Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters

The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25-fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells' migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumor-promoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells' ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface.     

Attraction rules: germ cell migration in zebrafish

The migration of zebrafish primordial germ cell towards the region where the gonad develops is guided by the chemokine SDF-1a. Recent studies show that soon after their specification, the cells undergo a series of morphological alterations before they become motile and are able to respond to attractive cues. As migratory cells, primordial germ cells move towards their target while correcting their path upon exiting a cyclic phase in which morphological cell polarity is lost. In the following stages, the cells gather at specific locations and move as cell clusters towards their final target. In all of these stages, zebrafish germ cells respond as individual cells to alterations in the shape of the sdf-1a expression domain, by directed migration towards their target - the position where the gonad develops.     

Lyb-5- B cells of CBA/N mice can be induced to synthesize DNA by culture with insolubilized but not soluble anti-Ig

The use of anti-immunoglobulin (anti-Ig) antibodies to stimulate B cell proliferation (1-4), and to stimulate B cell differentiation in the presence of T cell derived-lymphokines (5-8), has simplified investigations into the mechanisms of B cell growth and maturation that are dependent on the cross-linking of surface Ig (sIg). It is only the ontogenetically late appearing Lyb-5+ murine splenic B cells, however, that proliferate in response to anti-Ig antibodies, whereas B cells of the Lyb-5- phenotype obtained from neonatal mice or from mice with the xid immune defect cannot be induced to proliferate in response to this stimulus (1, 9, 10). Thus, the analysis of B lymphocyte physiology of the Lyb-5- B cell population has been hampered by the unavailability of B cell stimulants that mimic an antigen-induced sIg cross-linking event that leads to B cell activation. The inability of soluble anti-Ig antibodies to induce the proliferation of Lyb-5- cells has been particularly difficult to explain because these cells can be induced to increase in size (11) and to show an increase in their expression of surface Ia (sIa) after exposure to anti-Ig (12). Apparently, therefore, these cells are not entirely refractory to this stimulus but are simply unable to progress to the latter stages of cell activation. In view of our observations that the cells of CBA/N mice cannot respond to soluble trinitrophenyl-(TNP) dextran or TNP-polyacrylamide (13) but can respond to insolubilized forms of these antigens, we evaluated their ability to respond to insolubilized anti-Ig. In this paper we report that B cells from CBA/N mice can be stimulated to proliferate in response to anti-Ig conjugated to Sepharose beads, but in contrast to normal B cells they need to be stimulated with beads expressing a high-epitope density of anti-Ig antibodies.     

Memory and anergy: challenges to traditional models of T lymphocyte differentiation.

Traditional paradigms suggest that encounter with an antigen converts naive peripheral T cells into memory cells with less stringent requirements for activation and increased capacities for lymphokine production. Recent evidence argues that this view may be over-simplified in two ways. First, an encounter with antigen in the absence of certain costimulatory factors can render a T cell anergic--that is, unable to respond to antigen under normal conditions. Second, although cells of the memory T cell population are more responsive than naive cells to some stimuli, these cells are hyporesponsive in other situations. Intrinsic resistance of memory T cells to elevation of intracellular calcium ion concentrations may contribute to their poor responsiveness to agents that activate naive cells. Thus, aspects of the costimulatory environment can determine whether a resting T cell is activated or rendered anergic and may also influence the kinds of stimuli to which a memory T cell will respond.     

Cell death pathology: cross-talk with autophagy and its clinical implications

Autophagy is a self-digesting mechanism that cells adopt to respond to stressful stimuli. Morphologically, cells dying by autophagy show multiple cytoplasmic double-membraned vacuoles, and, if prolonged, autophagy can lead to cell death, “autophagic cell death”. Thus, autophagy can act both as a temporary protective mechanism during a brief stressful episode and be a mode of cell death in its own right. In this mini-review we focus on recent knowledge concerning the connection between autophagy and programmed cell death, evaluating their possible implications for therapy in pathologies like cancer and neurodegeneration.     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Response of CBA/N mice to human B cell activating factor.

The in vitro antibody responses of CBA mutant and normal mice were studied with respect to their relative abilities to respond to stimulation by purified B cell activating factor (BAF). It was found that mice carrying the X-linked inability to respond to T-independent antigens can respond to BAF. This result is discussed with respect to the possibility that BAF exerts its effect on a subset of B cells distinct from those cells responsive to non-mitogenic T-independent antigens.     

