrations of 25 nM and above (p < 0.01). Lovastatin added to mitomycin-C-treated cells resulted in a significant adjuvant effect, with cell growth slowed by an additional 20-30% by 1 microM lovastatin and by an additional 43-63% by 5 microM lovastatin, compared to mitomycin-C alone (p < 0.01). Lovastatin-treated cells also exhibited decreased adherence to substrate (p < 0.05). CONCLUSIONS: Lovastatin is effective alone and as an adjuvant to mitomycin-C in slowing the growth of HTC-4 cells. These in vitro results support further investigation of lovastatin as an adjuvant chemotherapeutic agent in animal models.

Cell adhesion and neurite extension in response to two proteolytic fragments of laminin.

Studies from several laboratories have suggested that laminin contains at least two domains that selectively mediate cell type-specific behavior. In this study, two proteolytic fragments of laminin are evaluated for their ability to interact with three different populations of embryonic chicken cells. A 600 kDa thrombin fragment, derived from the central portion of the laminin molecule, supports attachment of dorsal root ganglion (DRG), spinal cord (SC), and heart cells. Neurons from both DRGs and SCs extend neurites in response to this fragment. Quantitatively, both cell adhesion and neurite extension on the 600 kDa fragment are comparable to these responses to intact laminin. A 440 kDa chymotrypsin fragment, derived from either intact laminin or the 600 kDa fragment, does not support equivalent responses. Fewer DRG cells attach to this fragment and neurites are shorter than on the 600 kDa fragment. Heart and SC cell attachment is also reduced in comparison with activity of the 600 kDa fragment, and SC neurites do not form on the 440 kDa fragment. These results suggest that there are at least two cell binding domains in the laminin molecule, one with which a variety of cell types can interact and another that may mediate more restricted cellular responses. The latter site appears to be relatively inactive for SC and heart cell adhesion but supports limited attachment and neurite extension by DRG neurons.

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity.

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.

Growth cone migration across extracellular matrix components depends on integrin, but migration across glioma cells does not.

To promote neurite elongation, nerve growth cones must adhere to other surfaces. A complex of integral membrane glycoproteins mediates cell binding to the extracellular glycoproteins fibronectin and laminin (Horwitz et al., J Cell Biol 101:2134-2144, 1985). The receptor complex, named integrin, binds to fibronectin by recognition of a specific peptide sequence, Arg-Gly-Asp-Ser (RGDS), in the fibronectin molecule (Pierschbacher and Ruoslahti, Proc Natl Acad Sci USA 81:5985-5988, 1984). We have used antibodies to integrin and an RGDS synthetic peptide to probe the functions of integrin in the migration of growth cones extended from sensory and spinal cord neurons of chick embryos. Analyses of time lapse videotapes of growth cone migration before and after adding RGDS indicated that 2 mM RGDS rapidly inhibits growth cone movement on substrata coated with fibronectin or a fragment of fibronectin containing the RGDS sequence. RGDS has no effect on growth cone movement on laminin or on a surface coated with material deposited from heart conditioned medium. However, a monclonal antibody to the integrin complex (10 micrograms/ml CSAT) completely blocks growth cone movement on substrata treated with fibronectin, laminin, or heart conditioned medium. Thus integrin may be involved in growth cone adhesion to several extracellular molecules, although the selective effects of RGDS indicate that the integrin complex may have heterogeneous sites for interaction with different components of the extracellular matrix. CSAT antibody has no discernible effect, however, on growth cone migration across the upper surfaces of C6 glioma cells. These data indicate that the surfaces of nerve growth cones contain multiple binding molecules that mediate different adhesive interactions during migration.

The role of cell adhesion proteins--laminin and fibronectin--in the movement of malignant and metastatic cells.

Metastasizing tumor cells must traverse diverse extracellular matrices during dissemination. Extracellular matrices consist of two basic types, interstitial stroma and basement membranes. Extracellular matrices are chemically complex structures that interact with cell surfaces by a number of mechanisms. There has been a great deal of effort in recent years to understand the molecular nature of extracellular matrices, especially as it relates to the adhesion of normal and malignant cell types. Adhesive noncollagenous glycoproteins, such as laminin and fibronectin, serve pivotal roles in basement membrane and stromal matrices, respectively. These proteins participate in establishing the architecture of extracellular matrices as well as in attaching to the surface of cells and affecting cellular phenotype. This phenotypic effect ranges from adhesion and motility to growth and differentiation. Changes in adhesive characteristics and motility of cells have long been suspected to play a role in mediating the spread of malignant neoplasms. This article is designed to review extracellular matrix constituents that are currently known that can mediate the adhesion and motility of malignant neoplasms. The adhesion of normal and malignant cells to matrices is a complex process mediated by several distinct mechanisms which are initially manifested by changes in cytoskeletal architecture. The topic of normal and malignant cell adhesion to matrices will also be discussed in this regard, since any explanation of tumor cell migration must account for the complex dynamic interactions of the cell surface with the substratum as well as with the cytoskeleton. Finally, current efforts designed to understand the molecular nature of tumor cell:matrix interactions that contribute to metastatic behavior will also be discussed. The rationale behind these studies is that selective inhibition of specific tumor:extracellular matrix interactions can provide an avenue for therapeutic intervention of metastatic cancer.

