SMART MODE:

NORMAL GENOMIC

• Cui S, Hope RM, Rathjen J, Voyle RB, Rathjen PD
• Structure, sequence and function of a marsupial LIF gene: conservation of IL-6 family cytokines.
• Cytogenet Cell Genet. 2001; 92: 271-8
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• Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals. Copyright 2001 S. Karger AG, Basel.

• Bader A, Al-Dubai H, Weitzer G
• Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions.
• Circ Res. 2000; 86: 787-94
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• Cardiogenesis is a multistep process regulated by a hierarchy of factors defining each developmental stage of the heart. One of these factors, leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, is expressed in embryonic and neonatal cardiomyocytes and induces cardiomyocyte hypertrophy. Many aspects of embryogenesis are faithfully recapitulated during in vitro differentiation of embryonic stem cells in embryoid bodies. We exploited this model to study effects of growth factors on commitment and differentiation of cardiomyocytes and on maintenance of their phenotype. We identified LIF as a factor affecting commitment and differentiation of cardiomyocytes in an opposite manner. Diffusible LIF inhibited mesoderm formation and hampered commitment of cardiomyocytes. Lack of both the diffusible and matrix-bound isoforms of LIF in lif-/- embryoid bodies did not interfere with commitment, but it severely suppressed early differentiation of cardiomyocytes. Onset of differentiation was rescued by very low concentrations of diffusible LIF; however, consecutive differentiation was attenuated in a concentration-dependent manner by diffusible LIF both in wild-type and lif-/- cardiomyocytes. Differentiation of cardiomyocytes was severely hampered but not completely blocked in lifr-/- embryoid bodies, suggesting additional, LIF-receptor ligand independent pathways for commitment and differentiation of cardiomyocytes. At the fully differentiated state, both paracrine and autocrine LIF promoted proliferation and increased longevity of cardiomyocytes. These findings suggest that both paracrine and autocrine and both diffusible and matrix-bound isoforms of LIF contribute to the modulation of cardiogenesis in a subtle, opposite, and developmental stage-dependent manner and control proliferation and maintenance of the differentiated state of cardiomyocytes.

• Zandstra PW, Le HV, Daley GQ, Griffith LG, Lauffenburger DA
• Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation.
• Biotechnol Bioeng. 2000; 69: 607-17
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• A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.

• Haines BP, Voyle RB, Pelton TA, Forrest R, Rathjen PD
• Complex conserved organization of the mammalian leukemia inhibitory factor gene: regulated expression of intracellular and extracellular cytokines.
• J Immunol. 1999; 162: 4637-46
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• Leukemia inhibitory factor (LIF) is a member of the IL-6 family of pleiotropic cytokines, which are extensively involved in modulating hematopoiesis and immunity. We have undertaken a detailed analysis of LIF genomic organization and gene transcription and investigated the proteins expressed from alternate transcripts. Previously unidentified LIF transcripts, containing alternate first exons spliced onto common second and third exons, were cloned from murine embryonic stem cells, human embryonal carcinoma cells, and primary porcine fibroblasts. Based on sequence homology and position within the genomic sequence, this confirmed the existence of the LIF-M transcript in species other than the mouse and identified a new class of transcript, designated LIF-T. Thus, a complex genomic organization of the LIF gene, conserved among eutherian mammals, results in the expression of three LIF transcripts (LIF-D, LIF-M, and LIF-T) differentially expressed from alternate promoters. The first exon of the LIF-T transcript contained no in-frame AUG, causing translation to initiate downstream of the secretory signal sequence at the first AUG in exon two, producing a truncated LIF protein that was localized within the cell. Enforced secretion of this protein demonstrated that it could act as a LIF receptor agonist. Regulated expression of biologically active intracellular and extracellular LIF cytokine could thus provide alternate mechanisms for the modulation of hematopoiesis and immune system function.

• Tanaka M, Hara T, Copeland NG, Gilbert DJ, Jenkins NA, Miyajima A
• Reconstitution of the functional mouse oncostatin M (OSM) receptor: molecular cloning of the mouse OSM receptor beta subunit.
• Blood. 1999; 93: 804-15
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• Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor beta subunit (LIFRbeta), and the type II OSM receptor consists of gp130 and the OSM receptor beta subunit (OSMRbeta). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRbeta, which is 55.5% identical to the hOSMRbeta at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRbeta, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRbeta. mOSMRbeta alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRbeta and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRbeta constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRbeta gene was found to be located in the vicinity of the LIFRbeta locus in the proximal end of chromosome 15.

• Dani C et al.
• Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.
• Dev Biol. 1998; 203: 149-62
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• The propagation of pluripotential mouse embryonic stem (ES) cells is sustained by leukemia inhibitory factor (LIF) or related cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotency in vivo. In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of the lif gene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathway in vivo could play an important role in the regulation of pluripotency in the epiblast and account for the viability of lifr -/- and gp130 -/- embryos.

