EP0463004A1 - Metabolic effects of leukaemia inhibitory factor on bone - Google Patents
Metabolic effects of leukaemia inhibitory factor on boneInfo
- Publication number
- EP0463004A1 EP0463004A1 EP90904185A EP90904185A EP0463004A1 EP 0463004 A1 EP0463004 A1 EP 0463004A1 EP 90904185 A EP90904185 A EP 90904185A EP 90904185 A EP90904185 A EP 90904185A EP 0463004 A1 EP0463004 A1 EP 0463004A1
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- European Patent Office
- Prior art keywords
- lif
- bone
- cells
- animal
- polypeptides
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2093—Leukaemia inhibitory factor [LIF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
- A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates generally to the effect of leukaemia inhibitory factor (LIF) on bone in animals. More particularly, the present invention relates to the use of LIF and/or LIF-like polypeptides to induce, promote and/or enhance metabolic effects on bone in animals.
- LIF leukaemia inhibitory factor
- the cytokine, leukaemia inhibitory factor (LIF) is a protein that has previously been purified, cloned and produced in large quantities in purified recombinant form from both Escherichia coli and yeast cells
- LIF was originally isolated on the basis of its capacity to induce differentiation in and suppress the murine myeloid leukaemic cell line. M1. LIF has been shown to have a powerful differentiation suppressing action on normal embryonic stem cells. LIF has no apparent
- LIF receptors were detected on cells of the monocyte-macrophage lineage.
- OAF osteosteoclast activating factor'
- IL-1 interleukin 1
- TNF ⁇ tumour necrosis factors alpha and beta
- LIF has no significant structural or sequence homology with the aforementioned cytokines.
- LIF has a metabolic effect on bone by inducing, promoting and/or enhancing bone resorption and/or bone formation.
- LIF is an important paracrine regulator in bone and exerts a number of specific biochemical actions on osteoblasts. These include inhibition of plasminogen activator (PA) activity in osteoblasts, most likely through increased synthesis of PA inhibitor-1 formation. Furthermore, LIF itself is expressed in osteoblastic cells and primary osteoblasts. Thus several lines of evidence establish cells of the osteoblast lineage as targets for LIF action in a paracrine or autocrine manner.
- PA plasminogen activator
- one aspect of the present invention relates to a method of inducing, promoting and/or
- LIF leukaemia inhibitory factor
- Another aspect of the present invention is directed to a pharmaceutical composition useful in inducing, promoting and/or enhancing metabolic effects on bone in animals comprising an effective amount of
- LIF leukaemia inhibitory factor
- polypeptides in combination with one or more other molecules such as cytokines, inorganic or organic
- Still yet another aspect of the present invention contemplates the use of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides in the manufacture of a medicament useful in the induction, promotion and/or enhancement of metabolic effects on bone in animals.
- LIF leukaemia inhibitory factor
- LIF leukaemia inhibitory factor
- the animal is mammalian or avian and more preferably is human.
- GM-CSF Granulocyte-macrophage colony-stimulating factor PBS Phosphate buffered saline
- HEPES 4-(2-Hydroxyethyl)-1- piperazinoethanesulfonic acid The present invention arose, in part, from an analysis of the effects of LIF on certain responses in osteoblast-like cells and its effects on bone resorption in calvariae in organ culture. Furthermore, to test the effects of LIF in vivo and to delineate LIF's
- FDC-P1 continuous hemopoietic cell line FDC-P1
- FD continuous hemopoietic cell line
- retrovirus containing the gene which encodes LIF cloned sublines were developed that constitutively produce high levels of LIF and such cells were injected into mice to generate animals containing a continuous source of LIF-producing cells. Upon analysis of these animals,
- LIF is an important regulator of bone cell function causing the induction, promotion and/or enhancement of such activities as bone resorption and/or bone formation.
