Growth regulation of a cellular tumour antigen, p53, in nontransformed cells

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.     

Selective reconstitution of nude mice with long-term cultured and cloned specific helper T cells

An antibody response is the end result of complex interactions among T cells, adherent cells and B cells. An understanding of the interactions involved has proved difficult as pure populations of these cells have not been available. By making use of T-cell growth factor, we were able to grow normal helper T cells specific for heterologous erythrocytes. Because specificity and mechanism of action of these cells had been demonstrated solely in culture, we sought to establish their competence in the whole animal. We have therefore examined here whether antigen-specific helper T cells, maintained in culture over long periods, would enable syngeneic nude mice to respond to T-cell dependent antigens. The results show that specific helper T cells, propagated in serum-free medium in vitro for up to 15 months, can selectively and specifically reconstitute syngeneic C57BL/6J nu/nu mice. Depending on the specificity of the injected helper T cells, such nude mice could respond to sheep red blood cells (SRC) but not to horse red blood cells (HRC) and vice versa. The magnitude of the response was comparable to that of normal mice and could exceed it by almost 10-fold, depending on the source and number of injected helper T cells.     

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity

Collagen fibres form the stable architecture of connective tissues and their breakdown is a key irreversible step in many pathological conditions. The destruction of collagen is usually initiated by proteinases, the best known of which is the metalloproteinase collagenase (EC 3.4.24). Collagenase and related metalloproteinases are regulated at the level of their synthesis and secretion, through the action of specific stimuli such as hormones and cytokines, and also at the level of their extracellular activity through the action of a specific inhibitor, TIMP (tissue inhibitor of metalloproteinases), which irreversibly forms inactive complexes with metalloproteinases. Although the mechanisms governing the production of TIMP are unknown, immunologically identical forms of this glycoprotein have been detected in a wide variety of human body fluids and cell and tissue culture media. We therefore suggested that under physiological conditions this ubiquitous inhibitor predominates over active metalloproteinases and that tissue destruction may arise when any perturbation of this controlling excess arises. However, further progress towards testing this theory has been hindered by a lack of knowledge about the structure of TIMP and insufficient material for studying it in model systems. Here we describe the structure of TIMP predicted from its complementary DNA, its synthesis in Escherichia coli and transfected animal cells, and the finding that it is identical to a protein recently reported to have erythroid-potentiating activity (EPA).     

Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta

Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Protein biosynthesis in organelles requires misaminoacylation of tRNA

In the course of our studies on transfer RNA involvement in chlorophyll biosynthesis, we have determined the structure of chloroplast glutamate tRNA species. Barley chloroplasts contain in addition to a tRNA(Glu) species at least two other glutamate-accepting tRNAs. We now show that the sequences of these tRNAs differ significantly: they are differentially modified forms of tRNA(Gln) (as judged by their UUG anticodon). These mischarged Glu-tRNA(Gln) species can be converted in crude chloroplast extracts to Gln-tRNA(Gln). This reaction requires a specific amidotransferase and glutamine or asparagine as amide donors. Aminoacylation studies show that chloroplasts, plant and animal mitochondria, as well as cyanobacteria, lack any detectable glutaminyl-tRNA synthetase activity. Therefore, the requirement for glutamine in protein synthesis in these cells and organelles is provided by the conversion of glutamate attached to an 'incorrectly' charged tRNA. A similar situation has been described for several species of Gram-positive bacteria. Thus, it appears that the occurrence of this pathway of Gln-tRNA(Gln) formation is widespread among organisms and is a function conserved during evolution. These findings raise questions about the origin of organelles and about the evolution of the mechanisms maintaining accuracy in protein biosynthesis.     

Haemonectin, a bone marrow adhesion protein specific for cells of granulocyte lineage

There is substantial evidence that the haematopoietic microenvironment is crucial to the growth and differentiation of haematopoietic cells. This microenvironment is composed of stromal cells, soluble factors and extracellular matrix (ECM). We have shown that a complex extract of bone marrow ECM can stimulate the growth and differentiation of haematopoietic cells in vitro. Furthermore, the use of inhibitors or stimulators of ECM synthesis in long-term marrow culture affects cell proliferation. On a molecular level, however, the interactions between ECM and haematopoietic cells are not well understood. We have investigated the adhesion between specific bone marrow ECM components and haematopoietic cells, and found a protein, 'haemonectin', of relative molecular mass 60,000 in bone marrow ECM which is a lineage- and organ-specific attachment molecule for cells of granulocyte lineage. This specificity distinguishes haemonectin from previously described adhesion proteins which have a wider tissue distribution and cell type specificity.     

Stimulation of connective tissue cell growth by substance P and substance K

Connective tissue cells proliferate actively when cultured in the presence of serum. Platelet-derived growth factor (PDGF), a basic protein of relative molecular mass approximately 30,000, has been identified as the major serum mitogen for these cells; its main physiological/pathophysiological role may be to initiate wound healing in connection with tissue injury. However, growth of cultured cells is also influenced by several other factors, including epidermal growth factor, fibroblast growth factor, insulin and somatomedins. Furthermore, Rozengurt and Sinnett-Smith recently showed that bombesin, a neuroendocrine peptide isolated from frog skin, stimulates DNA synthesis and cell division in cultures of a specific subtype of 3T3 cells. Substance P and substance K (also known as neurokinin A or neuromedin L) are mammalian peptides belonging to the tachykinin family. Substance P has been studied extensively; it is distributed widely throughout the central and peripheral nervous system, including primary sensory neurones, and can be released in the periphery from axon collaterals of stimulated pain fibres and contribute to the inflammatory response. Substance K is a member of the tachykinin family isolated from mammalian spinal cord; Nawa et al. determined the primary structure of two types of substance P precursors, one of which contained a sequence homologous to substance K, as well as the sequence of substance P. We report here that substance P and substance K stimulate DNA synthesis in cultured arterial smooth muscle cells and human skin fibroblasts, and that this stimulation is inhibited by the substance P-antagonist spantide.     

