Note: Descriptions are shown in the official language in which they were submitted.
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Pharmaceutical Compositions and Methods of Using Secreted Frizzled
Related Protein
Field of the Invention
The present invention relates to compositions and methods for
regulating bone-forming activity. More particularly, the present invention
relates to methods and pharmaceutical compositions for regulating bone
forming activity with a secreted frizzled related protein (SFRP) derived form
an osteoblast cell line, portions thereof, as well as antibodies and nucleic
acids, including antisense, based thereon.
Background of the Invention
The topic of bone formation regulation and bone-related disorders has
recently gained considerable attention; for example in the women's health
area, there has been a particular focus on the bone-related disorder of
osteoporosis. Throughout life, there is a constant remodeling of skeletal
bone. In this remodeling process, there is a fragile balance between bone
formation by osteoblasts and subsequent bone resorption by osteoclasts.
As a normal part of the aging process the bone matrix undergoes
various structural changes, the nature, of which remains not fully determined.
The majority of studies on age-related changes in human bone have been
directed towards elucidating changes in bone on a morphological level or by
quantitatively comparing rates of bone loss. Identification of the
mechanisms involved in bone disorders is crucial for the understanding of
bone physiology and bone disorders. While numerous genes and gene
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families and the polypeptides encoded by them that participate in the
regulation of bone cells have been identified and cloned, their functions have
not been clearly delineated due to the complexities of the bone formation
pathways.
THE WNT GENE FAMILY
One group of genes and the proteins encoded by them that play an
important role in regulating cellular development is the Wnt family of
glycoproteins. Wnt proteins are a family of growth factors consisting of
more than a dozen structurally related molecules and are involved in the
regulation of fundamental biological processes like apoptosis,
embryogenesis, organogenesis, morphogenesis and tumorigenesis (reviewed
in Nusse and Varmus 1992 Cell 69: 1073-1087). These polypeptides are
multipotent factors and have similar biological activities to other secretory
proteins like transforming growth factor (TGF)-(3, fibroblast growth factors
(FGFsI, nerve growth factor (NGF) and bone morphogenetic proteins (BMPs).
One member of the Wnt growth factor family termed Wnt-x, is preferentially
expressed in bone tissue and in bone-derived cells and appears to be
involved in maintaining the mature osteoblast (bone-forming cell) phenotype
(PCT/US94/14708; WO 95/17416).
THE FRIZZLED FAMILY OF PROTEINS
Studies indicate that certain Wnt proteins interact with a family of
proteins named "Frizzled" that act as receptors for Wnt proteins or as
components of a Wnt receptor complex (reviewed in Moon et al. 1997 Cell
88: 725-728 and Barth et al. 1997 Curr. Opin. Cell Biol. 9: 683-690).
Frizzled proteins contain an amino terminal signal sequence for secretion, a
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cysteine-rich domain (CRD) that is thought to bind Wnt, seven putative
transmembrane domains that resemble a G-protein coupled receptor, and a
cytoplasmic carboxyl terminus.
The discovery of the first secreted frizzled-related protein (SFRP) was
reported by Hoang et al. in 1996 (J. Biol. Chem. 271: 26131-26137). This
protein, which was called "Frzb" for frizzled motif in bone development, was
purified and cloned from bovine articular cartilage extracts based on its
ability to stimulate in vivo chondrogenic activity in rats. The human
homologue of the bovine gene was also cloned. However, unlike the frizzled
proteins, Frzb did not contain a serpentine transmembrane domain. Thus,
this new member of the frizzled family appeared to be a secreted receptor
for Wnt. The Frzb cDNA encoded for a 325 aa/36,000 Dalton protein and
was predominantly expressed in the appendicular skeleton. The highest level
of expression was in developing long bones and corresponded to epiphyseal
chondroblasts; expression then declined and disappeared toward the
ossification center.
Recent studies indicate SFRPs participate in apoptosis and thus some
SFRPs have been identified as "SARPs" for secreted apoptosis related
proteins. Additional members of the SFRP family have also recently been
identified and shown to be antagonists of Wnt action. There are currently at
least five known human SFRP/SARP genes: SFRP-1 /FrzA/FRP-1 /SARP-2,
SFRP-2/SDF-5/SARP-1, SFRP-3/Frzb-1 /FrzB/Fritz, SFRP-4 and SFRP-5/SARP-
3 (Leimeister et al 1998 Mechanisms of Development 75: 29-42, which
sequences of this reference are incorporated herein). Although the precise
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role that SARPs/SFRPs play in apoptosis is not yet clear, these proteins
appear to either suppress or enhance the programmed cell death process.
In summary, a great need exists for the definitive identification of
targets for the treatment of bone disorders, including bone resorption
disorders such as osteoporosis and for regulation of bone formation in
humans.
Summary of the Invention
According to the present invention, there is provided methods and
pharmaceutical compositions for regulating bone-forming activity in a
mammal comprising a secreted frizzled related protein (SFRP) or regulating
portion thereof. Additional compositions of the present invention employ
antibodies formed from such proteins or portions thereof, and alternatively
can employ nucleic acids that encode such proteins or portions thereof,
including antisense sequences. In a preferred embodiment, the SFRP is from
human osteoblast cells. The bone forming activities regulated by the
composition of the present invention include the regulation of bone growth
and bone density. The SFRP has the amino acid sequence set forth in SEQ
ID NO: 2 which is obtained by the expression of the polynucleotide sequence
set forth in SEQ ID NO 1. In the most preferred embodiment, the SFRP is
SFRP-1 (SEQ ID NO 2~.
In another aspect, methods of the present invention include methods
for treating a bone disorder in a mammal, particularly a human comprising
the steps of administering a pharmaceutical composition described above.
As such, the methods for treating a bone disorder include but are not limited
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to disorders comprising the group consisting of (a) a bone formation disorder,
(b) a bone resorption disorder and (c) a bone density disorder.