- the in vivo exemplified aspects of the present invention are performed using a mouse model. This is done, however, with the understanding that the present invention extends to the effect of LIF on bone in all animals.
- the animals are mammalian or avian but more preferably mammalian.
- the present invention is directed to humans. Accordingly, one aspect of the present invention relates to a method of inducing, promoting and/or
- LIF and/or LIF-like polypeptides comprising administering to said animal an effective amount of LIF and/or LIF-like polypeptides alone or in combination with one or more other cytokines or one or more active substances, for a time and under conditions sufficient to effect bone anabolism.
- Metal effects include, although are not necessarily limited to, such processes as bone formation and/or bone resorption, increased protein synthesis in bone tissue and increased thymidine uptake in bone tissue.
- metabolism effects also encompasses anabolic and/or catabolic activities of LIF on bone.
- LIF and/or LIF-like polypeptides is meant derivatives and homologues of LIF and extends to the entire natural length LIF molecule or derivatives thereof carrying single or multiple amino acid substitutions, deletions and/or additions and includes substitution, deletion and/or addition of any other molecules
- LIF-like polypeptides extend to molecules having substantially similar activity as LIF while carrying amino acid rearrangements or alterations.
- LIF encompasses LIF-like polypeptides and vice verca.
- the present invention extends to naturally occurring but substantially pure LIF (i.e. greater than or equal to 70% by weight of LIF relative to other proteins or molecules and preferably greater than or equal to 85% and even more preferably greater than or equal to 90%), to recombinant LIF and to synthetic LIF made, for example, by chemical means.
- naturally occurring but substantially pure LIF i.e. greater than or equal to 70% by weight of LIF relative to other proteins or molecules and preferably greater than or equal to 85% and even more preferably greater than or equal to 90%
- synthetic LIF made, for example, by chemical means.
- LIF may be from a range of sources such as, but not limited to, human, mouse, dog, cow, pig, sheep or other ruminant. In some cases it will be preferable to use homologous LIF to the animal being treated, for example, human LIF on humans. But in other circumstances, heterologous LIF may be more convenient and/or more effective. The choice of source of LIF may depend on the exigency of the treatment required or the animal requiring treatment.
- LIF and/or LIF-like polypeptides are used alone. In another embodiment, they are used in a combination with one or more other cytokines, such as, but not limited to, IL-1, TNF ⁇ and/or TNF ⁇ , and which affect various aspects of bone resorption, and/or formation.
- cytokines such as, but not limited to, IL-1, TNF ⁇ and/or TNF ⁇
- the present invention also extends to the use of LIF and/or LIF-like polypeptides in combination with other active molecules such as agonists, antagonists, calcium or any inorganic or organic molecule which aids in the bone resorbing and/or forming process either directly or by enhancing the effect of LIF or LIF-like polypeptides.
- haemopoiesis in the spleen and liver haemopoiesis in the spleen and liver.
- Osteoblasts express receptors for LIF (27) and LIF is known to release calcium in vitro from bone tissue (Reid et al. Personal Communication). It is conceivable, therefore, that the osteoblast changes were due to direct actions of LIF which , if so, would suggest that LIF may be capable of influencing osteoblast precursor migration , osteoblast proliferation and bone formation. A well-documented association exists between osteoblast and osteoclast activity but, since osteoclasts lack LIF receptors (27), their altered behaviour may be secondary to changes in osteoblast activity. Although the perturbation in bone biology resulted in excess bone formation, many mice can be assumed to have had excess calcium levels because of calcium deposits particularly in muscle and liver tissue.
- LIF may, under appropriate conditions and requisite time, induce bone formation and may be extremely useful in the repair of fractured bones and to stimulate bone growth and should be particularly useful in the treatment of injuries and/or diseases where bone degeneration or fracture is a symptom, or a result, such as in increasing bone
- osteoporosis such as in post-menopausal osteoporosis, or in stimulating bone fracture repair.
