Sally L. Palm

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.

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

SummaryMetastasizing 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 understant 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.

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.

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.

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

AbstractBackground: 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 µM, 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 µM (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 µM lovastatin and by an additional 43–63% by 5 µM 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.

The Role of Growth Cone Adhesion in Neuronal Morphogenesis, as Demonstrated by Interactions with Fibronectin and Laminin

The embryonic formation of nerve fibers (axons and dendrites) by neurons is both complex and regular. For example, the arrangement of nerves at the brachial plexus and beyond in the vertebrate forelimb is predictable within members of a species, yet is distinct from the pattern of other species. Such precise axonal pathways are forged by the activities of extending nerve fiber tips, first named the growth cone by Santiago Ramon y Cajal (1890). As holds for all cell movements, growth cones can be studied in terms of distinct questions: what starts, what maintains, what regulates the directions of and what stops growth cone movements (Trinkaus, 1984)? Each question probes a different facet of growth cone behavior, and answers to each may involve different intrinsic and extrinsic factors. In the case of the growth cone, morphogenetic behavior can be divided into five distinct activities; neurite elongation, turning, branching, retraction and synaptogenesis. Consistent patterns of nerve fiber pathways, such as in the brachial plexus, arise from the readout of developmental programs that determine these five growth cone activities.

Peptide Fragments of Fibronectin and Laminin: Role in Cell Adhesion and Inhibition of Experimental Tumor Metastasis

Metastasis is the major cause of morbidity and mortality in cancer patients. This process is extremely complicated and involves numerous cell biological and biochemical reactions. For convenience, the spread of tumor cells via a hematogenous route can be discussed in terms of a number of discrete, yet related steps. Metastasizing tumor cells must interact with and spread through extracellular matrices consisting of a number of molecular constituents. The cells must enter the capillaries or vessels at the primary site, get into the blood stream, travel to a distant site, adhere to endothelial or subendothelial structures, migrate through the basement membrane, and move and grow at this secondary locus (see reviews 1–4).