Autocrine signals enable chondrocytes to survive in culture

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage-- which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.     

Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells.

We recently proposed that most mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD) and undergo PCD unless continuously signaled by other cells not to. Although some cells have been shown to work this way, the vast majority of cell types remain to be tested. Here we tested purified fibroblasts isolated from developing or adult rat sciatic nerve, a mixture of cell types isolated from normal or p53-null mouse embryos, an immortalized rat fibroblast cell line, and a number of cancer cell lines. We found the following: 1) All of these cells undergo PCD when cultured at low cell density in the absence of serum and exogenous signaling molecules but can be rescued by serum or specific growth factors, suggesting that they need extracellular signals to avoid PCD. (2) The mixed cell types dissociated from normal mouse embryos can only support one another's survival in culture if they are in aggregates, suggesting that cell survival in embryos may depend on short-range signals. (3) Some cancer cells secrete factors that support their own survival. (4) The survival requirements of a human leukemia cell line change when the cells differentiate. (5) All of the cells studied can undergo PCD in the presence of cycloheximide, suggesting that they constitutively express all of the protein components required to execute the death program.     

Does the lack of regression-associated mRNA expression render a rat ventral prostate epithelial cell line androgen independent?

A series of rapidly dividing epithelial (RDE) cell lines have been isolated from primary cultures of rat ventral prostate (RVP) epithelial cells. Unlike androgen-dependent secretory epithelial cells, the RDE cells in culture do not express the androgen-dependent secretory proteins, nor do they express the androgen-repressed cell death sequences (TRPM-2) found in the epithelial cells during prostatic regression. Screening of a cDNA clone library established from RDE cell mRNA has yielded a number of RDE cell-specific sequences. One of these, RDE-.25 is a 250-base mRNA. The sequence of RDE-.25 shows considerable homology with the rat growth hormone gene and two murine oncogene sequences. We believe that the absence of androgen-repressed cell death sequence expression confers androgen independence for survival and growth, while the expression of RDE-.25 may represent an autocrine growth stimulus which greatly increases the rate of cell division in these cells.     

THE UPPER CELL SURFACE: ITS INABILITY TO SUPPORT ACTIVE CELL MOVEMENT IN CULTURE

A variety of epithelial cells and fibroblasts fail to move over one another's upper surfaces in culture, resulting in monolayering. The failure of seeded fibroblasts to adhere to and spread on epithelial cell surfaces suggests that monolayering in culture is due to the lack of adhesion of the upper cell surface, at least of epithelial cells. Seeded fibroblasts and postmitotic, rounded fibroblasts likewise fail to spread on the upper surfaces of spread fibroblasts, suggesting that the inability of the upper cell surface to support spreading may be a general phenomenon. Inert particles and cell processes do not adhere directly to the upper cell surface. However, they can initiate adhesions to the surface at a cell's free margin, suggesting a variation of adhesive properties over a cell's surface.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Anchorage-independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties

Calf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life-span of the lens cells and to inhibit the increase in size that occurs after 2-3 weeks of culturing the untransformed cells.     

Cell interactions within nascent neural crest cell populations transiently promote death of neurogenic precursors

We have previously shown that cultured trunk neural crest cell populations irreversibly lose neurogenic ability when dispersal is prevented or delayed, while the ability to produce other crest derivatives is retained (Vogel, K. S. and Weston, J. A. (1988) Neuron 1, 569-577). Here, we show that when crest cells are prevented from dispersing, cell death is increased and neurogenesis is decreased in the population, as a result of high cell density. Control experiments to characterize the effects of high cell density on environmental conditions in culture suggest that reduced neurogenesis is the result of cell-cell interactions and not changes (conditioning or depletion) of the culture medium. Additionally, we show that the caspase inhibitor zVAD-fmk, which blocks developmentally regulated cell death, rescues the neurogenic ability of high density cultures, without any apparent effect on normal, low-density cultures. We conclude, therefore, that increased cell interaction at high cell densities results in the selective death of neurogenic precursors in the nascent crest population. Furthermore, we show that neurogenic cells in cultured crest cell populations that have dispersed immediately are not susceptible to contact-mediated death, even if they are subsequently cultured at high cell density. Since most early migrating avian crest cells express Notch1, and a subset expresses Delta1 (Wakamatsu, Y., Maynard, T. M. and Weston, J. A. (2000) Development 127, 2811-2821), we tested the possibility that the effects of cell contact were mediated by components of a Notch signaling pathway. We found that neurogenic precursors are eliminated when crest cells are co-cultured with exogenous Delta1-expressing cells immediately after they segregate from the neural tube, although not after they have previously dispersed. We conclude that early and prolonged cell interactions, mediated at least in part by Notch signaling, can regulate the survival of neurogenic cells within the nascent crest population. We suggest that a transient episode of cell contact-mediated death of neurogenic cells may serve to eliminate fate-restricted neurogenic cells that fail to disperse promptly in vivo.     

Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Morphological changes and growth stimulation of Bovine Epithelial Lens cells by a retinal extract in vitro

Adult Bovine Epithelial Lens cells (BEL cells) proliferate actively in vitro, acquiring several characteristics of spontaneous transformation. They do not differentiate into lens fibre cells under routine culture conditions but they can undergo a morphological change if treated by dexamethasone, as has been reported by van Venrooij et al. [1]. From adult bovine retina we obtained a saline phosphate buffer soluble extract which induced morphological and growth rate modifications of these cells either in primary cultures or in serially subcultured BEL cells. The morphology of the epithelial cells changes from a polygonal to a spindle shape. Experiments with cycloheximide indicate that this morphological change is protein synthesis dependent. It is inhibited by colchicine, suggesting an important participation of the microtubular apparatus. Cell morphology is reverted when the retinal extract is removed or by treatment with cycloheximide after a certain time lag. Cell proliferation is enhanced: the population doubling time, characteristic of the exponential phase, is conserved longer than in non-treated cultures and the density at confluency is increased. Thymidine uptake determinations have shown that DNA synthesis is increased, both in the presence and in the absence of serum. The morphological change can be uncoupled from cell division. These results do not prove that an endogenous morphogenic factor has been extracted, since differentiation into fibres has yet to be investigated, but they do show that, in vitro, the morphology of epithelial cells can be modified by a retinal extract (RE) and that the latter also contains a potent growth-promoting factor.     

THE DIFFERENTIATING ABILITY OF RAT LENS EPITHELIAL CELLS IN CELL CULTURE

Dissociated cells of lens epithelia of adult rats were monolayerly cultured in vitro. After about 15–20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gamma-crystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

DIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN TISSUE CULTURE (III) FUNCTIONS IN VITRO OF A TRANSFORMED RAT LENS EPITHELIAL CELL LINE

Epithelial cells isolated from one-day-old rat lens were neoplastically transformed using Rous Sarcoma Virus (Schmidt-Ruppin-D strain) and have been in continuous culture for over 30 months. This cell line displays some of the characteristics of lens epithelial cells, but can be shown to represent a permanent cell line in that it has been transferred more than 50 times and is continuing to grow, while primary lens cell cultures have never survived beyond 8 weeks. The infecting viral genome can be rescued from the cell line upon fusion with uninfected chick embryo cells. It is anticipated that this newly established cell line, designated RLE-R, will be useful in studies on cellular differentiation.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

High cell density provides potent survival signals for resting T-cells.

Activated T-cells are susceptible to apoptosis through two particularly important pathways: ligation of CD95 (Fas/Apo-1) or cytokine deprivation. Resting T-cells have until recently been considered to be relatively resistant to apoptosis. In this report we show that resting T-cells die rapidly by apoptosis when deprived of serum or cell contact. Primed CD45RO+ cells were more susceptible than naive CD45RA+ cells, consistent with their relative expression of Bcl-2. CD4+, CD8+ and gammadelta T-cells were equally prone to apoptosis under all studied conditions. A linear relationship between cell survival and serum concentration was observed for cells cultured between 0.5-2x10(6)/ml. T-cells cultured at low density died even in high concentrations of serum. However, resting T-cells cultured at high cell density (4x10(6)/ml) survived for extended periods in the absence of serum or other survival factors. This effect was mediated by the production of soluble factors and independent of integrin mediated signals. These results suggest that T-cells at sites of high density such as the lymph node paracortex are independent of external survival factors, while those trafficking through the peripheral circulation are highly dependent on serum derived factors for survival.     

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

The present study aims to understand the mechanism of the lens epithelial cell’s strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.Subject terms: Tumour-necrosis factors, Apoptosis     

Cell death in the oligodendrocyte lineage.

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues.     

Survival in vitro of motoneurons identified or purified by novel antibody-based methods is selectively enhanced by muscle-derived factors

Motoneurons were identified in vitro by a new method using the SC1 monoclonal antibody. They constituted up to 30% of total neurons in cultures of whole spinal cord from 4.5-day chicken embryos, and survived for at least 5 days in the presence of muscle extract, but not in its absence. By contrast, other neurons and floor-plate cells survived without muscle-derived factors. Motoneurons were purified to homogeneity by 'panning' on dishes coated with SC1 antibody; they developed rapidly even in the absence of other spinal cells. Concentrations of muscle extract required for half-maximal motoneuron survival were indistinguishable in pure and mixed cultures, suggesting that muscle-derived factors act directly on motoneurons. Other purified growth factors tested, including ciliary neurotrophic factor, did not have the survival-promoting activity of muscle.     

STUDIES ON DROSOPHILA EMBRYONIC CELLS IN VITRO I. CHARACTERISTICS OF CELL TYPES IN CULTURE *

Embryonic cells from Drosophila melanogaster were cultured in medium K-17 supplemented with 0.1 mg/ml of fetuin and 15% fetal bovine serum, and the behavior of embryonic cells, the characteristics of some cell types and the maintenance of the phenotypic properties of the cells were examined. Embryos at the stage with a saclike midgut (12 hr eggs) were found to be the best materials for cultivation. Muscle cells, epithelial cells, fibroblastic cells and small cells were observed as distinct migrating cell types. Muscle cells pulsated synchrónously when two or more cells made contact with each other and this pulsation continued for more than six weeks under the culture conditions employed. Epithelial cells matured with increase in size and in the number of cytoplasmic granules. Fibroblastic cells had conspicuous motile activity. Small cells were peculiar for their size of about 3 μm diameter and formed groups of about forty to fifty cells. Their characteristic shape and size suggested that they were imaginal disc cells. Cellular spheres, which consisted of epithelial cells and fibroblastic cells, and nerve fibers of nerve cells were also observed. Some mitotic figures, suggesting the occurrence of cell division, were observed in cultures of epithelial cells.