- LIF LIF
- Administration of LIF will be by standard procedures, for example, intravenous, interperitoneal, intramuscular, or topically to the extent that it is applied to the
- LIF LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes.
- LIF LIF or its pharmaceutical composition comprising LIF or LIF-like polypeptides are encompassed by the present invention.
- the present invention modifications to LIF or its pharmaceutical composition comprising LIF or LIF-like polypeptides are encompassed by the present invention.
- modifications may comprise adding protective compounds (e.g. enzyme inhibitors) to pharmaceutical compositions.
- protective compounds e.g. enzyme inhibitors
- the effective amount of LIF or LIF-like polypeptides required in pharmaceutical compositions or in vivo to induce metabolic effects will vary according to the animal and condition being treated. For example, an effective amount of from 0.01 to 1 million units may be necessary but in some circumstances a range of 0.1 to 10,000 units may be required. In other circumstances it may be propitious to administer LIF at 0.01 ⁇ g to 100,000 mg per kg of body weight of animal. Another preferred range may be 0.1 jug to 10,000 mg of LIF or LIF-like polypeptides per kg of body weight of animal.
- the aforementioned effective amounts may, depending on the situation, be per hour or per day or possibly per week or month. Administration may be single or multiple application. Accordingly, "effective amount” refers to the amount necessary to give the desired effect.
- compositions useful in the disclosure are:
- induction of bone formation or increasing bone resorption comprising a bone formation-inducing or bone resorption-effective amount of LIF and/or LIF-like polypeptides may be prepared according to standard practices.
- the present invention extends to the use of LIF and/or LIF-like polypeptides in the manufacture of a medicament for the induction, promotion and/or
- the present invention contemplates the use of LIF and/or LIF-like polypeptides in the manufacture of a medicament for treating injuries and/or diseases in animals, and in particular humans, where bone degeneration or fracture is a symptom or a result such as in osteoporosis or bone fracture.
- the present invention extends to parts or derivatives of LIF and/or LIF-like polypeptides defined by their ability to interact with osteoblasts.
- LIF interaction may comprise the binding of the LIF fragment to a LIF receptor on an osteoblast cell.
- LIF and/or LIF-like polypeptides would be isolated by any number of means such as chemically treating the LIF molecule, isolating cleaved or altered sections and testing whether the sectioned or fragmented LIF has activity in bioassays as described in
- LIF-like polypeptides extends to fragments or parts of LIF capable of interacting with osteoblasts.
- osteoblasts Several bone-resorbing hormones stimulate PA activity in osteoblast-like cells in culture. In order to localize the proteolytic activity of the PA-plasmin system at sites where it is required, precise regulatory control is needed. This is provided by the stimulating hormones and paracrine factors and also by PA inhibitors (PAIs), which bind to and limit the activity of both tissue-type PA (tPA) and urokinase.
- PAIs PA inhibitors
- PAI-1 and PAI-2 which bind to and inactivate PA, are the products of separate genes.
- LIF is shown to enhance production of PAI-1 protein and mRNA, an effect which is most likely
- LIF LIF
- LIF lin-1-phosphate phosphatase
- TGF ⁇ alkaline phosphatase
- LIF exhibits a regulatory effect on the PA-PA inhibitor system, further evidencing the potential of LIF as an important regulator in the microenvironment of bone cells and, therefore, important in bone remodelling.
- the effect of LIF on PA does not appear to be acting by the generation of TGF ⁇ .
- the present invention is also described by the following non-limiting Figures and Examples. Although the Examples use mice as the animal model, the techniques used and results obtained will be equally applicable to other animals including avian and mammalian (e.g. human) species, and, hence, the present invention encompasses the use of LIF in inducing, promoting and/or enhancing bone formation and resorption and other metabolic
- Figure 1 is a graphical representation showing survival of mice, with or without exposure to radiation, following injection of FD cells expressing LIF.