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1alpha (HIF-1alpha) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis.     

Localized production of TGF-beta mRNA in tumour promoter-stimulated mouse epidermis

Tumour promoters induce a wide spectrum of morphological and biochemical alterations when applied to mouse epidermis in vivo. These include the induction of RNA, DNA and protein synthesis during discrete phases of proliferation and differentiation. This constitutes an ideal model for studying molecular events underlying the disruption of epidermal homeostasis by TPA, and its subsequent re-establishment. Transforming growth factor-beta (TGF-beta) can induce either growth stimulation, inhibition, or differentiation, depending on the target cell. A function has been proposed for TGF-beta in wound healing and in tumour promotion, but the main source of TGF-beta is generally thought to be platelets, macrophages or lymphocytes, and a direct role for this growth factor in regulating tissue homeostasis in vivo has not been demonstrated. We show here that when the tumour promoter 12-tetradecanoyl-phorbol-13-acetate (TPA) is applied to the skin of mice, very high levels of TGF-beta messenger RNA are induced in the epidermal cells. In situ hybridization techniques show that the main site of TGF-beta synthesis is in the suprabasal differentiating epidermal cells. These results suggest that TGF-beta may be a natural regulator of epidermal homeostasis which is important in tumour promotion.     

Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome)

There have been many reports that eukaryotic cells contain ring-shaped 19S or 20S particles which are composed of numerous polypeptide subunits ranging in size between 25 and 35 kilodaltons. Because these particles seemed to copurify with inactive mRNA, they were assumed to function in regulating mRNA translation and hence were named 'prosomes' (for 'programmed-o-some'). A number of properties have been reported for these structures, including an association with specific RNA species or with certain heat-shock proteins and involvement in tRNA processing or aminoacyl tRNA synthesis. However, these proposed activities have not been supported by definitive evidence. During studies of the proteolytic systems in mammalian tissues, we noted many similarities between these 19S particles and the high molecular weight protease complexes that are present in most or all eukaryotic cells. This (700 kilodalton) enzyme complex, designated here as LAMP for 'large alkaline multi-functional protease', contains three distinct endoproteolytic sites which function at neutral or alkaline pH and are specific for hydrolysis of proteins, hydrophobic peptides, or basic peptides. This protease also exists in a latent form which can be activated by polylysine, fatty acids, or ATP. In this report, we show that the prosomes and these protease complexes are very similar or identical with respect to their size, polypeptide composition, immunological cross-reactivity, appearance in the electron microscope, radial symmetry of subunits, subcellular localization, and proteolytic activities. Therefore, the 'prosome' probably plays a critical role in intracellular protein breakdown, and we propose that it be renamed 'proteasome'.     

Production and auto-induction of transforming growth factor-alpha in human keratinocytes

Transforming growth factor-alpha (TGF-alpha) is a polypeptide which is structurally related to epidermal growth factor (EGF) and binds to the EGF receptor. TGF-alpha synthesis occurs in a variety of neoplastic cells and during early fetal development but has not been reported in normal cells of the adult organisms. TGF-alpha has therefore been regarded as an embryonic growth factor which is inappropriately expressed during neoplasia. Here we report that primary cultures of normal human keratinocytes synthesize TGF-alpha. Furthermore, we show that addition of EGF or TGF-alpha to these cultures induces TGF-alpha gene expression, suggesting that a mechanism of auto-induction exists. Analysis of normal skin biopsies using in situ hybridization and immunohistochemistry demonstrates the in vivo presence of TGF-alpha messenger RNA and protein in the stratified epidermis.     

Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei

The protozoan parasite Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly (Glossina spp.). Trypanosomes ingested by the fly undergo a number of changes in the insect midgut during differentiation to procyclic forms. These include the loss of the variant specific glycoprotein (VSG) coat and the appearance of a common set of procyclic surface antigens. In order to investigate genes other than VSG genes which are expressed only at certain stages of the life cycle, the first cDNA specific to procyclic culture form trypanosomes (equivalent to the stage found in the insect midgut) has been characterized. The encoded polypeptide shows several characteristics of membrane proteins, but its most striking feature is the presence of a repetitive amino-acid sequence in which there are 22 tandem repeats of the dipeptide-Glu-Pro-. Related genes are also found in other trypanosome species and in leishmania. This gene shows many similarities to a number of surface antigen genes described in malaria and, more recently, Trypanosoma cruzi. This is the first example of a repetitive sequence in a parasite protein which is present only in the insect vector, and which therefore cannot be implicated in the mammalian host immune response.     

Immunoradiometric assay of human leukocyte interferon using monoclonal antibody

Interferon research has been hampered by problems in its assay. The only widespread assays use tissue culture cells and compare some parameter of viral growth (such as viral RNA synthesis or host cell death) in the presence and absence of interferon. These complex biological assays, though sensitive, are laborious and subject to inherent variability. In particular, components other than interferon present in the assay sample often influence viral growth. There is clearly a need for a simple, direct, interferon assay; I now describe such an assay--an immunoradiometric assay utilizing monoclonal antibody.     

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.     

Derivation of pluripotent epiblast stem cells from mammalian embryos

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.