Journal of Cellular Physiology

Reactive oxygen species (ROS), whether produced endogenously as a consequence of normal cell functions or derived from external sources, pose a constant threat to cells living in an aerobic environment as they can result in severe damage to DNA, protein, and lipids. The importance of oxidative damage to the pathogenesis of many diseases as well as to degenerative processes of aging has becoming increasingly apparent over the past few years. Cells contain a number of antioxidant defenses to minimize fluctuations in ROS, but ROS generation often exceeds the cell's antioxidant capacity, resulting in a condition termed oxidative stress. Host survival depends upon the ability of cells and tissues to adapt to or resist the stress, and repair or remove damaged molecules or cells. Numerous stress response mechanisms have evolved for these purposes, and they are rapidly activated in response to oxidative insults. Some of the pathways are preferentially linked to enhanced survival, while others are more frequently associated with cell death. Still others have been implicated in both extremes depending on the particular circumstances. In this review, we discuss the various signaling pathways known to be activated in response to oxidative stress in mammalian cells, the mechanisms leading to their activation, and their roles in influencing cell survival. These pathways constitute important avenues for therapeutic interventions aimed at limiting oxidative damage or attenuating its sequelae. Published 2002 Wiley‐Liss, Inc.

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU‐S), production of granulocyte precursor cells (CFU‐C), and extensive granulopoiesis can be maintained in vitro for several months. Such cultures consist of adherent and non‐adherent populations of cells. The adherent population contains phagocytic mononuclear cells, “epithelial” cells, and “giant fat” cells. The latter appear to be particularly important for stem cell maintenance and furthermore there is a strong tendency for maturing granulocytes to selectively cluster in and around areas of “giant fat” cell aggregations. By “feeding” the cultures at weekly intervals, between 10 to 15 “population doublings” of functionally normal CFU‐S regularly occurs. Increased “population doublings” may be obtained by feeding twice weekly. The cultures show initially extensive granulopoiesis followed, in a majority of cases, by an accumulation of blast cells. Eventually both blast cells and granulocytes decline and the cultures contain predominantly phagocytic mononuclear cells.

Culturing at 33°C leads to the development of a more profuse growth of adherent cells and these cultures show better maintenance of stem cells and increased cell density.

When tested for colony stimulating activity (CSA) the cultures were uniformly negative. Addition of exogenous CSA caused a rapid decline in stem cells, reduced granulopoiesis and an accumulation of phagocytic mononuclear cells.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods–;proliferation, extracellular matrix maturation, and mineralization–;and 2) two restriction points to which the cells can progress but cannot pass without further signal–;the first when proliferation is down‐regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle‐and cell growth‐regulated genes, produce a fibronectin/type I collagen extracel‐lular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phos‐phatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid‐induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Liver regeneration after partial hepatectomy is a very complex and well‐orchestrated phenomenon. It is carried out by the participation of all mature liver cell types. The process is associated with signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. Liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body homeostasis during the entire regenerative process. In situations when hepatocytes or biliary cells are blocked from regeneration, these cell types can function as facultative stem cells for each other. J. Cell. Physiol. 213: 286–300, 2007. © 2007 Wiley‐Liss, Inc.

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death—metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980's to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed. J. Cell. Physiol. 213: 374–383, 2007. © 2007 Wiley‐Liss, Inc.