- Figure 2 is a graphical representation showing serum levels of LIF in irradiated and non-irradiated mice following injection of FD cells and FD cells expressing LIF.
- Figure 3 is a pictorial representation of the femur from mouse injected with FD cells (A) and FD/LIF cells (B). Note the excess new bone formation and abnormal trabeculae in FD/LIF recipient.
- Figure 6 is a graphical representation showing that the increase in bone resorption is associated with an increase in osteoclast numbers in the calvaria.
- Figure 7 is a graphical representation showing the ability of 10 -7 M indomethacin to block LIF stimulated bone resorption.
- Figure 8 is a graphical representation showing that [ 3 H]-thymidine incorporation into the calvaria is stimulated by LIF.
- Figure 9 is a graphical representation showing that stimulation by LIF of [ 3 H]-thymidine incorporation into the calvaria is not blocked by the presence of indomethacin.
- Figure 10 is a graphical representation showing PA activity in calvarial osteoblasts (A) and UMR 106-01 cells (B).
- UMR 106-01 cells were incubated in the presence ( ⁇ ) and absence (o) of a submaximal (20ng/ml) dose of PTH. Results are the means ⁇ SEM of triplicate determinations.
- Figure 11 is a photographic respresentation showing (A) Time courses of PAI-1 mRNA levels following treatment of UMR 106-01 cells with LIF (1000 U/ml).
- Figure 12 is a photographic representation of an autoradiograph of a Northern blot showing the effect of LIF on mRNA for ⁇ 1(I) collagen (A) and osteonectin (B) in UMR201 cells.
- the 6 lanes on the left in each case are the LIF treatment samples.
- the cells were incubated with LIF for 24 hours in 2% v/v uv irradiated fetal calf serum.
- An aliquot of 20 ⁇ g total RNA was electrophoresed as described in Example 5.
- Figure 13 is a photographic representation showing autoradiographs of cells from newborn rat long bones labelled with 125 I-LIF. Specifically, the photograph shows osteoblasts and an osteoclast in the absence of
- Figure 14 is a diagrammatic representation showing (A) saturation binding isotherm of total (o), specific
- Figure 15 is a photographic representation showing the detection of LIF transcripts in osteoblastic cells.
- Equivalent amounts of RNA from UMR 106-06, UMR 201 or osteoblast-rich primary cultures (1°Ca) were probed for LIF or GAP-DH transcripts (as a positive control) by PCR amplification as described in Example 5.
- the negative control track represents a "no cell" sample carried in parallel through RNA extraction, cDNA synthesis and PCR reaction.
- the + and - symbols refer to treatment with or without TGF ⁇ (2ng/ml) for the number of hours indicated.
- mice that had previously been given 6 Gy whole-body irradiation began to show evidence of weight loss, ruffled hair and restricted movement. Within days, such mice became moribund and by 28 days after injection 80% of these mice had become moribund (Figure 1). Irradiated mice injected with control FD cells remained in good health during this period. Unirradiated mice injected with FD/LIF cells developed an identical clinical syndrome but with a delayed onset. Unirradiated mice first became moribund 30 days after injection but
- mice exhibited circulatory collapse and a reduced
- mice injected with FD/LIF cells exhibited moderate spleen enlargement that was more evident in TABLE 2
- Marrow cells were not able to be recovered from 10 of 19 irradiated and 11 of 19 unirradiated mice.
- mice injected with FD/LIF cells showed a remarkable overgrowth of bone, seen as thickening of the bone cortex and the formation of
- hemopoietic tissue unirradiated and irradiated recipients and was depleted of hemopoietic cells, those remaining being mainly granulocytic cells although in some but not all marrows, megakaryocytes were present. Replacing the hemopoietic tissue,
- osteoblasts particularly in the ends of the long bones were elongated cells oriented in parallel bundles. These cells were enlarged close to bone surface and merged with the developed bone. From the calcification evident in the cytoplasm of these cells, they appeared to be osteoblasts but, although the total number seemed well in excess of the number that could have been present in the normal marrow cavity, no obvious migration was seen through bone foramina. In some bones, osteoclasts were prominent and adjacent bone had irregular scalloped edges. Irregular foramina were also present in many bones particularly at the ends of long bones. These could have been the consequence of increased osteoclastic activity. The open channels between the marrow and the tissues outside the bone could have allowed free entry of osteoblast precursors or exit of marrow cells.