• Taupin JL, Pitard V, Dechanet J, Miossec V, Gualde N, Moreau JF
• Leukemia inhibitory factor: part of a large ingathering family.
• Int Rev Immunol. 1998; 16: 397-426
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• Leukemia Inhibitory Factor (LIF) has a wide variety of biological activities. It regulates the differentiation of embryonic stem cells, neural cells, osteoblasts, adipocytes, hepatocytes and kidney epithelial cells. It also triggers the proliferation of myoblasts, primordial germ cells and some endothelial cells. Many of these biological functions parallel those of interleukin-6, Oncostatin M, ciliary neurotrophic factor, interleukin-11 and cardiotrophin-1. These structurally related cytokines also share subunits of their receptors which could partially explain the redundancy in this system of soluble mediators. In vivo LIF proves important in regulating the inflammatory response by fine tuning of the delicate balance of at least four systems in the body, namely the immune, the hematopoietic, the nervous and the endocrine systems. Although we are far from its therapeutic applications, the fast increasing knowledge in this field may bring new insights for the understanding of the cytokine biology in general.

• Sumikawa H, Suzuki E
• Tertiary structural models of human interleukin-6 and evaluation by comparison with X-ray and NMR structures.
• Chem Pharm Bull (Tokyo). 1998; 46: 136-8
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• Tertiary structure models of interleukin-6 were constructed using a routine prediction method based on the X-ray crystal structures of granulocyte colony-stimulating factor (GCSF) and leukemia inhibitory factor (LIF). Those models were evaluated using a sequence-structure compatibility (3D-1D) method program Compass and a limited amount of NMR distance information when it was concluded that the model based on GCSF (IBGC) was preferable to that from LIF (Sumikawa et al., FEBS Lett., 404, 234 (1997)). We evaluated the quality of this model (IBGC) by comparing with X-ray (Somers et al., EMBO., 16, 989 (1997)) and NMR (Xu et al., J. Mol. Biol., 268, 468 (1997)) structures. Consequently, normal mode calculations were carried out for this model, giving conformation fluctuations similar to the C alpha deviation pattern between X-ray and NMR structures.

• Sumikawa H, Suzuki E, Fukuhara K, Nakajima Y, Kamiya K, Umeyama H
• Dynamic structures of granulocyte colony-stimulating factor proteins studied by normal mode analysis: two domain-type motions in low frequency modes.
• Chem Pharm Bull (Tokyo). 1998; 46: 1069-77
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• In this study, granulocyte colony-stimulating factor (GCSF) proteins were chosen as subjects for normal mode analysis. As helical cytokines with a four helix bundled type topology, they were classified into long chain and short chain groups by Sprang and Bazan. Normal mode calculations were also carried out with leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and growth hormone (GH) as members of the long chain group and GCSF and IL-2 and IL-4 as members of the short chain group. For the GCSF families it was found that the fluctuations in the helical region are smaller than in the loop region, and it is clear that on the whole the smaller fluctuation residues belong to a large hydrophobic core region. Thus, it can be imagined how the receptor binding sites approach the receptor within the normal time-scale of pico seconds. In addition, two similar domain-type motions in low frequency modes were found with proteins in the long chain group, although we never observed any sequence similarity in the two separate two-domain regions in each protein of the long chain group. On the other hand, these two domain-type motions were not clear in proteins of the short chain group.

• Nemetz C, Hocke GM
• Transcription factor Stat5 is an early marker of differentiation of murine embryonic stem cells.
• Differentiation. 1998; 62: 213-20
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• Embryonic stem (ES) cells are pluripotent descendants of the inner cell mass of blastocysts capable of differentiating into progenitor cells of most if not all tissues. The pluripotency of ES cells is maintained by leukemia inhibitory factor (LIF), a member of the family of interleukin-6-type cytokines. These cytokines activate Janus tyrosine kinases and signal transducer and activator of transcription factors (Stat) via the signalling receptor component gp130. Pluripotent ES1 cells proliferating in the presence of LIF were known from previous studies to contain Stat3 and Stat1 capable of transcriptional activation. Here we report that the level of tyrosine-phosphorylated Stat3 decreases rapidly during differentiation induced by treatment of ES1 cells either with retinoic acid (RA) or by withdrawal of LIF. In line with this finding, the DNA-binding activity of Stat3 decreased during differentiation. In contrast, Stat5 was absent from pluripotent proliferating ES cells, but appeared early after induction of differentiation. The positive correlation between induction of differentiation and expression of Stat5 mRNA was confirmed for three independent ES cell lines. Stat5 transcripts were detectable in ES1 cells as early as 12 h after treatment with RA and 36 h after withdrawal of LIF. Stat5 protein was detectable 2 days after the onset of differentiation. These results establish Stat5 as a novel marker of very early stages of differentiation of ES cells.

• Kitchen D, Hoffman RC, Moy FJ, Powers R
• Homology model for oncostatin M based on NMR structural data.
• Biochemistry. 1998; 37: 10581-8
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• Oncostatin M (OM) is a member of the cytokine family which regulates the proliferation and differentiation of a variety of cell types and includes interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and granulocyte-colony stimulating factor (G-CSF). This family of proteins adopts a four-helix bundle fold with up-up-down-down topology and contains intramolecular disulfide bonds. Since an X-ray or NMR structure for OM is not currently available, a homology model for OM was determined from the X-ray structures of human growth hormone (hGH), LIF, and G-CSF where the alignment was based on secondary structure instead of sequence. The OM secondary structure was determined from NMR structural data, and the secondary structures for hGH, LIF, and G-CSF were obtained from the reported X-ray structures. The resulting homology model was refined using sequential NOE distance 13C restraints, chemical shift information, and a conformational database.