The Sp/KLF family contains at least twenty identified members which include Sp1‐4 and numerous krüppel‐like factors. Members of the family bind with varying affinities to sequences designated as ‘Sp1 sites’ (e.g., GC‐boxes, CACCC‐boxes, and basic transcription elements). Family members have different transcriptional properties and can modulate each other's activity by a variety of mechanisms. Since cells can express multiple family members, Sp/KLF factors are likely to make up a transcriptional network through which gene expression can be fine‐tuned. ‘Sp1 site’‐dependent transcription can be growth‐regulated, and the activity, expression, and/or post‐translational modification of multiple family members is altered with cell growth. Furthermore, Sp/KLF factors are involved in many growth‐related signal transduction pathways and their overexpression can have positive or negative effects on proliferation. In addition to growth control, Sp/KLF factors have been implicated in apoptosis and angiogenesis; thus, the family is involved in several aspects of tumorigenesis. Consistent with a role in cancer, Sp/KLF factors interact with oncogenes and tumor suppressors, they can be oncogenic themselves, and altered expression of family members has been detected in tumors. Effects of changes in Sp/KLF factors are context‐dependent and can appear contradictory. Since these factors act within a network, this diversity of effects may arise from differences in the expression profile of family members in various cells. Thus, it is likely that the properties of the overall network of Sp/KLF factors play a determining role in regulation of cell growth and tumor progression. © 2001 Wiley‐Liss, Inc.

A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply‐defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 10⁷ cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r²/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

The complex interactions of cells with extracellular matrix (ECM) play crucial roles in mediating and regulating many processes, including cell adhesion, migration, and signaling during morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Many of these interactions involve transmembrane integrin receptors. Integrins cluster in specific cell–matrix adhesions to provide dynamic links between extracellular and intracellular environments by bi‐directional signaling and by organizing the ECM and intracellular cytoskeletal and signaling molecules. This mini review discusses these interconnections, including the roles of matrix properties such as composition, three‐dimensionality, and porosity, the bi‐directional functions of cellular contractility and matrix rigidity, and cell signaling. The review concludes by speculating on the application of this knowledge of cell–matrix interactions in the formation of cell adhesions, assembly of matrix, migration, and tumorigenesis to potential future therapeutic approaches. J. Cell. Physiol. 213:565–573. © 2007 Wiley‐Liss, Inc.

The ability of heparin or that of hexuronyl hexosaminoglygan sulfate (HHS‐4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK‐21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at −80°C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65°C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS‐4, basic and acidic FGF retained their potency. The effect of heparin and HHS‐4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS‐4 concentrations. This indicates that heparin and HHS‐4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.

Increasing evidence suggests that mechanical cues inherent to the extracellular matrix (ECM) may be equally as critical as its chemical identity in regulating cell behavior. We hypothesized that the mechanical properties of the ECM directly regulate the motility of vascular smooth muscle cells (SMCs) and tested this hypothesis using polyacrylamide substrates with tunable mechanical properties. Quantification of the migration speed on uniformly compliant hydrogels spanning a range of stiffnesses (Young's moduli values from 1.0 to 308 kPa for acrylamide/bisacrylamide ratios between 5/0.1% and 15/1.2%, respectively) revealed a biphasic dependence on substrate compliance, suggesting the existence of an optimal substrate stiffness capable of supporting maximal migration. The value of this optimal stiffness shifted depending on the concentration of ECM protein covalently attached to the substrate. Specifically, on substrates presenting a theoretical density of 0.8 μg/cm² fibronectin, the maximum speed of 0.74 ± 0.09 μm/min was achieved on a 51.9 kPa gel; on substrates presenting a theoretical density of 8.0 μg/cm² fibronectin, the maximum speed of 0.72 ± 0.06 μm/min occurred on a softer 21.6 kPa gel. Pre‐treatment of cells with Y27632, an inhibitor of the Rho/Rho‐kinase (ROCK) pathway, reduced these observed maxima to values comparable to those on non‐optimal stiffnesses. In parallel, quantification of TritonX‐insoluble vinculin via Western blotting, coupled with qualitative fluorescent microscopy, revealed that the formation of focal adhesions and actin stress fibers also depends on ECM stiffness. Combined, these data suggest that the mechanical properties of the underlying ECM regulate Rho‐mediated contractility in SMCs by disrupting a presumptive cell‐ECM force balance, which in turn regulates cytoskeletal assembly and ultimately, cell migration. © 2005 Wiley‐Liss, Inc.