- the red pulp of the spleen was enlarged and was packed with hemopoietic cells, the lymphoid follicles being severely depleted of cells.
- hemopoietic cells In many livers, there were also prominent foci of hemopoietic cells and some livers showed areas of necrosis and/or calcification. Less commonly, cirrhotic changes were present but there was no mitotic activity in parenchymal cells and there was consistent reduction in the volume of individual parenchymal cells. Areas of calcification were seen in striated muscle and the heart in some but not all animals but no foci of cellular infiltration were observed in these locations. In several mice, prominent macroscopic areas of calcification were visible in the liver.
- mice injected with FD cells showed none of the above pathological changes and the only condition evident in some of these mice was the presence of small focal
- Bone resorption was measured as the release of 45 Ca from bones which had been pre-labelled in vivo. Hemicalvaria were dissected as previously described (3) and preincubated for 24 hours in minimal essential media plus 1% v/v fetal calf serum. Bones were then changed to media containing the experimental compounds and incubated for a further 48 hours, at the end of which time media and bone were harvested for measurement of 45 Ca content.
- Calvarial osteoblasts and UMR 106-01 cells were subcultured onto 125-I fibrin coated plates as described in the Methods section. Cells were preincubated for 8 h in serum free conditions as described in the legend in Table 4. The cells were incubated in the presence and absence of LIF (1000 U/ml ) and indomethacin (1.5 x 10 -5 M ).
- LIF has significant effects on [ 3 H]-thymidine incorporation in neonatal mouse calvaria even at doses as low as 100 units/ml (Table 4) .
- calvaria Its action on thymidine incorporation probably reflects responses of osteoblasts, and does not appear to involve local prostaglandin production since it is
- LIF may be an important regulator of bone cell function.
- PA plasminogen activator
- Synthetic human parathyroid hormone (PTH) (1-34) was from Beckman Pty. Ltd., Palo Alto, CA, U.S.A., while human recombinant TGF ⁇ , TNF ⁇ and TNF ⁇ , and full-length rat actin cDNA were gifts from Genentech Inc., San Francisco, CA, U.S.A. 1,25-dihydroxyvitamin D 3 was generously provided by Dr. M. Uskokovich, Hoffman La Roche Inc., New Jersey, and
- prostaglandin E 2 was purchased from Upjohn Company,
- Rabbit antiserum against PAI-2 prepared form U937 cells, was by courtesy of Professor E.K.O. Kruithof, Centre Hospitaller Universitaire, Vaudois, Lausanne, Switzerland.
- Rabbit anti-rat hepatoma cell (HTC)PAI-1 (S) and rat PAI-1 cDNA cloned into the EcoRl site of pBluescript SK(-)(6) were kindly provided by Drs. R. Zeheb and T.D. Gelehrter,
- the cDNA probe for IGF ⁇ was provided by Genentech, San Francisco.
- the cDNA probe for murine LIF was the plasmid pLIF7.2b which spans the coding region of the murine LIF mRNA.
- 125 I-fibrinogen, [ ⁇ 32 P]UTP, [ ⁇ 32 P]CTP and a T7/SP6 paired promoter system were from Amersham International, T 3 RNA polymerase was from
- SDS-PAGE M r standards were from Bio-Rad Laboratories, Richmond, CA 94804, U.S.A.
- the UMR 106-01 and UMR106-06 cells are a subclone of the UMR106 cells which are a clonal line of rat
- osteogenic sarcoma cells in which many features of the osteoblast phenotype are preserved (1, 8, 9).