• Takahama Y et al.
• Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells.
• Oncogene. 1998; 16: 3189-96
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• Embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo. Maintenance of the stem-cell phenotype of ES cells in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) such as leukemia inhibitory factor (LIF). Here we report the cloning of complete rat LIF cDNA and its nucleotide sequence so as to facilitate studies of rat ES cell technologies on tumor biology. The nucleotide sequence of the rat LIF cDNA indicated that the rat LIF has 91% amino acid sequence identity with murine LIF. The cloned rat LIF cDNA has a putative biological activity as a differentiation-inducing factor on the murine myeloid leukemia cell line M1 cells. Culture supernatant of the rat LIF cDNA-transduced rat fibroblast cell line could maintain the stem-cell phenotype of rat ES cells which showed alkaline phosphatase activity, and this effect was much stronger than that by murine LIF. The availability of rat LIF which shows DIA will assist the in vitro analysis of rat ES cells, and culture of these cells is a route for the generation of gene targeting in rat.

• Sumikawa H, Fukuhara K, Suzuki E, Matsuo Y, Nishikawa K
• Tertiary structural models for human interleukin-6 and evaluation by a sequence-structure compatibility method and NMR experimental information.
• FEBS Lett. 1997; 404: 234-40
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• Tertiary structure models of Interleukin-6 were constructed using a routine prediction method based on the X-ray crystal structures of granulocyte colony-stimulating factor (GCSF) and leukemia inhibitory factor (LIF). The models were evaluated with the aid of the sequence-structure compatibility (3D-1D) method program compass and NMR experimental information. The model constructed from GCSF gained higher scores on compass examination than did that from LIF, and the NOE data [Nishimura et al. (1996) Biochemistry 35, 273-281] also turned to be more consistent with the former model.

• Yamasaki K, Naito S, Anaguchi H, Ohkubo T, Ota Y
• Solution structure of an extracellular domain containing the WSxWS motif of the granulocyte colony-stimulating factor receptor and its interaction with ligand.
• Nat Struct Biol. 1997; 4: 498-504
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• We have determined the NMR structure of a ligand-binding domain of the granulocyte colony-stimulating factor (G-CSF) receptor, containing the highly conserved WSxWS motif. The domain consists of seven beta-strands with the fibronectin type III-like topology seen in several cytokine receptors. Comparisons between the spectra of the 15N-labelled domain with and without G-CSF indicate that the major ligand-recognition site is on the FG loop just upstream of the WSxWS sequence, and not on the BC loop which is mainly used in the growth hormone system. The WSxWS residues are suggested to contribute to ligand-recognition and to the protein architecture of the G-CSF receptor.

• Berger CN, Sturm KS
• Self renewal of embryonic stem cells in the absence of feeder cells and exogenous leukaemia inhibitory factor.
• Growth Factors. 1997; 14: 145-59
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• To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the beta-galactosidase transgenic Kli ES cells, revealed that LIF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.

• Gendall AR, Dunn AR, Ernst M
• Isolation and characterization of a leukemia inhibitory factor-independent embryonic stem cell line.
• Int J Biochem Cell Biol. 1997; 29: 829-40
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• Leukemia inhibitory factor (LIF) is a mammalian cytokine that has a wide range of physiological activities, including the inhibition of differentiation of embryonic stem (ES) cells. We have used insertional mutagenesis in an attempt to isolate molecules that participate in LIF signal transduction via the LIF receptor. Using a robust screen for undifferentiated cells, we have isolated one ES cell line, Poly 27, that does not require exogenous LIF to remain undifferentiated in vitro. We present evidence that Poly 27 is not irreversibly committed to an undifferentiated phenotype, but can differentiate in vitro if cultured in the presence of chemical differentiating agents, while in syngeneic mice Poly 27 cells form tumours which are composed largely of undifferentiated cells. We have characterized the mechanism of factor independence in Poly 27, and shown it to be a result of autocrine LIF production. This LIF production is potentially the result of a mutation in a gene critically involved in regulating LIF production in ES cells.

• Du XX, Shi WK
• [Study on growth and differentiation of ES cells transfected with LIF gene]
• Shi Yan Sheng Wu Xue Bao. 1996; 29: 413-27
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• We constructed plasmids pSVLD(+) and pSVLD(-) containing human D-form Leukemia Inhibitory Factor (LIF) cDNA sequence in sense or antisense orientation, transfected them into cells of an embryonic stem cell line ES-5, and isolated 248 pSVLD(+)-transfected and 93 pSVLD(-)-transfected G 418-resistant clones. By stepwise reducing LIF concentration in the medium, we obtained 3 pSVLD(+)-transfected clones (A 1-3) that could grow in 15% BRL-CM, including ESL(+)A 2 that could grow without LIF: we also obtained 13 pSVLD(-)-transfected clones (B 1-13) which would differentiate in 60% BRL-CM, including ESL(-)B 3 and B 5 that could not be passaged without LIF. ESL(+)A 2 and ESL(-)B 5 cells had the relatively stronger LIF mRNA or antisense LIF RNA expression, and LIF overexpression in ESL(+)A 2 cells was shown by biological assay for ES cell differentiation inhibition. ESL(+)A 2 cells could be continuously passaged for at least 13 passages without addition of exogenous LIF, retained undifferentiated morphology as well as a high growth rate, and resembled ES-5 cells in terms of stem cell characteristics and pluripotent properties, as analyzed for alkaline phosphatase activity and with staining the paraffin sections of tumor formed by inoculating ESL(+) A 2 cells into mouse. On the contrary, ESL(-) cells should be cultured in higher concentration of LIF than ES-5 cells, otherwise, would undertake extensive differentiation. By hanging drop culture for 3 days in the presence of 10(-6) mol/L RA then observing the differentiation of the formed embryonic bodies (EBs), we found that ESL(+) A 2 and ES-5 cells underwent similar morphologically differentiation, with round and epitheliallike cells occurring around the EBs; while ESL(-) B 5 cells, despite initial differentiation to round cells, differentiate into fibroblast-like and spindle shaped cells. The above results indicate that LIF overexpression in ESL(+) A 2 cells is able to completely free ES cells from the dependence on LIF-conditioned medium, and endogenous LIF gene expression, although is very low, may be indispensable for inhibiting the differentiation in vitro of ES cells; LIF overexpression might not obviously change the differentiation way of ES-5 cells, however, blocking endogenous LIF expression gives rise to the increased sensitivity of ES-5 cells to differentiate, with an altered differentiation pattern. The establishment of ESL(+) and ESL (-) cell lines provides models for further study of the growth and differentiation of ES-5 cells.