The molecular control of DNA damage-induced cell death

Because of the singular importance of DNA for genetic inheritance, all organisms have evolved mechanisms to recognize and respond to DNA damage. In metazoans, cells can respond to DNA damage either by undergoing cell cycle arrest, to facilitate DNA repair, or by undergoing cell suicide. Cell death can either occur by activation of the apoptotic machinery or simply be a consequence of irreparable damage that prevents further cell division. In germ cells, mechanisms for limiting alterations to the genome are required for faithful propagation of the species whereas in somatic cells, responses to DNA damage prevent the accumulation of mutations that might lead to aberrant cell proliferation or behavior. Several of the genes that regulate cellular responses to DNA damage function as tumor suppressors. The clinical use of DNA damaging agents in the treatment of cancer can activate these tumor suppressors and exploits the cellular suicide and growth arrest mechanisms that they regulate. It appears that in some but not all types of tumors the propensity to undergo apoptosis is a critical determinant of their sensitivity to anti-cancer therapy. This review describes current understanding of the molecular control of DNA damage-induced apoptosis with particular attention to its role in tumor suppression and cancer therapy.     

Pathogenic stimulation of intestinal stem cell response in drosophila

Stem cell‐mediated tissue repair is a promising approach for many diseases. Mammalian intestine is an actively regenerating tissue such that epithelial cells are constantly shedding and underlying precursor cells are constantly replenishing the loss of cells. An imbalance of these processes will lead to intestinal diseases including inflammation and cancer. Mammalian intestinal stem cells (ISCs) are located in bases of crypts but at least two groups of cells have been cited as stem cells. Moreover, precursor cells in the transit amplifying zone can also proliferate. The involvement of multiple cell types makes it more difficult to examine tissue damage response in mammalian intestine. In adult Drosophila midgut, the ISCs are the only cells that can go through mitosis. By feeding pathogenic bacteria and stress inducing chemicals to adult flies, we demonstrate that Drosophila ISCs in the midgut can respond by increasing their division. The resulting enteroblasts, precursor cells for enterocytes and enteroendocrine cells, also differentiate faster to become cells resembling enterocyte lineage. These results are consistent with the idea that Drosophila midgut stem cells can respond to tissue damage induced by pathogens and initiate tissue repair. This system should allow molecular and genetic analyses of stem cell‐mediated tissue repair. J. Cell. Physiol. 220: 664–671, 2009. © 2009 Wiley‐Liss, Inc.     

TLRs mediate IFN-gamma production by human uterine NK cells in endometrium

The human endometrium (EM) contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay between these different cell types and their roles in defense against pathogen invasion in this specialized tissue are important for controlling infection and reproduction. TLRs are a family of receptors able to recognize conserved pathogen-associated molecular patterns. In this study, we determined the expression of TLRs on uterine NK (uNK) cells from the human EM and the extent to which uNK cells responded to TLR agonist stimulation. uNK cells expressed TLRs 2, 3, and 4, and produced IFN-gamma when total human endometrial cells were stimulated with agonists to TLR2 or TLR3 (peptidoglycan or poly(I:C), respectively). Activated uNK cell clones produced IFN-gamma upon stimulation with peptidoglycan or poly(I:C). However, purified uNK cells did not respond directly to TLR agonists, but IFN-gamma was produced by uNK cells in response to TLR stimulation when cocultured with APCs. These data indicate that uNK cells express TLRs and that they can respond to TLR agonists within EM by producing IFN-gamma. These data also indicate that the uNK cells do not respond directly to TLR stimulation, but rather their production of IFN-gamma is dependent upon interactions with other cells within EM.     

Stem cell dynamics in response to nutrient availability

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.     

Application of high-density optical microwell arrays in a live-cell biosensing system

In this paper, we use optical imaging fibers to fabricate a chemical and biochemical sensor that utilizes the ability of living cells to respond to biologically significant compounds. The sensor is created by randomly dispersing single NIH 3T3 mouse fibroblast cells into an optically addressable fiber-optic microwell array such that each microwell accommodates a single cell. The cells are encoded to identify their location within the array and to correlate changes or manipulations in the local environment to responses of specific cell types. The entire array can be simultaneously measured, yielding a rapid, repetitive, and high-density analysis method.     