- the UMR201 clonal cell line which was derived from nerborn rat calvaria (10), has properties consistent with those of an osteoblast precursor. Treatment with retinoic acid leads to substantial increase in alkaline phosphatase activity and mRNA, and the cells produce predominantly type I collagen and have other osteoblastic features (10, 11).
- Osteoblast-rich calvarial cells were prepared as described previously (7, 8) and were used on the first subculture. Conditions of culture maintenance for UMR201 and osteoblast-rich cells were as described for osteogenic sarcoma cells.
- Osteoclasts were isolated from newborn long rat bones as previously described (24), and allowed to settle onto glass coverslips for periods of greater than 30 mins before washing, to ensure significant contamination with osteoblasts (25). (iii) RNA Preparation and Hybridization
- RNA filters After removing DNA bound to the RNA by boiling for 5 min in 0.1 x SSC, 0.1% w/v SDS filters were probed a second time with a nick-translated full-length rat actin cDNA probe. The filters were washed to a stringency of 0.1 x SSC, 0.1% w/v SDS at 42oC and exposed as above. Similar hybridization conditions were used in detecting mRNA for osteonectin and type I collagen. In each case the probes were labelled by nick translation.
- Alkaline Phosphatase Cells were subcultured at 20,000/200 ⁇ 1 into 125 I-fibrin-coated wells of 96 well tissue culture plates (13). After 24 hours in maintenance medium the cells were washed twice with Dulbecco's PBS and the medium was replaced with MEM and 0.1% w/v BSA. After 8 hours the medium was replaced with fresh Eagle's MEM and 0.1% w/v BSA together with human plasminogen (1.56 ug/ml). Plasminogen-independent activity was measured in parallel in the absence of added human plasminogen. After 24 hours an aliquot of medium was removed and counted for solubilized radioactivity.
- Plasminogen dependent fibrinolytic activity is expressed as percentage of total radioactivity released from the plate by trypsin.
- UMR 201 cells were subcultured into 9.6cm 2 six-plate multiwell dishes in alpha modified MEM ( ⁇ -MEM)
- UMR 106-01 and 106-06 cells were subcultured, centrifuged in Hanks' BSS and kept on ice after which cells were resuspended and layered over 180 ⁇ l fetal calf serum (FCS) in tapered flexible plastic tubes. Cell-associated and free 125 I-LIF were separated by centrifugation of the cells through FCS. The tip of the tube containing the cell pellet was cut off and the pellet and supernatant were counted in a ⁇ -counter (Packard Crystal Multi-Detecter,
- Cells were subcultured at 500,000 cells/well (9.6 cm 2 ) in MEM and 5% v/v FCS, After 25 h the medium was aspirated, cells washed twice with PBS and the medium replaced with 1 ml MEM and 0.1% w/v BSA. After 8 h the medium was aspirated and 1 ml of fresh MEM and 0.1% w/v BSA added together with treatment. At 25 h the medium was aspirated and centrifuged to remove any cell debris. The cells were washed twice with PBS and lysed in 1ml. 0.1% v/v triton X 100.
- CM Conditioned medium
- cell extracts prepared as described above, were resolved by SDS-PAGE on 10% w/v slab gels under non-reducing conditions as described (16). PA and PAI activity were visualized by fibrin (17) and reverse fibrin (18) gel autograph, respectively.
- 106-01 cells and calvarial osteoblasts incubated for 24 h in serum-free MEM were concentrated by ultrafiltration with a 30,000 M r cutoff, while cells were washed twice with PBS, scraped, centrifuged and lysed in 0.1% v/v triton X 100.
- the proteins were resolved by SDS-PAGE as above and
- PAI-1 was detected by incubation of the nitrocellulose with rabbit polyclonal anti-rat HTC PAI-1 (5) followed by anti-rabbit Ig conjugated with alkaline phosphatase and visualization with 5' bromo-4-chloro-3-indolyl phosphate (19).
- each dNTP contained 200 ⁇ M of each dNTP, 1 ⁇ m of each specific primer, buffer as supplied in the GeneAmp kit (Cetus Corp., USA) and 1.25 units Taq polymerase.
- the primers used for PCR contained 200 ⁇ M of each dNTP, 1 ⁇ m of each specific primer, buffer as supplied in the GeneAmp kit (Cetus Corp., USA) and 1.25 units Taq polymerase.
- CTCAGTGTAGCCCAGGATGC defining a 500 bp fragment.
- the PCR reaction conditions were: 1.5 min at 94°C; 2 min at 60°C; 3 min at 72°C for 25 cycles in a Perkin-Elmer-Cetus DNA
- the hybridization probes were the gel-purified cDNA inserts of plasmids pLIF7.2b and hGAP-DH, radiolabeled to a specific activity of approximately 2x10 8 cpm/ug by nick translation and included in the hybridization at approximately 2x10 7 cpm/ml.
- FIG. 11 shows a time course up to 24 hours of the PAI-1 mRNA with response to LIF. As early as 1 hour after the beginning of LIF treatment there was a clear increase in PAI-1 mRNA. This effect of LIF on inhibition of PA activity and promotion of PAI-1 mRNA is very similar to the effect of transforming growth factor ⁇ (TGF ⁇ ) in the same cells. To exclude the possibility that LIF might be acting through the generation of TGF ⁇ in the cells, two experiments were carried out. In the first, an antibody against TGF ⁇ was used to determine whether it blocked the action of LIF.
- TGF ⁇ transforming growth factor ⁇
- TGF ⁇ (0.5 ng/ml) 3.2 ⁇ 0.1
- LIF tumour necrosis factor ⁇
- LIF The effect of LIF on the osteoblast-like cells is largely anabolic, in that there is clear evidence of a LIF effect to increase synthesis of some of the important specific components of bone protein, and a regulatory effect on the PA-PA inhibitor system, which give LIF the potential of being an important regulator in the microenvironment of bone cells, and therefore important in bone remodelling.
- TGF ⁇ also promotes resorption in the mouse calvarial organ culture system, but it is
- TGF ⁇ is not a promoter of resorption in a system using fetal rat long bones.
- the mouse calvarial system is one in which the generation of prostaglandins is virtually certain to result in increased resorption.
- TGF ⁇ has its predominant effect on promoting formation of bone, as does LIF. In other types of experimental systems there is good evidence that prostaglandins in fact stimulate bone formation.
- Receptor autoradiography with 125 I-LIF was carried out to determine which cells of newborn rodent bone bound LIF specifically. Osteoclast preparations from newborn rat long bones were prepared so as to ensure that the cultures were heavily contaminated with osteoblasts, macrophages and other cells. This was achieved by allowing a long settling time onto coverslips after isolation of cells (25). In these experiments, specific binding of 125 I-LIF was achieved.
- osteoblasts Figure 14 and to macrophages as the inventors have shown previously (22), but no evidence of LIF binding to osteoclasts or to mononuclear tartrate-resistant acid phosphatase-positive cells (osteoclast precursors) could be found.
- Cells were subcultured on to 125 -I-fibrin-coated plates as described in the Me thods section. Cells were preincubated for 8 h in MEM plus 0.1% BSA before the addition of fresh medium containing the bone resorbing factors. The incubation was carried out for 24 h.
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Abstract
La présente invention se rapporte généralement à l'effet du facteur inhibiteur de leucémie (FIL) dans les os des animaux et plus particulièrement à l'usage du FIL et/ou de polypeptides de même type pour induire, promouvoir et/ou renforcer les effets métaboliques sur les os des animaux.The present invention generally relates to the effect of the leukemia inhibiting factor (IDF) in the bones of animals and more particularly to the use of IDF and / or of polypeptides of the same type to induce, promote and / or reinforce the effects. metabolic on animal bones.
Description
Claims
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU3091/89 | 1989-03-07 | ||
AUPJ309189 | 1989-03-07 | ||
AUPJ474289 | 1989-06-15 | ||
AU4742/89 | 1989-06-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0463004A1 true EP0463004A1 (en) | 1992-01-02 |
EP0463004A4 EP0463004A4 (en) | 1992-04-01 |
Family
ID=25643646
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900904185 Withdrawn EP0463004A4 (en) | 1989-03-07 | 1990-03-06 | Metabolic effects of leukaemia inhibitory factor on bone |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0463004A4 (en) |
JP (1) | JP3055802B2 (en) |
CA (1) | CA2048673A1 (en) |
NZ (1) | NZ232826A (en) |
WO (1) | WO1990010454A1 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL97779A (en) * | 1990-04-10 | 2000-01-31 | Genentech Inc | Compositions for cytoprotection against radiation and chemotherapy injury or risk thereof |
WO1994021272A1 (en) * | 1993-03-17 | 1994-09-29 | Amrad Corporation Limited | A method for treating microbial infection in an animal by administering a composition comprising lif and a cytokine |
CA2385498C (en) * | 1999-09-30 | 2013-01-08 | Kaken Pharmaceutical Co., Ltd. | Method to enhance healing of sternum after sternotomy |
WO2014138628A1 (en) | 2013-03-07 | 2014-09-12 | Case Western Reserve University | Bioadhesive hydrogel |
US11273236B2 (en) | 2016-09-07 | 2022-03-15 | Case Western Reserve University | Engineered tissue constructs |
EP3551199A4 (en) * | 2016-12-11 | 2020-07-22 | Case Western Reserve University | Compositions and methods of modulating endochondral ossification and bone formation |
WO2019040224A1 (en) | 2017-08-21 | 2019-02-28 | Case Western Reserve University | Hydrogel for tissue engineering and bioprinting |
US20190298883A1 (en) | 2018-03-30 | 2019-10-03 | Case Western Reserve University | Insoluble native collagen fibers and their use in cell aggregates and tissue constructs |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4438032A (en) * | 1981-01-30 | 1984-03-20 | The Regents Of The University Of California | Unique T-lymphocyte line and products derived therefrom |
US4675295A (en) * | 1981-10-28 | 1987-06-23 | Denki Kagaku Kogyo Kabushiki Kaisha | Process for producing subculturable lymphokine-producing human T cell hybridomas |
AU609128B2 (en) * | 1987-04-02 | 1991-04-26 | Amrad Operations Pty. Limited | Leukaemia-inhibitory factor |
-
1990
- 1990-03-06 CA CA002048673A patent/CA2048673A1/en not_active Abandoned
- 1990-03-06 JP JP2504497A patent/JP3055802B2/en not_active Expired - Fee Related
- 1990-03-06 WO PCT/AU1990/000092 patent/WO1990010454A1/en not_active Application Discontinuation
- 1990-03-06 EP EP19900904185 patent/EP0463004A4/en not_active Withdrawn
- 1990-03-07 NZ NZ232826A patent/NZ232826A/en unknown
Non-Patent Citations (2)
Title |
---|
JOURNAL OF BONE AND MINERAL RESEARCH, vol. 3, no. 6, December 1988, pages 635-645, Mary Ann Liebert, Inc., New York, US; E. ABE et al.: "A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation" * |
See also references of WO9010454A1 * |
Also Published As
Publication number | Publication date |
---|---|
NZ232826A (en) | 1992-09-25 |
WO1990010454A1 (en) | 1990-09-20 |
JP3055802B2 (en) | 2000-06-26 |
EP0463004A4 (en) | 1992-04-01 |
CA2048673A1 (en) | 1990-09-08 |
JPH04503958A (en) | 1992-07-16 |
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