Neuron-specific interactions with two neurite-promoting fragments of fibronectin.

Proteolytic fragments of fibronectin were used to identify regions of the molecule that support neurite extension and to investigate further the differential behavior of central and peripheral nervous system neurons in response to fibronectin (Rogers, S. L., P. C. Letourneau, S. L. Palm, J. B. McCarthy, and L. T. Furcht (1983) Dev. Biol. 98: 212-220). Fibronectin fragments with differing biological activities were produced by proteolytic digestion with trypsin and cathepsin D and sequential affinity chromatography on gelatin-agarose and heparin-Sepharose. The resulting fragments (described by Smith, D. E., D. F. Mosher, R. B. Johnson, and L. T. Furcht (1982) J. Biol. Chem. 257: 5831-5838; Smith, D. E., and L. T. Furcht (1982) J. Biol. Chem. 257: 6518-6523 included an NH2-terminal 27,000-dalton peptide that weakly binds heparin, a 46,000-dalton gelatin-binding fragment, a series of fragments (80,000 to 125,000 daltons) from the center of the molecule containing previously described cell-binding activity, two major peptides of Mr = 33,000 and 66,000 that bind heparin strongly and are thought to originate from the A and B chains, respectively, of plasma fibronectin, and a 31,000-dalton COOH-terminal peptide containing a free sulfhydryl from the A chain of the molecule. Tissue culture dishes were treated with these proteolytic preparations, and dissociated embryonic chick peripheral (PNS) and central nervous system (CNS) cells were cultured on each experimental substratum in serum-free medium. The fibronectin fragments were evaluated for ability to promote cell attachment, neurite initiation, and maintenance of neurite growth. The 27,000-, 46,000-, and 31,000-dalton preparations did not promote cell attachment or neurite extension. Both PNS and CNS neurons attached to and extended stable neurites upon the COOH-terminal heparin-binding preparation containing the 33,000- and 66,000-dalton peptides. A differential response of the neurons to the 80,000- to 125,000-dalton "cell-binding" peptides was observed: whereas PNS neurons maintained neuritic growth on this preparation for at least 48 hr, CNS neurons extended neurites during the first 24 hr of culture but, by 48 hr, withdrew these neurites and became increasingly clumped. On the basis of (1) the observed neuronal responses to the heparin binding and "cell binding" regions, and (2) the different ligand-binding properties of these regions, we propose that cell attachment and neurite extension can be mediated and/or modulated by two separate regions of fibronectin and that cellular response to the intact molecule may involve multivalent interactions.

Immunohistochemical localization of laminin in the basement membranes of normal, hyperplastic, and neoplastic human prostate.

We examined the effects of fixatives and antibody sources on the immunohistologic localization of laminin in normal and cancer-containing human prostates and studied the localization patterns in carcinomas of varying degrees of histologic differentiation. Two different polyclonal antibodies were localized in paraffin-embedded or cryostat sections of fixed (alcohol, formalin, and paraformaldehyde) or unfixed tissue, using the immunofluorescence (IF) or immunoperoxidase (IP) techniques, with positive and negative controls. We found that the IF reactions were more intense in unfixed or alcohol-fixed sections than in paraformaldehyde-fixed specimens. IP reactions were very weak or absent in fixed and paraffin-embedded sections, but pepsin treatment of these sections resulted in more intense and uniform IP reaction products, stronger than in unfixed or ethanol-fixed cryostat sections. With the IP technique, laminin localization was intense and uniform in the basement membranes (BM) of acini, blood vessels, smooth muscle, and nerve fibers in normal prostate, benign hyperplasia (BPH), and well-differentiated carcinomas. The BM of poorly differentiated carcinomas showed widespread absence of laminin reactivity. In normal BPH and well-differentiated tumors, occasional epithelial cells and their surface and acinar lumina had laminin reactivity. However, in higher grade tumors, numerous neoplastic cells had laminin reactivity in cytoplasm, their surface, and secretory material. Some macrophages and neutrophils also contained laminin reactivity, presumably of degraded laminin. In some moderately and poorly differentiated tumors, the BM of small capillaries did not contain laminin. The BM of larger vessels always had laminin reactivity, even in the higher grade tumors.