In another aspect of the invention, the bone disorder is a degenerative
bone disorder wherein the degenerative bone disorder is selected from the
group consisting of neurodegeneration, myodegeneration, and
osteodegeneration disorders. The osteodegeneration disorder is selected
from the group consisting of osteopenia, osteoarthritis, osteoporosis.
In an additional embodiment, the invention includes methods for
identifying test compounds regulating SFRP activity. In the method, the
compounds regulating the bone-forming activity in a mammal are assayed by
first incubating a sample comprising a SFRP in a test medium containing the
test compound. The next step is to determine the SFRP activity, wherein an
increase in activity relative to SFRP alone indicates the compound is a SFRP
activator and a decrease in activity indicates the compound is a SFRP
inhibitor.
In a further embodiment, the invention includes methods of
modulating Wnt-mediated signaling in a cell comprising contacting said cell
with the SFRP described above, wherein said Wnt activity is regulated.
The present invention additionally relates to a method of facilitating
bone formation or repair in a bone cell culture, comprising isolating the
cells
from a bone culture, introducing a recombinant construct expressing SFRP
having the amino acid sequence set forth in SEQ ID NO: 2, and returning
said cells into the bone culture. Preferably, the construct expresses an
antisense sequence for a nucleic acid sequence which encodes all or a
portion of a SFRP protein.
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role that SARPs/SFRPs play
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Another embodiment relates to a polynucleotide probe capable of
hybridizing with the polynucleotide having the nucleic acid sequence set
forth in SEQ ID NO: 1. Such probe is used in a diagnostic process for
detecting a SFRP polynucleotide in a sample derived from a mammalian host
comprising detecting the presence or absence of the SFRP in the sample.
IV. Brief Description of the Drawings
Figure 1 shows the osteogenic RADE results that lead to the
discovery of the human osteoblast (hOB) secreted frizzled-related protein
(SFRP) cDNA. The figure shows autoradiograms of differential-display
polymerase chain reaction (DD-PCR) gels from the experiments performed
with the three hOB cell lines (hOB-03-C5, hOB-03-CE6 and hOB-01-C1 ) in
three different stages of differentiation (proliferative-stage, maturation-
stage
and pre-osteocyticl. The arrow points to the 276 base pair (bp) hOB SFRP
gene fragment (i.e., RADE fragment) that is up-regulated by PGEZ treatment
in the hOB-03-C5 and hOB-03-CE6 cells, but is down-regulated by TGF-(31
treatment in the hOB-01-C1 cells. It can also be seen that the hOB-01-C1
cells express high basal levels of this gene. A basic local alignment search
tool (BLAST) search of this gene fragment against the public databases
indicated that this cDNA was homologous to the mouse SFRP-1 and bovine
FrzA genes.
Figure 2 shows an autoradiogram of a Northern blot of poly A + RNA
isolated from the hOB-03-C5 cells after treatment with control, PTH, PGEZ
and TGF-[31 for 24 hr. In this experiment, both the excised hOB SFRP RADE
gene fragment and a cloned glyceraldehyde phosphate dehydrogenase
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(GAPDH) cDNA were used as probes. The arrow points to the hOB SFRP
mRNA (--4.4 kb) which is completely up-regulated by treatment of the cells
with PGE2.
Figure 3 shows an autoradiogram of a Northern blot of poly A + RNA
isolated from the hOB-03-C5 cells after treatment with control, PGEZ and
TGF-(31 for 24 hr. In this experiment, both the cloned hOB SFRP RADE gene
fragment and a cloned GAPDH cDNA were used as probes. Once again, the
hOB SFRP mRNA is completely up-regulated by treatment of the cells with
PGEz.
Figure 4 shows an autoradiogram of a Northern blot of poly A+ RNA
isolated from either the hOB-03-CE6 or hOB-01-C1 cells after treatment with
control, PTH, PGE2 and TGF- (31 for 24 hr. In this experiment, both the
excised hOB SFRP RADE gene fragment and a cloned GAPDH cDNA were
used as probes. The arrow points to the hOB SFRP mRNA that is completely
up-regulated by treatment of the hOB-03-CE6 cells with PGE2, but is down-
regulated by treatment of the hOB-01-C1 cells with PTH. It can also be seen
that the hOB-01-C1 cells express high basal levels of the mRNA.
Figure 5 shows an autoradiogram of a Northern blot of poly A + RNA
isolated from the hOB-01-C1 cells after treatment with control and TGF-(31
for 24 hr. In this experiment, both the full-length (1.1 kb) cloned hOB SFRP
cDNA and a cloned beta-Actin cDNA were used as probes. The results
show that the hOB SFRP mRNA is down-regulated by treatment of the cells
with TGF-(31. Once again, it can also be seen that the hOB-01-C1 cells
express high basal levels of the mRNA.
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Figure 6 shows an autoradiogram of a Northern blot of poly A+ RNA
isolated from 23 different human tissues. In this experiment, both the
excised hOB SFRP RADE gene fragment and a cloned beta-Actin cDNA were
used as probes. The arrow points to the hOB SFRP mRNA that is highly
expressed in heart and kidney, moderately expressed in placenta and uterus,
expressed at lower levels in brain, pancreas and other tissues, but not
expressed in thymus and lymphocytes.
Ficture 7 shows an autoradiogram of a Northern blot of poly A + RNA
isolated from SaOS-2 human osteosarcoma osteoblast-like cells and explant
cultures of normal human osteoblasts (hOB) after treatment with control,
PTH, PGEZ and TGF-(31 for 24 hr. In this experiment, both the full-length
cloned hOB SFRP cDNA and a cloned GAPDH cDNA were used as probes.
The results show that the SaOS-2 cells express low basal levels of hOB
SFRP mRNA which is not regulated by these agents. In contrast, hOB cells
express moderate levels of this message which is up-regulated by treatment
with PGEZ. TaqMan quantitative RT-PCR analysis of hOB cells treated with
PGE2 indicated that PGEZ upregulated SFRP message levels 10-fold.
Figure 8 shows an autoradiogram of a Southern blot of reverse
transcriptase (RT)-PCR products of total RNA isolated from either human
placenta or hOB-03-CE6 cells after treatment with control or PGE2 for 24 hr.
RT-PCR was performed with oligonucleotide primers that were specific for
human FRP-1 /SARP-2, and the Southern blot was hybridized with an internal
oligonucleotide probe specific for human FRP-1 /SARP-2. The expected size
for this RT-PCR product was 1.1 kb. The results shown that (as expected)
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placenta expresses FRP-1 /SARP-2 mRNA, and that PGEz treatment of the
hOB-03-CE6 cells strongly upregulates the expression of this message.
Figure 9 shows the results of a cell viability experiment with the hOB-
03-C5, hOB-03-CE6 and hOB-01-C1 cells using the Coulter cell counter. The
results are presented as the % relative to the day 0 control (i.e., ~ 200,000
cells per well of a 6 well platel. The results of this experiment indicate
that
the hOB-03-C5 cells proliferate slowly at 39°C in serum-containing
medium,
while the hOB-03-CE6 cells stop dividing but are viable for the 6 day
incubation. In contrast, the hOB-01-C1 cells undergo accelerated cell death,
which correlates with the high basal expression of hOB SFRP mRNA in these
cells.
Figure 10 shows the results of a cell viability experiment with the
hOB-03-C5 cells (panel A), hOB-03-CE6 cells (panel B) and hOB-01-C1 cells
(panel C) using the Coulter cell counter. For these experiments, the cells
were treated with either control or PGEZ (panels A & B) or control and TGF-
(31 (panel C) in serum-free medium at 39°C for 3 or 6 days. The results
are
presented as the % relative to the day 0 control /i.e., - 200,000 cells per
well of a 6 well plate). The results of this experiment indicate that hOB cell
viability declines over time in serum-free medium. In addition, for the hOB-
03-C5 and hOB-03-CE6 cells, this rate of decline is accelerated by treatment
with PGE2. This enhanced rate of cell death correlates with the upregulation
of hOB SFRP mRNA levels in these cells following PGEZ treatment. In
contrast, treatment of the hOB-01-C1 cells with TGF-[31, which
downregulates hOB SFRP message levels, increases cell viability.
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Figure 11 shows that treatment of hOB-03-C5 cells with PGEZ
induces apoptosis or programmed cell death. Apoptosis was measured by
flow cytometry using annexin V-FITC. Panel A shows that the number of
viable cells, which do not stain with either annexin V or propidium iodide (a
stain for necrotic cells), declines with increasing PGEZ concentrations. In
contrast, panel B shows that the number of apoptotic cells, which stain with
annexin V but not propidium iodide, increases with increasing PGEZ
concentrations. Similarly, panel C shows that the number of necrotic cells,
which stain with both annexin V and propidium iodide, increases with
increasing PGEZ concentrations.
Figure 12 shows the results of another cell viability experiment with
the hOB-03-C5 cells (panel A) and hOB-03-CE6 cells (panel B) using the
Coulter cell counter. These experiments were performed in a similar manner
to the ones depicted in Figure 10. However, for these experiments, the cells
were co-treated with either vehicle control /i.e., 0.1 % ethanol) or PGEZ in
the
absence or presence of sense (control) or antisense initiation-site directed
phosphorothioate oligonucleotides to human SARP-2. The results are
presented as either the % relative to the day 0 control (i.e., -200,000 cells
per well of a 6 well plate) or as the % relative to the vehicle treated
control.
The results of this experiment indicate that co-treatment of the hOB cells
with the antisense oligonucleotide to SARP-2 reverses the ability of PGE2 to
accelerate the rate of cell death, while co-treatment with the sense (control)
oligonucleotide as no effect on this process.
Figure 13 shows the results of a cell viability experiment with the
hOB-01-C1-PS-09 cells using the Coulter cell counter (panel A). For these
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experiments, the cells were stably transfected with either an hOB SFRP
cDNA (i.e., SFRP-1 /FRP-1 /SARP-2) mammalian expression plasmid or the
empty vector /i.e., pcDNA3.1 ). The results are presented as the % of the
day 0 control cells. The results of this experiment indicates that
overexpression of hOB SFRP/SFRP-1 /FRP-1 /SARP-2 by the hOB cells
accelerates the rate of cell death when compared to the empty vector which
has no effect on this process. Panel B shows the results of a Northern
hybridization of poly A+ RNA isolated from either the empty vector cells (V)
or the SARP-2 overexpressing cells (S). This analysis demonstrates that the
SARP-2 cells express substantially more SFRP-1 /FRP-1 /SARP-2 mRNA than
the vector cells.
Figure 14 shows the results of another cell viability experiment
performed with the empty vector expressing hOB-01-C1-PS-09 cells
(pcDNA3.1 ) and a subclone of the SFRP-1 /FRP-1 /SARP-2 overexpressing
cells (SARP-2 Clone #1 ). Panel A shows the results of a TaqMan
quantitative RT-PCR analysis of RNA isolated from the cells. This analysis
indicates that the SARP-2 Clone #1 cells express 50-60 times more human
SFRP-1 /FRP-1 /SARP-2 mRNA than the empty vector expressing cells.
Likewise, as shown in panel B, the SARP-2 Clone #1 cells die at a rate that
is 3-times faster than the empty vector control cells using the Coulter cell
counter to measure cell number.
Ficture 15 is an autoradiogram of a Western blot of whole cell extracts
isolated from hOB-03-CE6 cells treated with either control or PGEZ for 24 hr.
The immunoblot was probed with a monoclonal antibody to ~3-catenin, which
has a molecular weight of 92,000. The results show that treatment of the
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hOB cells with PGEZ downregulates (3-catenin levels, which is consistent with
an antagonism of the Wnt signaling pathway by hOB SFRP/SFRP-1 /FRP-
1 /SARP-2.
Figure 16 shows the results of a transient transfection experiment
with the hOB-01-C1-PS-09 cells (panel A) and hOB-02-C1-PS-02 cells (panel
B). The results show that transfection of either human [h] or rat [r] SARP-2
or human Frzb-1 expression plasmids downregulates Wnt signaling (when
compared to the pcDNA3.1 empty vector control) in the hOB cells as
measured by the TCF-luciferase reporter gene assay. This assay is an
authentic measurement of Wnt signaling and (3-catenin nuclear activity.
Figure 17 summarizes a screening paradigm for anabolic bone agents
using the hOB cells and hOB SFRP/SFRP-1 /FRP-1 /SARP-2. This screening
paradigm would identify compounds that regulate SFRP-1 /FRP-1 /SARP-2
function.
Figure 18 depicts an example of a high-throughput screening (HTS)
assay for compounds that inhibit SFRP-1 /FRP-1 /SARP-2 function in
osteoblastic/osteocytic cells. The results show that the hOB-01-C 1-PS-09
cells overexpressing SFRP-1 /FRP-1 /SARP-2 (SARP-2 #1 cells) die faster than
the empty vector expressing cells (pcDNA3.1 ) using the CyQuant DNA
fluorescence assay. Moreover, an antipeptide antiserum to SFRP-1 /FRP-
1 /SARP-2 (SARP-2 AS) blocks the cell death caused by overexpression of
this gene.
Figure 19 outlines the strategy that was used to create the SFRP-
1 /SARP-2 knock-out mice. Exon 1 of the mouse SFRP-1 /SARP-2 gene,
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which encodes for the entire cysteine-rich domain (CRD) was replaced with
the expression cassette for (3-galactosidase and neomycin resistance.
Figure 20 depicts a Northern blot of poly A + RNA isolated from
female and male kidneys obtained from the wild-type (WT) and knock-out
(KO) SFRP-1 mice. This blot shows that the WT kidneys express high levels
of SFRP-1 mRNA (4.4 kbl, while the KO kidneys do not express this gene.
Figure 21 shows the results of a micro-computerized tomography
(micro-CT) analysis of femurs obtained from the male (panel A) and female
(panel B) wild-type (+/+) and knock-out (-/-) SFRP-1 mice. When compared
to the +/+ control mice, the data demonstrate that the -/- mice exhibit
increased parameters of bone formation (i.e., BV/TV, Tb. Th., Conn. Den.,
Tb. N. & Tb. Sp.) as determined by this method.
Figure 22 is a set of graphs showing (A) cell proliferation of hOB cells
(B) the ability/enhancement in hOB cells for vitamin D3 - treatment to up-
regulate alkaline phosphatase activity at 39° C, and (C) the inability
of
vitamin D3 to induce osteocalcin secretion from hOB cells at 34° C, as
detailed in Example A.
Figure 23 depicts the results from Example A regarding the
expression of PTH-1 receptor mRNA expression; wherein incubation of the
hOB cells ells for 48 hours at 39° C increases the steady-state message
levels for PTH-1 receptor by 7-fold when compared to cells maintained at
34°C.
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Figure 24 depicts the results from Example A for the effect on
intracellular cyclic adenosine monohosphate (CAMP) in response to
increasing concentrations of PTH 1-34 in the hOB cells.
Figure 25 is a graph which depicts the effect of hOB cells when
treated with synthetic glucorticoid dexamethasone, which up-regulates
alkaline phosphatase activity, as detailed in Example A.
Figure 26 depicts the results from experiments on the effect of
mechanosensory stimulation on pre-osteocytic hOB cells, when subject to a
Flexerall Strain Unit.
V. Detailed Description of the Invention
The present invention relates to a gene whose expression is regulated
by osteogenic or bone-forming agents in three different human osteoblast
(hOB) cell lines in vitro. In one aspect of the present invention, the
expression of this gene is upregulated during hOB differentiation, suggesting
it may be involved in the bone formation process. DNA sequence analysis
indicated that this gene fragment shared significant sequence identity to a
mouse cDNA called secreted frizzled-related protein (SFRP)-1 (Rattner et al.
1997 Proc. Natl. Acad. Sci. USA 94: 2859-2863). Subsequent cDNA
cloning and additional sequence analysis indicated that this gene, which is
referred to as the hOB SFRP, was identical to human FRP-1 /SARP-2 (Finch et
al. 1997 Proc. Natl. Acad. Sci. USA 94: 6770-6775; Melkonyan et al. 1997
Proc. Natl. Acad. Sci. USA 94: 13636-136411. Characterization of the hOB
SFRP contemplates its use as a novel osteoporotic drug target and in a novel
drug screen for identifying anabolic bone agents.
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Definitions
The term "bone formation" is the process of bone synthesis and
mineralization. The osteoblast cell modulates the process.
The term "bone growth" is the process of skeletal expansion. This
process occurs by one of two ways: (1 ) intramembraneous bone formation
arises directly from mesenchymal or bone marrow cells; (2) longitudinal or
endichindual bone formation arises where bone from cartilage.
The term "osteogenesis" is synonymous with the term bone
formation, defined above.
The terms "secreted frizzled related proteins" or "SFRP" is a secreted
receptor of the Wnt signaling pathway and exhibits a number of
characteristics that make it a useful tool for studying cell growth and
differentiation. The frizzled like gene family encodes cell membrane proteins
having transmembrane domains with unknown functions.
The terms "secreted apoptosis related protein" or "SARP" are
synonymous with the term secreted frizzled related proteins or SFRPs
defined above.
