Terminal Deoxynucleotidyl Transferase-mediated dUTP-Biotin Nick End Labeling

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 ceils 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. M ANY typeS of vertebrate cells require signals from other cells to survive, at least in culture. Developing neurons, for example, require neurotrophic factors (Hamburger and Levi-Montalcini, 1949; Korshing and Thoenen, 1983; Cowan et al., 1984; Levi-Montalcini, 1987; Purves, 1988; Barde, 1989; Oppenlaeim, 1991), my_ eloid cells require colony stimulating factors (Metcalf, 1989; Wdliams et al., 1990; Koury and Bondurant, 1990), endocrine-dependent cells require specific hormones (Kerr and Searle, 1973; Krypaniou and Issacs, 1988; Wyllie et al., 1973, 1980), developing oligodendrocytes and their precursor cells require specific growth factors and cytokines (Barres et al., 1992; Barres et al., 1993b), and so on. If deprived of survival factors, these cells seem to activate an intrinsic death program and kill themselves-a process called programmed cell death (PCD). ~ Cells that die in this Address all correspondence to Dr, Martin C. Raft, Developmental Neurobioiogy Programme, Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom. Telephone: (071) 380-7016; FAX: (071) 380-7805. 1. Abbreviations used in this paper: BSO, buthionine-sulfoximine; IGE insulin-like growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCD, programmed cell death; T3, triiodothyronine; Tin, melting point; TUNEL, terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling. way normally undergo a characteristic set of morphological changes referred to as apoptosis, where the cells shrink and often fragment (Kerr et al., 1972; Wyllie et al., 1980; Searle et al., 1982). Naturally occurring cell deaths in developing tissues (Glucksmann, 1951) are also thought to be examples of PCD (Saunders, 1966; Wyllie et al., 1980). As they can sometimes be prevented or postponed by treatment of the developing animal with exogenous survival factors (Hamburger et al., 1981; Hofer and Barde, 1988; Oppenheim et al., 1988; Barres et al., 1992, 1993a; Coles et al., 1993), it is likely that at least some of these deaths occur because the cells fail to get enough endogenous survival factors. We recently proposed that most mammalian cells are programmed to kill themselves unless they are continuously signaled by other cells not to (Raft, 1992). Such "social" controis on cell survival would ensure that cells only survive when and where they are needed, just as similar social controis on cell proliferation are thought to ensure that cells only proliferate when new cells are needed (Baserga, 1985). But do most mammalian cells need signals from other cells to survive? If there are cells (other than blastomeresBiggers et al., 1971) that can survive without signals from other cells, lens and cartilage cells might be expected to be among them, as both lens and cartilage are unusual in that they contain only a single cell type and are not innervated, vascularized, © The Rockefeller University Press, 0021-9525/94/08/1069/9 $2.00 Tbe Journal of Cell Biology, Volume 126, Number 4, August 1994 1069-1077 1069 on F ebuary 0, 2013 jcb.rress. org D ow nladed fom Published August 15, 1994