• Shellard J, Perreau J, Brulet P
• Role of leukemia inhibitory factor during mammalian development.
• Eur Cytokine Netw. 1996; 7: 699-712
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• Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferative, survival and differentiation activities on a wide range of cell types. A role for LIF in embryonic development is suggested by: i) its ability to stimulate the proliferation of embryonic stem (ES) cells in vitro, while maintaining their totipotency and ii) by both its maternal and embryonic expression at the time of blastocyst implantation. Functional studies of LIF and its receptor during mouse embryogenesis have been performed using the techniques of targeted gene replacement and transgene expression in ES cells to produce transgenic mice bearing either loss- or gain-of-function mutations for LIF activity. Whereas, the phenotype observed in the LIF gain-of-function mutant mice supports a role for LIF in early embryogenesis, the loss-of-function phenotypes point to more specialized functions for LIF in development and further reveal the redundant feature of the LIF cytokine/receptor family.

• Piekorz RP, Blasius R, Fey GH, Hocke GM
• Kinetics of the activation of the LIF-response factor in M1 myeloid leukemic cells.
• Ann N Y Acad Sci. 1995; 762: 452-4

• Malik N, Haugen HS, Modrell B, Shoyab M, Clegg CH
• Developmental abnormalities in mice transgenic for bovine oncostatin M.
• Mol Cell Biol. 1995; 15: 2349-58
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• Oncostatin M belongs to the subfamily of hematopoietin cytokines that binds a receptor complex containing gp130. To date, only the human form of oncostatin M has been identified, and its evolutionary conservation is unresolved. We have isolated a bovine gene whose open reading frame encodes a precursor protein that is 58% identical to human oncostatin M. A comparison of the bovine and human amino acid sequences predicts significant similarity, including the four-alpha-helical-bundle structure and the placement of disulfide bridges. As with the human protein, bovine oncostatin M binds specific receptors on human H2981 cells and inhibits the proliferation of human A375 tumor cells and mouse M1 leukemia cells. To identify activities regulated in vivo, we injected bovine oncostatin M fusion genes containing various tissue-specific promoters into mouse embryos. The frequencies of transgenic mice were reduced significantly, suggesting that overexpression of the bovine cytokine is detrimental to normal mouse development. In addition to deaths associated with expression in neurons and keratinized epithelia, bovine oncostatin M caused abnormalities in bone growth and spermatogenesis, stimulated fibrosis surrounding islets in the pancreas, and disrupted normal lymphoid tissue development. This work establishes the existence of a nonprimate oncostatin M gene and provides the first demonstration that this cytokine can function in a pleiotropic manner in vivo. Information regarding bovine oncostatin M may help characterize the structure and function of this cytokine in other vertebrate species.

• Pennica D et al.
• Cardiotrophin-1. Biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex.
• J Biol Chem. 1995; 270: 10915-22
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• Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and oncostatin M. These cytokines induce a pleiotropic set of growth and differentiation activities via receptors that use a common signaling subunit, gp130. In this work we determine the activity of CT-1 in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-1 is inactive in stimulating the growth of the hybridoma cell line, B9 and inhibits the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the differentiation of mouse embryonic stem cells. CT-1 and LIF cross-compete for binding to M1 cells, Kd [CT-1] approximately 0.7 nM, and this binding is inhibited by an anti-gp130 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M1 cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-1 binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-1 has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp130 signaling subunit.

• Tomida M
• Analysis of recombinant soluble mouse D-factor/LIF receptor.
• J Biochem (Tokyo). 1995; 117: 1228-31
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• The recombinant soluble mouse D-factor/LIF receptor (sD-factor-R) was expressed in COS-7 cells. Scatchard analysis of the bindings of mouse 125I-D-factor and human 125I-D-factor to the sD-factor-R indicated dissociation constants (Kd) of 12 and 0.267 nM, respectively, which were comparable to those of the binding protein in mouse serum. The apparent molecular masses of the sD-factor-R and human D-factor observed by gel filtration chromatography were 150 and 50 kDa, respectively. The size of the sD-factor-R.human D-factor complex was approximately 200 kDa, indicating that D-factor forms a 1:1 complex with the sD-factor-R. The sD-factor-R inhibited the induction of differentiation of mouse myeloid leukemic M1 cells by mouse D-factor by blocking the binding of this factor to the cells.

• Samal BB et al.
• High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.
• Biochim Biophys Acta. 1995; 1260: 27-34
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• LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

• Hocke GM, Cui MZ, Fey GH
• The LIF response element of the alpha 2 macroglobulin gene confers LIF-induced transcriptional activation in embryonal stem cells.
• Cytokine. 1995; 7: 491-502
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• Leukaemia Inhibitory Factor (LIF), an interleukin 6 (IL-6)-type cytokine, is an essential growth factor for murine embryonal stem cells. The LIF-receptor was known in these cells, but the cell-internal part of the signal cascade and the transcription factors through which LIF controls its growth-promoting target genes in embryonal stem cells, had not been identified. This study shows that the type II IL-6-response element of the rat alpha 2 macroglobulin (alpha 2M) gene, which mediates IL-6- and LIF-responses in hepatic cells, also functioned as a LIF-response element (LIF-RE) in ES1 embryonal stem cells and P19 embryonal carcinoma cells. It conferred transcriptional activation by LIF of transfected reporter constructs in these cells. A characteristic DNA-binding activity interacting with this LIF-RE was induced by treatment of these cells with LIF. The complex between this activity and the LIF-RE had identical electrophoretic mobility, sequence-specificity and kinetics of induction as the complex with the corresponding LIF-response factor (LIF-RF) from hepatic cells. The transcription factor STAT3 was part of this complex, as shown by its reactivity with anti-STAT3 antibodies. Withdrawal of LIF from ES1 cells caused the induction of differentiation and the disappearance of this DNA-binding activity. Simultaneously, the surface density of high-affinity LIF receptors was reduced approximately 10-fold.

• Rose TM, Weiford DM, Gunderson NL, Bruce AG
• Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro.
• Cytokine. 1994; 6: 48-54
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• Oncostatin M (OSM) is a cytokine which shares a common gene structure and amino acid sequence similarity with leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), suggesting evolution from a common ancestral gene. These four cytokines share several biological activities including the ability to induce the differentiation of the murine M1 myeloid leukemic cell line. To further define the functional similarities within this family, we have investigated whether OSM can substitute for LIF in the maintenance in vitro of the undifferentiated state of pluripotent embryonic stem (ES) cells. In this study, we demonstrate that human recombinant OSM is similar to LIF in its ability to inhibit the differentiation of MBL-5 murine ES cells cultured in vitro. The level of differentiation was determined by morphological criteria and by the continued expression of the embryonic stem cell-specific surface antigen defined by the ECMA-7 monoclonal antibody. Competition binding studies demonstrate that OSM binds to the LIF receptor on MBL-5 ES cells. Our results implicate OSM as a developmental regulatory factor for embryonic stem cells in vivo.

• Alexander HR, Billingsley KG, Block MI, Fraker DL
• D-factor/leukaemia inhibitory factor: evidence for its role as a mediator in acute and chronic inflammatory disease.
• Cytokine. 1994; 6: 589-96

• Maurer T et al.
• NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF). Comparison with human LIF.
• Growth Factors. 1994; 11: 271-6
• Display abstract

• Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine active on many cell types. We present here 1H NMR studies on the solution properties and stability of MH35, a chimera of murine and human LIF which can be expressed at high levels in Escherichia coli, thus enabling efficient labelling of the protein with the stable isotopes 13C and 15N. MH35 has 85% sequence identity with human LIF and similar activity in biological assays. The 1H chemical shifts of the 12 conserved aromatic residues and the pKa values of the five conserved histidine residues in MH35 and human LIF are very similar. Temperature dependence studies indicate that both proteins are stable, with significant conformational changes occurring only above 70 degrees C. These results show that these proteins have a similar overall structure and stability and that MH35 is therefore a suitable analogue of human LIF for structural studies in solution.

• Yoshida K et al.
• Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways.
• Mech Dev. 1994; 45: 163-71
• Display abstract

• Propagation of the undifferentiated pluripotential phenotype of embryonic stem (ES) cells is dependent on the cytokine differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). The DIA/LIF receptor complex is a heterodimer of DIA/LIF receptor (DIA/LIF-R) and gp130. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gp130 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gp130 alone, bypassing DIA/LIF-R, and therefore implicate gp130 as the key component in the signalling pathway responsible for stem cell renewal.

• Gearing DP
• The leukemia inhibitory factor and its receptor.
• Adv Immunol. 1993; 53: 31-58

• Escary JL, Perreau J, Dumenil D, Ezine S, Brulet P
• Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.
• Nature. 1993; 363: 361-4
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• Leukaemia inhibitory factor (LIF) has a variety of effects on different cell types in vitro, inhibiting the differentiation of embryonic stem cells and promoting the survival and/or proliferation of primitive haematopoietic precursors and primordial germ cells. Here we show that LIF-deficient mice derived by gene targeting techniques have dramatically decreased numbers of stem cells in spleen and bone marrow. Injection of spleen and marrow cells from these mice promotes long-term survival of lethally irradiated wild-type animals, however, showing that the LIF- stem cells remain pluripotent. The numbers of committed progenitors are also reduced in the spleen but not the bone marrow, suggesting that stem cells interact differently with the splenic and medullary microenvironment. Heterozygous animals are intermediate in phenotype, implying that LIF has a dosage effect, and defects in stem cell number can be compensated by exogenous LIF. LIF thus appears to be required for the survival of the normal pool of stem cells, but not their terminal differentiation.