Early cellular events in systemic autoimmunity driven by chromatin-reactive T cells

In vivo exposure of the thymus of normal mice to procainamide-hydroxylamine, a lupus-inducing drug, causes development of chromatin-reactive T cells. Autoantibodies subsequently appear, but their origin and significance are unknown. The current studies were undertaken to determine the specificities of B cells that respond to chromatin-reactive T cells at the initiation of this autoimmune process. Three days after adoptive transfer of 6 x 10(6) chromatin-reactive T cells, B cells with the capacity to secrete IgM anti-chromatin antibodies were detected in 1/10(6) splenocytes, and these became 10- to 50-fold more numerous if either the donor T cells or the recipient had defective Fas due to the lpr allele. Five days later these mice developed IgG anti-chromatin-secreting B cells at a precursor frequency of 3-6 x 10(-5). B cells with dDNA-binding activity isolated from mice primed in vivo to a complex of methylated pigeon cytochrome c and dDNA could stimulate naive, cytochrome c-reactive T cells in vitro, demonstrating that B cells can internalize dDNA-bound proteins through their dDNA immunoblobulin receptor and can functionally present a T cell epitope. However, no capacity of chromatin for binding anti-dDNA antibodies was detected, and IgM dDNA-specific B cells did not expand when challenged with chromatin-reactive T cells in vivo. The rapid and robust expansion of anti-chromatin-secreting B cells indicates that the normal immune repertoire includes nontolerant autoreactive B cells that respond to strong T cell drive and are readily manifested if Fas-mediated activation-induced cell death is inhibited.     

Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl-phorbol-acetate

The potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) can stimulate quiescent, nonproliferating 3T3 cells to reenter the cell cycle and divide. We have previously used a slection technique developed in our laboratory to isolate variant cell lines which no longer divide in response to epidermal growth factor. We have now utilized the same selection procedure to isolate, from 3T3 cells, two variant cell lines, TNR-2 and TNR-9, which retain growth control and divide in response to elevated serum or fibroblast growth factor, but which do not respond to TPA. The variants do not incorporate precursors into DNA in response to TPA, demonstrating that the cells do not enter the S phase of the cell cycle. The TPA nonresponsive variant TNR-2 cannot respond to epidermal growth factor; TNR-9 responds to this mitogen. TNR-2 variant cells, which do not respond to EGF, do not bind ¹²⁵I-EGF. TPA can modulate ¹²⁵I-EGF binding to TNR-9 cells in a manner similar to its action on parental 3T3 cells. This TPA-induced alteration of EGF binding indicates that TNR-9 cells still interact with TPA, despite their inability to mount a mitogenic response.     

Stiffness of MDCK II Cells Depends on Confluency and Cell Size

Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. In the steady state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments and in response to external chemical and mechanical stimuli. However, a major problem exists in determining mechanical properties because many techniques, such as atomic force microscopy, that assess these properties of adherent cells locally can only address a limited number of cells and provide elastic moduli that vary substantially from cell to cell. The origin of this spread in stiffness values is largely unknown and might limit the significance of measurements. Possible reasons for the disparity are variations in cell shape and size, as well as biological reasons such as the cell cycle or polarization state of the cell. Here, we show that stiffness of adherent epithelial cells rises with increasing projected apical cell area in a nonlinear fashion. This size stiffening not only occurs as a consequence of varying cell-seeding densities, it can also be observed within a small area of a particular cell culture. Experiments with single adherent cells attached to defined areas via microcontact printing show that size stiffening is limited to cells of a confluent monolayer. This leads to the conclusion that cells possibly regulate their size distribution through cortical stress, which is enhanced in larger cells and reduced in smaller cells.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Matriptase Autoactivation Is Tightly Regulated by the Cellular Chemical Environments

The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl₂ and CdCl₂. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl₂-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.     

Investigating the limits of filopodial sensing: a brief report using SEM to image the interaction between 10 nm high nano-topography and fibroblast filopodia

Having the ability to control cell behaviour would be of great advantage in tissue engineering. One method of gaining control over cell adhesion, proliferation, guidance and differentiation is use of topography. Whilst it has be known for some time that cells can be guided by micro‐topography, it is only recently becoming clear that cells will respond strongly to nano‐scale topography. The fact that cells will take cues from their micro‐ and nano‐environment suggests that the cells are in some way ‘spatially aware’. It is likely that cells probe the shape of their surroundings using filopodia, and that this initial filopodia/topography interaction may be critical to down‐stream cell reactions to biomaterials, or indeed, the extracellular matrix. One intriguing question is how small a feature can cells sense? In order to investigate the limits of cell sensing, high‐resolution scanning electron microscopy has been used to simultaneously view cell filopodia and 10 nm high nano‐islands. Fluorescence microscopy has also been used to look at adhesion formation. The results showed distinct filopodial/nano‐island interaction and changes in adhesion morphology.     

Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis

Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence.