Rudy L. Juliano

Clinging to life: cell to matrix adhesion and cell survival

Cell to matrix adhesion regulates cellular homeostasis in multiple ways. Integrin attachment to the extracellular matrix mediates this regulation through direct and indirect connections to the actin cytoskeleton, growth factor receptors, and intracellular signal transduction cascades. Disruption of this connection to the extracellular matrix has deleterious effects on cell survival. It leads to a specific type of apoptosis known as anoikis in most non-transformed cell types. Anchorage independent growth is a critical step in the tumorigenic transformation of cells. Thus, breaching the anoikis barrier disrupts the cells defenses against transformation. This review examines recent investigations into the molecular mechanisms of anoikis to illustrate current understanding of this important process.

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context.

ADHESION MOLECULES IN CANCER : THE ROLE OF INTEGRINS

It has been known for some time that cell adhesion receptors, including members of the integrin family, play an important role in the biology of tumors. Until recently, most of the emphasis in this area of research has concerned the functioning of integrins as adhesive molecules in the invasive and metastatic behavior of malignant cells. Now it has become clear that integrins can function as true receptors capable of transducing signals to the cell interior. Tyrosine phosphorylation seems to be a key aspect of integrin-mediated signal transduction, and a new tyrosine kinase has been described that seems to be important in this process. Evidence is accumulating that integrin-mediated signals can induce gene expression and affect transit through the cell cycle. Thus, the role of integrins in cancer seems not only to involve cell adhesion events, but may also involve the regulation of tumor cell growth and differentiation.

Signal transduction by cell adhesion receptors

Over the last few years, it has become clear that cell adhesion receptors function in signal transduction processes leading to the regulation of cell growth and differentiation. Signal transduction by both integrins and CAMs has been shown to involve activation of tyrosine kinases, while CAM signaling in neural cells involves G proteins as well. In the case of integrins, some of the downstream signaling events intersect with the Ras pathway, particularly the activation of MAP kinases. In fibroblasts, integrin mediated anchorage to the substratum regulates cell cycle traverse, while in epithelial cells, loss of anchorage can trigger programmed cell death. In many cell types, but particularly monocytic cells, integrin ligation has a profound impact on gene expression. Preliminary evidence also implicates CAMs and selectins in gene regulation. A consistent theme in signal transduction mediated by adhesion receptors concerns the role of the cytoskeleton. Integrin mediated signaling processes are interrupted by cytoskeletal disassembly. Identification of the APC and neurofibromatosis type 2 tumor suppressors suggest that cytoskeletal complexes also play a key role in signaling by cadherins and CD44, respectively. Thus, signaling by cell adhesion receptors may involve aspects that impinge on previously known signaling pathways including the RTK/Ras pathway and serpentine receptor/G protein pathways. However, novel aspects of signal transduction involving cytoskeletal assemblies may also be critical.

Integrins and GTPases in tumour cell growth, motility and invasion

One of the most exciting aspects of recent research on cell-adhesion receptors is the realization that these molecules can participate in bidirectional signal-transduction processes. Integrins can convey signals from the extracellular matrix to the cell interior, and intracellular events can influence the affinity of integrins for their ligands. Another important emerging concept is that integrins and small GTPases of the Rho family work together to coordinate both cytoskeletal organization and signalling processes. In this review, the authors examine integrin signalling and integrin-GTPase interplay in the context of cancer cell growth and behaviour.

Signal transduction by cell adhesion receptors in leukocytes.

Leukocytes usually pass through the blood stream as nonadherent cells, but during an immune response and inflammation, they become adherent in order to migrate through tissues. Three families of adhesion molecules, the immunoglobulin family, the selectins, and the integrins, participate in interactions between leukocytes and tissues. Near sites of inflammation, leukocytes initially interact with the endothelium via selectins, causing them to slow down and to roll along the walls of blood vessels. Next, chemoattractans induce the activation of integrins on leukocytes. Finally, activated integrins mediate leukocyte migration through the endothelium into the inflamed site. Interactions of leukocytes with other cells and various extracellular matrix (ECM) proteins lead to different functional cell responses, including changes in growth, behavior, and differentiation. Many of these interactions are mediated by integrins, which “integrate” ECM protein signals with the cytoskeleton and which also act as true receptors that generate biochemical signals within the cell. Changes in pH, cytoplasmic Ca2+ concentration, phosphorylation, and gene induction have all been observed after integrin engagement. Adhesion‐mediated gene induction in monocytes is perhaps the best example that integrins initiate signaling cascades in the cell to deliver information from the ECM all the way to the nucleus. J. Leukoc. Biol. 57: 189–198: 1995.

Pharmacokinetics of liposome-encapsulated anti-tumor drugs. Studies with vinblastine, actinomycin D, cytosine arabinoside, and daunomycin.

Abstract We have investigated the effects of encapsulation within liposomes (phospholipid vesicles) on the plasma clearance kinetics and tissue disposition of four anti-tumor drugs, namely vinblastine. cytosinc arabinoside, actinomycin- d and daunomycin. In each case, subsequent to intravenous administration, the liposome-encapsulated drugs were cleared from the plasma much more slowly than were the free drugs. For example, the major portion of daunomycin injected in free form had a plasma half-life of less than 5 min, while liposome-encapsulated daunomycin had a plasma half-life in excess of 150 min. Encapsulation also caused a marked alteration in the tissue disposition of the injected drugs. Thus, encapsulation within liposomes resulted in a large increase in the total amount of drug equivalents retained by the tissues at various times after injection. In the case of cytosine arabinoside, for example, the level of drug equivalents in the liver at 16 hr post injection was 68-fold greater for liposome-encapsulated drug than for free drug. Encapsulation also altered the relative distribution of drugs in the tissues, with tissues rich in reticuloendothelial cells, such as liver and spleen, being the favored sites of uptake.

Factors affecting the clearance kinetics and tissue distribution of liposomes, microspheres and emulsions

Abstract Upon injection into the circulation, microparticulate drug carriers including liposomes, polymer microspheres and lipid emulsions tend to be rapidly cleared and to accumulate at tissue sites, especially liver and spleen. The cells of the mononuclear phagocyte system (MPS) play an important role in the clearance and disposition of injected microparticles. These cells are actively endocytic and possess cell-surface receptors for a number of the plasma proteins which are likely to adsorb to injected microparticles. Some of the blood proteins known to interact with liposomes include immunoglobulin, complement components, clotting factors, fibronectin and lipoproteins; these proteins may also adsorb to other types of microparticles. The chemical composition of the microparticles plays an important role in determining their interaction with plasma proteins, subsequent interactions with MPS cells, and stability in the blood environment.

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin.

Distinct mechanisms mediate the initial and sustained phases of integrin-mediated activation of the Raf/MEK/mitogen-activated protein kinase cascade

Integrin-mediated adhesion to the extracellular matrix activates the canonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activation of Raf-1, an upstream regulator of mitogen-activated protein kinase, occurs in two phases. Efficient early activation of Raf required Raf-Ras interaction but was not affected by protein kinase C (PKC) inhibitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PKC inhibitors. The combination of PKC inhibition and lack of Ras binding completely blocked integrin-mediated Raf activity. The activity of a membrane-bound Raf mutant that is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by adhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapidly stimulated to wild-type levels by cell adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation of wild-type and mutant Raf proteins was ablated by cytochalasin D, demonstrating that cytoskeletal organization is required for activation of Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respectively, and that integrin-mediated adhesion may regulate a cytoskeleton-associated factor(s) responsible for Raf activation.