• Nicola NA, Cross B, Simpson RJ
• The disulfide bond arrangement of leukemia inhibitory factor: homology to oncostatin M and structural implications.
• Biochem Biophys Res Commun. 1993; 190: 20-6
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• Murine leukemia inhibitory factor (LIF) (the fully active recombinant form produced in E. coli) was digested in the unreduced state with trypsin and Staphylococcal V8 protease in 0.05% sodium dodecyl sulfate. Disulfide-bonded peptides were identified by altered mobility on reverse-phase high-performance liquid chromatography in the presence or absence of dithiothreitol and subjected to amino acid sequencing. Peptides containing more than one disulfide bond were subjected to further proteolysis and disulfide-bonded subfragments identified and sequenced. The three disulfide bonds are CYS13-CYS135, CYS19-CYS132 and CYS61-164 and the first and third of these are clearly homologous to the two disulfide bonds in oncostatin M. The spatial organization of the cysteine residues in the predicted four alpha-helical bundle structure of LIF (Bazan, Neuron 7,197;1991) is compatible with these disulfide assignments.

• Conquet F, Peyrieras N, Tiret L, Brulet P
• Inhibited gastrulation in mouse embryos overexpressing the leukemia inhibitory factor.
• Proc Natl Acad Sci U S A. 1992; 89: 8195-9
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• Leukemia inhibitory factor (LIF) is a cytokine active in vitro on different target cells. It is detected in vivo during mouse gestation in both extraembryonic membranes and maternal tissues. Two isoforms have been described maintaining embryonic stem cells in culture in a pluripotent state. However, overexpression of their cDNAs in chimeric mouse embryos observed between 6.5 and 9.5 days postcoitus gave strikingly different phenotypes. Embryos overexpressing the diffusible form of LIF cDNA looked essentially normal. Chimerae expressing LIF associated with the extracellular matrix cDNA showed an abnormal proliferation of tissues and the absence of differentiated mesoderm. They have not undertaken the normal pathway of gastrulation.

• Smith AG, Nichols J, Robertson M, Rathjen PD
• Differentiation inhibiting activity (DIA/LIF) and mouse development.
• Dev Biol. 1992; 151: 339-51
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• Analysis of the differentiation in culture of murine embryonic stem (ES) cells has resulted in the identification and characterization of the regulatory factor differentiation inhibiting activity (DIA). DIA specifically suppresses differentiation of the pluripotential ES cells without compromise of their developmental potential. DIA is identical to the pleiotropic cytokine leukaemia inhibitory factor (LIF) which has a broad range of biological activities in vitro and in vivo. It is produced in both diffusible and matrix-localised forms whose expression is differentially regulated. The compartmentalization of DIA/LIF and the modulation of its expression during stem cell differentiation and by other cytokines may be significant elements in the control of early embryo development. These features may also indicate general principles of the regulatory networks which govern stem cell renewal and differentiation in later development.

• Fry RC
• The effect of leukaemia inhibitory factor (LIF) on embryogenesis.
• Reprod Fertil Dev. 1992; 4: 449-58
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• Leukaemia inhibitory factor (LIF) was originally identified as a haemopoetic factor that induced the differentiation of certain myeloid leukaemia cell lines. In contrast to this action, LIF was subsequently shown to inhibit the spontaneous differentiation of murine embryonic stem cells in culture, thus maintaining their pluripotency and ability to contribute to the germline of chimaeric mice. In the mouse, mRNA for LIF is expressed by the endometrial glands of the uterus coincident with the time of blastocyst implantation and receptors have been found on the preimplantation blastocyst. The signal for LIF expression appears to be of maternal origin, perhaps regulated by oestradiol. Recombinant LIF improves the development of murine and ovine blastocysts in culture although there is some species specificity with respect to the type of LIF that is bioactive. It is proposed here that LIF acts on the trophectoderm of the rapidly expanding blastocyst and improves the implantation rate of otherwise compromised embryos. Further studies in livestock should elicit therapeutic uses for LIF in embryo culture, embryo transfer and embryo survival in vivo.

• Gearing DP et al.
• Reconstitution of high affinity leukaemia inhibitory factor (LIF) receptors in haemopoietic cells transfected with the cloned human LIF receptor.
• Ciba Found Symp. 1992; 167: 245-55
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• cDNA clones encoding the human leukaemia inhibitory factor (hLIF) receptor were isolated by screening a placental cDNA expression library in COS-7 cells with 125I-hLIF. The cloned LIF receptor is a member of the haemopoietin receptor family and comprises a signal sequence (44 amino acids), an extracellular region of two haemopoietin receptor domains and three fibronectin type III domains (789 amino acids), a transmembrane domain (26 amino acids) and a cytoplasmic domain (238 amino acids). The LIF receptor is expressed in COS-7 cells as a 190 kDa glycoprotein that specifically binds human LIF with low affinity, but does not bind mouse LIF. Clones encoding a soluble form of the homologous mouse LIF receptor have been isolated, suggesting complex interactions between the various forms of LIF ligand and receptor in vivo. The LIF receptor is most related to the gp130 signal-transducing component of the IL-6 receptor, a feature that may provide a molecular basis for the intertwined biologies of LIF and IL-6 in the absence of obvious structural similarly between the ligands. Mouse B9 plasmacytoma cells transfected with the human LIF receptor display novel high affinity LIF receptors that are presumed to consist of transfected receptors in association with endogenous mouse high affinity-converting subunits. Unlike the low affinity human LIF receptor, the mixed species high affinity receptor is capable of binding mouse LIF.

• Brown GS, Brown MA, Hilton D, Gough NM, Sleigh MJ
• Inhibition of differentiation in a murine F9 embryonal carcinoma cell subline by leukemia inhibitory factor (LIF).
• Growth Factors. 1992; 7: 41-52
• Display abstract

• Leukemia inhibitory factor (LIF) is a cytokine previously shown to maintain pluripotent embryonic stem cells in their undifferentiated state. We have examined the effects of LIF in nullipotent embryonal carcinoma cell lines, and have found that LIF blocks differentiation induced by retinoic acid and at low temperature in OTF9 cells. LIF did not block differentiation in a parent F9 cell line. For OTF9 cells, LIF acts early in differentiation, inhibiting the appearance of parietal endoderm-type product cells. However, it acts subsequent to retinoic acid, and at least one early retinoic acid-induced event is unaltered in the presence of LIF. This finding provides both a means of dissecting the cascade of events leading to EC cell differentiation, and a well-characterised target cell type for studying the mechanism of action of LIF.

• Layton MJ, Cross BA, Metcalf D, Ward LD, Simpson RJ, Nicola NA
• A major binding protein for leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor.
• Proc Natl Acad Sci U S A. 1992; 89: 8616-20
• Display abstract

• A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (Kd = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the alpha chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 microgram/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

• Van Vlasselaer P
• Leukemia inhibitory factor (LIF): a growth factor with pleiotropic effects on bone biology.
• Prog Growth Factor Res. 1992; 4: 337-53
• Display abstract

• Historically, growth factors are denominated based on a specific biological activity. In many cases, these factors display a much broader spectrum of activities, especially when their effect is tested on various cell or tissue types. Consequently, names of certain factors are quite deceptive. A textbook example is leukemia inhibitory factor (LIF). LIF was initially described based on its ability to induce differentiation in the murine myeloid leukemia cell line M1. Later, LIF turned out to be a synonym for at least nine different factors defined on the basis of their effects on a variety of cell types including lymphomas, liver cells, embryonic stem cells and carcinoma cells, neurons, melanomas and osteoclasts. Apart from its differential effect on unrelated cell types and tissues. LIF induces biphasic effects on cells of the same "lineage" as well. Needless to say, LIF activity in these circumstances largely depends on the developmental stage of the target cells. An example is LIF activity on bone cells. Osteoclast as well as osteoblast activity is stimulated or suppressed by LIF depending on the developmental stage of the respective cells. This concept is of utmost importance in the evaluation of the seemingly opposing or contradictory effects of LIF in vitro as well as in vivo.

• Gearing DP, Bruce AG
• Oncostatin M binds the high-affinity leukemia inhibitory factor receptor.
• New Biol. 1992; 4: 61-5
• Display abstract

• Oncostatin M (OSM) is a glycoprotein cytokine that was recently demonstrated to be structurally and functionally related to the leukemia inhibitory factor (LIF). We have investigated the binding of each cytokine to a variety of cellular receptors including those on solid tumor lines, leukemic cells, endothelial cells, macrophages, and cells transfected with the recently cloned low-affinity LIF receptor, and to a soluble form of the LIF receptor. LIF is incapable of binding either high- or low-affinity OSM receptors, yet OSM is capable of binding the high-affinity but not the low-affinity LIF receptor. Since the presence of high-affinity LIF receptors correlates with the biological activity of LIF on a wide range of target cells, we predict that OSM should have similar effects on LIF-responsive cells.

• Tomida M
• [Function, molecular structure and gene expression regulation of receptor for D-factor/LIF]
• Nippon Rinsho. 1992; 50: 1956-61
• Display abstract

• Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) is a cytokine inducing differentiation of mouse myeloid leukemic M1 cells. IL-6, oncostatin M (OSM) and G-CSF also induce differentiation of M1 cells. These four cytokines are suggested to be members of a single cytokine family. The LIF receptor is structurally related to the gp130 signal-transducing component of the IL-6 receptor and to the G-CSF receptor. The high-affinity receptors for LIF, OSM and IL-6 share the common subunit, gp130. This provides an explanation for the functional redundancy of those cytokines.

• Metcalf D
• The leukemia inhibitory factor (LIF).
• Int J Cell Cloning. 1991; 9: 95-108
• Display abstract

• Leukemia inhibitory factor (LIF) is a glycoprotein able to enforce differentiation and/or suppress clonogenic self-renewal in a number of myeloid leukemic cell lines. When acting on normal embryonic stem cells, it has the opposite action of preventing differentiation commitment. LIF is not a proliferative factor when acting alone on normal hemopoietic cells, but can potentiate the action of interleukin 3 on blast cell and megakaryocyte precursors. When injected in vivo, LIF stimulates rises in megakaryocyte numbers and platelet levels. LIF also exhibits striking functional effects on a wide range of other cells including hepatic parenchymal cells, neurones, adipocytes, osteoblasts and gonadal cells. The polyfunctionality of LIF suggests strongly that it is normally intended to be produced locally and act as a local regulator. Despite its wide range of actions, LIF remains a promising candidate for clinical use in thrombocytopenia and myeloid leukemia.

• Nichols J, Evans EP, Smith AG
• Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity.
• Development. 1990; 110: 1341-8
• Display abstract

• The regulatory factor Differentiation Inhibiting Activity/Leukaemia Inhibitory Factor (DIA/LIF) suppresses the differentiation of cultured embryonic stem (ES) cells. In the present study, it is shown that ES cell lines can be derived and maintained in the absence of feeder layers using medium supplemented with purified DIA/LIF. These cells can differentiate normally in vitro and in vivo and they retain the capacity for germ-line transmission. DIA/LIF therefore fulfils the essential function of feeders in the isolation of pluripotential stem cells.

• Heath JK, Smith AG, Hsu LW, Rathjen PD
• Growth and differentiation factors of pluripotential stem cells.
• J Cell Sci Suppl. 1990; 13: 75-85
• Display abstract

• The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.

• Hozumi M et al.
• Protein factors that regulate the growth and differentiation of mouse myeloid leukaemia cells.
• Ciba Found Symp. 1990; 148: 25-33
• Display abstract

• We have purified and characterized several protein factors that regulate the growth and differentiation of mouse myeloid leukaemia M1 cells. The differentiation factor (D-factor) from conditioned medium (CM) of Ehrlich ascites tumour cells is a glycoprotein of Mr 40,000-50,000. Its amino acid sequence was found to be almost identical to that of leukaemia inhibitory factor (LIF) from Krebs II ascites cells. The differentiation inhibitory factor (I-factor) from the CM of variant M1 cell clones which were resistant to several differentiation inducers is a basic protein of apparent Mr 68,000. The growth inhibitory factor (GI-factor) that specifically inhibits the partially differentiated and still growing monocytic leukaemia M1 cells was isolated from the CM of a clone of M1 cells resistant to the differentiation inducers. This GI-factor is a basic protein with an Mr of 25,000. Regulation by these protein factors together with other known cytokines of growth and differentiation of M1 cells is reported.

• Yamamori T, Fukada K, Aebersold R, Korsching S, Fann MJ, Patterson PH
• The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor.
• Science. 1989; 246: 1412-6
• Display abstract

• A protein secreted by cultured rat heart cells can direct the choice of neurotransmitter phenotype made by cultured rat sympathetic neurons. Structural analysis and biological assays demonstrated that this protein is identical to a protein that regulates the growth and differentiation of embryonic stem cells and myeloid cells, and that stimulates bone remodeling and acute-phase protein synthesis in hepatocytes. This protein has been termed D factor, DIA, DIF, DRF, HSFIII, and LIF. Thus, this cytokine, like IL-6 and TGF beta, regulates growth and differentiation in the embryo and in the adult in many tissues, now including the nervous system.

• Gough NM, Williams RL
• The pleiotropic actions of leukemia inhibitory factor.
• Cancer Cells. 1989; 1: 77-80
• Display abstract

• Leukemia inhibitory factor (LIF) is a glycoprotein regulator that induces the differentiation and suppresses the clonogenicity of M1 myeloid leukemic cells. M1 cells require only a brief exposure to LIF to become irreversibly committed to differentiation. In combination with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, LIF is also able to suppress HL-60 and U937 cell proliferation. Normal monocytes and macrophages have LIF receptors, but the function of LIF on such cells is unclear. Recently it was discovered that LIF also acts on embryonic stem (ES) cells, where its continuous presence is required to prevent ES cell differentiation.

• Williams RL et al.
• Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.
• Nature. 1988; 336: 684-7
• Display abstract

• Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.

• Moreau JF, Donaldson DD, Bennett F, Witek-Giannotti J, Clark SC, Wong GG
• Leukaemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells.
• Nature. 1988; 336: 690-2
• Display abstract

• Leukaemia inhibitory factor (LIF) is a cytokine that induces macrophage differentiation of the murine M1 myeloid leukaemia cell line. We have isolated a cDNA clone encoding a novel human haemopoietic growth factor, human interleukin for DA cells (HILDA) that supports the proliferation of the murine interleukin-3-dependent leukaemic cell line, DA-la (refs 3-5). HILDA proved to be identical to LIF. The demonstration that the differentiation factor LIF will also serve as a growth factor for at least one myeloid leukaemic cell line provides further evidence that the distinction between growth-promoting and differentiation-inducing activities are largely determined by the target cell type.

• Gearing DP et al.
• Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).
• EMBO J. 1987; 6: 3995-4002
• Display abstract

• Leukaemia inhibitory factor (LIF) can induce macrophage differentiation in M1 murine myeloid leukaemic cells and suppress their proliferation in vitro. It does not stimulate the proliferation of normal progenitor cells and is apparently distinct from known colony-stimulating factors. We have used oligo-nucleotides complementary to partial amino acid sequence of LIF to isolate a LIF clone from a T lymphocyte cDNA library. When this cDNA was coupled to a yeast expression vector (YEpsec1) and introduced into yeast cells, a molecule with the biological properties characteristic of native LIF was secreted into the growth medium. The amino acid sequence of LIF established it to be a unique molecular entity, distinct from the other known haemopoietic growth factors. Since LIF is encoded by a unique gene, two biochemically separable forms of LIF probably represent post-transcriptional or posttranslational variants of the same gene product. In contrast to several other haemopoietic regulators, the 0.8- to 1-kb LIF mRNA was expressed constitutively in two murine T lymphocyte cell lines examined, and its abundance was not enhanced by stimulation with concanavalin A. Cloning, sequencing and expressing LIF has resolved several discrepancies in the literature concerning the identity of factors capable of inducing differentiation of murine myeloid leukaemic cells in vitro.

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