The terms "proteins", "peptides" and "polypeptides" are used
interchangeably and are intended to include purified and recombinantly
produced SFRP molecules containing amino acids linearly coupled through
peptide bonds. The amino acids of this invention can be in the L or D form
so long as the biological activity of the polypeptide is maintained. The SFRP
proteins of this invention may also include proteins that are post-
translationally modified by reactions that include glycosylation, acetylation
and phosphorylation. Such polypeptides also include analogs, alleles and
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allelic variants that can contain amino acid derivatives or non-amino acid
moieties that do not affect the biological or functional activity of the SFRP
protein as compared to wild-type or naturally occurring protein. The term
amino acid refers both to the naturally occurring amino acids and their
derivatives, such as TyrMe and PheCl, as well as other moieties
characterized by the presence of both an available carboxyl group and an
amine group. Non-amino acid moieties that can be contained in such
polypeptides include, for example, amino acid mimicking structures.
Mimicking structures are those structures that exhibit substantially the same
spatial arrangement of functional groups as amino acids but do not
necessarily have both the amino and carboxyl groups characteristic of amino
acids.
"Muteins" are SFRP proteins or polypeptides that have minor changes
in amino acid sequence caused, for example, by site-specific mutagenesis or
other manipulations; by errors in transcription or translation; or which are
prepared synthetically by rational design. These minor alterations result in
amino acid sequences wherein the biological activity of the protein or
polypeptide is altered as compared to wild-type or naturally occurring
polypeptide or protein. Examples of muteins include the SFRP-1 of SEQ. ID.
No. 2 described herein.
As used herein, the term "hydrophobic" is intended to include those
amino acids, amino acid derivatives, amino acid mimics and chemical
moieties that are non-polar. Hydrophobic amino acids include Phe, Val, Trp,
Ile, and Leu. As used herein, the term "positively charged amino acid" refers
to those amino acids, amino acid derivatives, amino acid mimics and
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chemical moieties that are positively charged. Positively charged amino
acids include, for example, Lys, Arg and His.
"Purified" when referring to a SFRP protein or polypeptide, is
distinguishable from native or naturally occurring proteins or polypeptides
because they exist in a purified state. These "purified" SFRP proteins or
polypeptides, or any of the intended variations as described herein, shall
mean that the compound or molecule is substantially free of contaminants
normally associated with the compound in its native or natural environment.
The terms "substantially pure" and "isolated" are not intended to exclude
mixtures of polynucleotides or polypeptides with substances that are not
associated with the polynucleotides or polypeptides in nature.
"Native" SFRP polypeptides, proteins, or nucleic acid molecules refer
to those SFRP recovered from a source occurring in nature or "wild-type".
A "composition" is intended to mean a combination of active agent,
i.e., the SFRP of the present invention and another compound or
composition, inert (for example, a detectable agent or label) or active, such
as an adjuvant.
A "pharmaceutical composition" is intended to include the
combination of SFRP, particularly SFRP-1 as the active. agent with a carrier,
inert or active, making the composition suitable for diagnostic or therapeutic
use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water, and emulsions, such as an
oil/water or water/oil emulsion, and various types of wetting agents. The
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compositions also can include stabilizers and preservatives. For examples of
carriers, stabilizers and adjuvants, see Martin, Reminaton's Pharm. Sci., 15th
Ed. (Mack Publ. Co., Easton (197511.
The term "nucleic acid" as it relates to the SFRP described herein
means single and double stranded DNA, cDNA, genome-derived DNA, and
RNA, as well as the positive and negative strand of the nucleic acid that are
complements of each other, including anti-sense RNA. A "nucleic acid
molecule" is a term used interchangeably with "polynucleotide" and each
refers to a polymeric form of nucleotides of any length, either
ribonucleotides
or deoxyribonucleotides, or analogs thereof. It also includes known types of
modifications, for example labels which are known in the art (e.g., Sambrook
et al. 11989) infra.l, methylation, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
carbamate, etc.), those containing pendant moieties, such as for example,
proteins (including, e.g., nuclease, toxins, antibodies, signal peptides,
etc.l,
those with intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.l,
those containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.l, as well as unmodified forms of the
polynucleotide. The polynucleotide can be chemically or biochemically
modified or contain non-natural or derivatized nucleotide bases. The
nucleotides may be complementary to the mRNA encoding the polypeptides.
These complementary nucleotides include, but are not limited to, nucleotides
capable of forming triple helices and antisense nucleotides. Recombinant
18
chemical moieties that are
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polynucleotides comprising sequences otherwise not naturally occurring are
also provided by this invention, as are alterations of wild-type polypeptide
sequences, including but not limited to, those due to deletion, insertion,
substitution of one or more nucleotides or by fusion to other polynucleotide
sequences.
A SFRP polynucleotide is said to "encode" a SFRP polypeptide if, in
its native state or when manipulated by methods well-known to those skilled
in the art, it can be transcribed and/or translated to produce a polypeptide
or
mature protein. Thus, the term polynucleotide shall include, in addition to
coding sequences, processing sequences and other sequences that do not
code for amino acids of the mature protein. The anti-sense strand of such a
polynucleotide is also said to encode the sequence.
The term "recombinant" polynucleotide or DNA refers to a
polynucleotide that is made by the combination of two otherwise separated
segments of sequence accomplished by the artificial manipulation of isolated
segments of DNA by genetic engineering techniques or by chemical
synthesis. In so doing, one may join together DNA segments of desired
functions to generate a desired combination of functions.
An "analog" of a SFRP DNA, RNA or a polynucleotide, refers to a
macromolecule resembling naturally occurring polynucleotides in form and/or
function (particularly in the ability to engage in sequence-specific hydrogen
bonding to base pairs on a complementary polynucleotide sequence) but
which differs from DNA or RNA in, for example, the possession of an
unusual or non-natural base or an altered backbone. See for example,
Uhlmann et al. 11990) Chemical Reviews 90:543-584.
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"Isolated when referring to a SFRP nucleic acid molecule, means
separated from other cellular components normally associated with native or
wild-type SFRP DNA or RNA intracellularly.
"Hybridization" refers to hybridization reactions that can be performed
under conditions of different "stringency". Conditions that increase the
stringency of a hybridization reaction are widely known and published in the
art: see, for example, Sambrook et al., infra. Examples of relevant
conditions include (in order of increasing stringency): incubation
temperatures of 25°C, 37°C, 50°C, and 68°C; buffer
concentrations of 10
x SSC, 6 x SSC, 1 X SSC, 0.1 x SSC (where SSC is 0.15 M NaCI and 15
mM citrate buffer) and their equivalent using other buffer systems;
formamide concentrations of 0%, 25%, 50%, and 75%, incubation times
from 5 minutes to 24 hours and washes of increasing duration, increasing
frequency, or decreasing buffer concentrations.
"Tm" is the temperature in degrees Centigrade at which 50% of a
polynucleotide duplex made of complementary strands hydrogen bonded in
an antiparallel direction by Watson-Crick base paring dissociates into single
strands under the conditions of the experiment. Tm may be predicted
according to standard formula, for example:
Tm=81.5 + 16.6 log [Na+] + 0.41 (%G/C) - 0.61 (%F) -
600/L
where Na+ is the cation concentration (usually sodium ion) in mol/L; (%G/C)
is the number of G and C residues as a percentage of total residues in the
duplex; (%F) is the percent formamide in solution (wt/vol); and L is the
number of nucleotides in each strand of the duplex.
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A "stable duplex" of polynucleotides, or a "stable complex" formed
between any two or more components in a biochemical reaction, refers to a
duplex or complex that is sufficiently long lasting to persist between the
formation of the duplex or complex, and its subsequent detection. The
duplex or complex must be able to withstand whatever conditions exist or
are introduced between the moment of formation and the moment of
detection, these conditions being a function of the assay or reaction which is
being performed. Intervening conditions which may optionally be present
and which may dislodge a duplex or complex include washing, heating,
adding additional solutes or solvents to the reaction mixture (such as
denaturantsl, and competing with additional reacting species. Stable
duplexes or complexes may be irreversible or reversible, but must meet the
other requirements of this definition. Thus, a transient complex may form in
a reaction mixture, but it does not constitute a stable complex if it
dissociates spontaneously or as a result of a newly imposed condition or
manipulation introduced before detection.
When stable duplexes form in an antiparallel configuration between
two single-stranded polynucleotides, particularly under conditions of high
stringency, the strands are essentially "complementary". A double-stranded
polynucleotide can be "complementary" to another polynucleotide, if a stable
duplex can form between one of the strands of the first polynucleotide and
the second. A complementary sequence predicted from the sequence of
single stranded polynucleotide is the optimum sequence of standard
nucleotides expected to form hydrogen bonding with the single-stranded
polynucleotide according to generally accepted base-pairing rules.
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A "sense" strand and an "antisense" strand when used in the same
context refer to single-stranded SFRP polynucleotides which are
complementary to each other. They may be opposing strands of a double-
stranded polynucleotide, or one strand may be predicted from the other
according to generally accepted base-pairing rules. Unless otherwise
specified or implied, the assignment of one or the other strand as "sense" or
"antisense" is arbitrary.
A linear sequence of SFRP nucleotides is "identical" to another linear
sequence, if the order of nucleotides in each sequence is the same, and
occurs without substitution, deletion, or material substitution. It is
understood that purine and pyrimidine nitrogenous bases with similar
structures can be functionally equivalent in terms of Watson-Crick base-
pairing; and the inter-substitution of like nitrogenous bases, particularly
uracil
and thymine, or the modification of nitrogenous bases, such as by
methylation, does not constitute a material substitution. An RNA and a DNA
polynucleotide have identical sequences when the sequence for the RNA
reflects the order of nitrogenous bases in the polyribonucleotide, the
sequence for the DNA reflects the order of nitrogenous bases in the
polydeoxyribonucleotide, and the two sequences satisfy the other
requirements of this definition. Where at least one of the sequences is a
degenerate oligonucleotide comprising an ambiguous residue, the two
sequences are identical if at least one of the alternative forms of the
degenerate oligonucleotide is identical to the sequence with which it is being
compared. For example, AYAAA (SEO ID NO 3) is identical to ATAAA (SEQ
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ID NO 4), if AYAAA (SEQ ID NO 5) is a mixture of ATAAA (SEQ ID N06 )
and ACAAA (SEQ ID NO 7).
When comparison is made between polynucleotides, it is implicitly
understood that complementary strands are easily generated, and the sense
or antisense strand is selected or predicted that maximizes the degree of
identity between the polynucleotides being compared. For example, where
one or both of the polynucleotides being compared is double-stranded, the
sequences are identical if one strand of the first polynucleotide is identical
with one strand of the second polynucleotide. Similarly, when a
polynucleotide probe is described as identical to its target, it is understood
that it is the complementary strand of the target that participates in the
hybridization reaction between the probe and the target.
A linear sequence of nucleotides is "essentially identical" or the
"equivalent" to another linear sequence, if both sequences are capable of
hybridizing to form duplexes with the same complementary polynucleotide.
It should be understood, although not always explicitly stated that
Applicants refer to a specific nucleic acid molecule, its equivalents are also
intended. Sequences that hybridize under conditions of greater stringency
are more preferred. It is understood that hybridization reactions can
accommodate insertions, deletions, and substitutions in the nucleotide
sequence. Thus, linear sequences of nucleotides can be essentially identical
even if some of the nucleotide residues do not precisely correspond or align.
Sequences that correspond or align more closely to the invention disclosed
herein are comparably more preferred. Generally, a polynucleotide region of
about 25 residues is essentially identical to another region, if the sequences
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are at least about 80% identical; more preferably, they are at least about
90% identical; more preferably, they are at least about 95% identical; still
more preferably, the sequences are 100% identical. A polynucleotide region
of 40 residues or more will be essentially identical to another region, after
alignment of homologous portions if the sequences are at least about 75%
identical; more preferably, they are at least about 80% identical; more
preferably, they are at least about 85% identical; even more preferably, they
are at least about 90% identical; still more preferably, the sequences are
100% identical.
In determining whether polynucleotide sequences are essentially
identical, a sequence that preserves the functionality of the polynucleotide
with which it is being compared is particularly preferred. Functionality can
be determined by different parameters. For example, if the polynucleotide is
to be used in reactions that involve hybridizing with another polynucleotide,
then preferred sequences are those which hybridize tot he same target under
similar conditions. In general, the Tm of a DNA duplex decreases by about
10°C for every 1 % decrease in sequence identity for duplexes of 200 or
more residues; or by about 50°C for duplexes of less than 40 residues,
depending on the position of the mismatched residues (see, e.g. Meinkoth et
al.). Essentially identical or equivalent sequences of about 100 residues will
generally form a stable duplex with each other's respective complementary
sequence at about 20°C less than Tm; preferably, they will form a
stable
duplex at about 15°C less; more preferably, they will form a stable
duplex at
about 10°C less; even more preferably, they will form a stable duplex
at
about 5°C less; still more preferably, they will form a stable duplex
at about
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Tm. In another example, if the polypeptide encoded by the polynucleotide is
an important part of its functionality, then preferred sequences are those
which encode identical or essentially identical polypeptides. Thus,
nucleotide differences which cause a conservative amino acid substitution
are preferred over those which can cause a non-conservative amino acid
substitution are preferred over those which cause a non-conservative
substitution, nucleotide differences which do not alter the amino acid
sequence are more preferred, while identical nucleotides are even more
preferred. Insertions or deletions in the polynucleotide that result in
insertions or deletions in the polypeptide are preferred over those that
result
in the down-stream coding regions being rendered out of phase;
polynucleotide sequences comprising no insertions or deletions are even
more preferred. The relative importance of hybridization properties and the
encoded polypeptide sequence of a polynucleotide depends on the
application of the invention.
A polynucleotide has the same characteristics or is the equivalent of
another polynucleotide if both are capable of forming a stable .duplex with a
particular third polynucleotide under similar conditions of maximal
stringency. Preferably, in addition to similar hybridization properties, the
polynucleotides also encode essentially identical polypeptides.
"Conserved" residues of a polynucleotide sequence are those residues
that occur unaltered in the same position of two or more related sequences
being compared. Residues that are relatively conserved are those that are
conserved amongst more related sequences than residues appearing
elsewhere in the sequences.
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"Related" polynucleotides that share a significant proportion of
identical residues.
As used herein, a "degenerate" oligonucleotide sequence is a
designed sequence derived from at least two related originating
polynucleotide sequences as follows: the residues that are conserved in the
originating sequences are preserved in the degenerate sequence, while
residues that are not conserved in the originating sequences may be
provided as several alternatives in the degenerate sequence. For example,
the degenerate sequence AYASA (SEQ ID NO 8lmay be assigned from
originating sequences ATACA (SEQ ID NO 9) and ACAGA (SEQ ID NO 101,
where Y is C or T and S is C or G. Y and S are examples of "ambiguous"
residues. A degenerate segment is a segment of a polynucleotide containing
a degenerate sequence.
It is understood that a synthetic oligonucleotide comprising a
degenerate sequence is actually a mixture of closely related oligonucleotides
sharing an identical sequence, except at the ambiguous positions. Such an
oligonucleotide is usually synthesized as a mixture of all possible
combinations of nucleotides at the ambiguous positions. Each of the
oligonucleotides in the mixture is referred to as an "alternative form".
A polynucleotide "fragment" or "insert" as used herein generally
represents a sub-region of the full-length form, but the entire full-length
polynucleotide may also be included.
Different polynucleotides "correspond" to each other if one is
ultimately derived from another. For example, messenger RNA corresponds
to the gene from which it is transcribed. cDNA corresponds to the RNA
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from which it has been produced, such as by a reverse transcription
reaction, or by chemical synthesis of a DNA based upon knowledge of the
RNA sequence. cDNA also corresponds to the gene that encodes the RNA.
Polynucleotides also "correspond" to each other if they serve a similar
function, such as encoding a related polypeptide, in different species,
strains
or variants that are being compared.
A "probe" when used in the context of SFRP polynucleotide
manipulation refers to an oligonucleotide that is provided as a reagent to
detect a target potentially present in a sample of interest by hybridizing
with
the target. Usually, a probe will comprise a label or a means by which a
label can be attached, either before or subsequent to the hybridization
reaction. Suitable labels include, but are not limited to radioisotopes,
fluorochromes, chemiluminescent compounds, dyes, and proteins, including
enzymes.
A "primer" is an oligonucleotide, generally with a free 3'-OH group,
that binds to a target potentially present in a sample of interest by
hybridizing with the target, and thereafter promotes polymerization of a
polynucleotide complementary to the target.
Processes of producing replicate copies of the same polynucleotide,
such as PCR or gene cloning, are collectively referred to herein as
"amplification" or "replication". For example, single or double-stranded DNA
may be replicated to form another DNA with the same sequence. RNA may
be replicated, for example, by and RNA-directed RNA polymerase, or by
reverse-transcribing the DNA and then performing a PCR. In the latter case,
the amplified copy of the RNA is a DNA with the identical sequence.
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A "polymerase chain reaction" ("PCR") is a reaction in which replicate
copies are made of a target polynucleotide using one or more primers, and a
catalyst of polymerization, such as a reverse transcriptase or a DNA
polymerase, and particularly a thermally stable polymerase enzyme.
Generally, a PCR involves reiteratively forming three steps: "annealing", in
which the temperature is adjusted such that oligonucleotide primers are
permitted to form a duplex with the polynucleotide to be amplified;
"elongating", in which the temperature is adjusted such that oligonucleotides
that have formed a duplex are elongated with a DNA polymerase, using the
polynucleotide to which they have formed the duplex as a template; and
"melting", in which the temperature is adjusted such that the polynucleotide
and elongated oligonucleotides dissociate. The cycle is then repeated until
the desired amount of amplified polynucleotide is obtained. Methods for
PCR are taught in USP No. 4,683,195 to Mullis and USP 4,683,202 to
Mullis et al.
Elements within a gene include but are not limited to promoter
regions, enhancer regions, repressor binding regions, transcription initiation
sites, ribosome binding sites, translation initiation sites, protein encoding
regions, introns and exons, and termination sites for transcription and
translation. An "antisense" copy of a particular polynucleotide refers to a
complementary sequence that is capable of hydrogen bonding to the
polynucleotide and can therefor, be capable of modulating expression of the
polynucleotide. These are DNA, RNA or analogs thereof, including analogs
having altered backbones, as described above. The polynucleotide to which
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the antisense copy binds may be in single-stranded form or in double-
stranded form.
As used herein, the term "operatively linked" means that the DNA
molecule is positioned relative to the necessary regulation sequences, e.g., a
S promoter or enhancer, such that the promoter will direct transcription of
RNA off the DNA molecule in a stable or transient manner.
"Vector" means a self-replicating nucleic acid molecule that transfers
an inserted nucleic acid molecule into and/or between host cells. The term is
intended to include vectors that function primarily for the replication of
nucleic acid and expression vectors that function for transcription and/or
translation of the DNA or RNA. Also intended are vectors that provide more
than one of the above functions.
"Host cell" is intended to include any individual cell or cell culture that
can be or have been recipients for vectors or the incorporation of exogenous
nucleic acid molecules and/or proteins. It also is intended to include progeny
of a single cell, and the progeny may not necessarily be completely identical
(in morphology or in genomic or total DNA complement) to the original
parent cell due to natural, accidental, or deliberate mutation.
An "antibody" is an immunoglobulin molecule capable of binding an
antigen. As used herein, the term encompasses not only intact
immunoglobulin molecules, but also anti-idiotypic antibodies, mutants,
fragments, fusion proteins, humanized proteins and modifications of the
immunoglobulin molecule that comprise an antigen recognition site of the
required specificity.
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An "antibody complex" is the combination of antibody (as defined
above) and its binding partner or ligand.
A "suitable cell" for the purposes of this invention is one that includes
but is not limited to a cell expressing the SFRP, e.g., a bone marrow cell,
preferentially an hOB cell.
A "biological equivalent" of a nucleic acid molecule is defined herein
as one possessing essential identity with the reference nucleic acid
molecule. A fragment of the reference nucleic acid molecule is one example
of a biological equivalent.
A "biological equivalent" of an SFRP polypeptide or protein is one that
retains the same characteristic as the reference protein or polypeptide. It
also includes fragments of the reference protein or polypeptide.
The SFRP proteins and polypeptides also can be obtained by chemical
synthesis using a commercially available automated peptide synthesizer such
as those manufactured by Applied Biosystems, Inc., Model 430A or 431 A,
Foster City, CA and the amino acid sequence provided in SEQ. ID NO 2. The
synthesized protein or polypeptide can be precipitated and further purified,
for example by high performance liquid chromatography (HPLC).
Accordingly, this invention also provides a process for chemically
synthesizing the proteins of this invention by providing the sequence of the
protein /e.g., SEQ. ID NO 2) and reagents, such as amino acids and enzymes
and linking together the amino acids in the proper orientation and linear
sequence.
Alternatively, the proteins and polypeptides can be obtained by well-
known recombinant methods as described, for example, in Sambrook et al.,
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Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor
Laboratory (19891) using, for example, the host cell and vector systems
described and exemplified below. This invention further provides a process
for producing a SFRP, analog, mutein or fragment thereof, by growing a host
cell containing a nucleic acid molecule encoding the desired protein, the
nucleic acid being operatively linked to a promoter of RNA transcription. The
desired protein may be introduced into the host cell by use of a gene
construct which contains a promoter and termination sequence for the
nucleic acid sequence of the desired protein. The host cell is grown under
suitable conditions such that the nucleic acid is transcribed and translated
into protein. In a separate embodiment, the protein is further purified.
The proteins of this invention also can be combined with various
liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically
acceptable carriers, suspensions and emulsions. Examples of non-aqueous
solvents include propyl ethylene glycol, polyethylene glycol and vegetable
oils. When used to prepare antibodies, the carriers also can include an
adjuvant that is useful to nonspecifically augment a specific immune
response. A skilled artisan can easily determine whether an adjuvant is
required and selects one. However, for the purpose of illustration only,
suitable adjuvants include, but are not limited to Freund's Complete and
Incomplete, mineral salts and polynucleotides.
Therapeutic Applications for SFRPs/SARPs
Although the SFRPs/SARPs gene family has only recently been
discovered and there is still much to learn about its biology, there are
nevertheless several potential therapeutic applications for these proteins.
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Since Wnts have been implicated as proto-oncogenes, SFRPs/SARPs may
serve as tumor suppressors due to their ability to antagonize Wnt activity.
These proteins may also be utilized in tissue regeneration. For example,
since FrzB-1 stimulated ectopic chondrogenic activity in vivo, it could be
used to accelerate fracture repair or the healing of joints after hip and knee
replacement (patent application number WO 98/16641 A1 ). Finally, since
SFRPs/SARPs appear to control apoptosis, these proteins could also be
utilized to treat a variety of degenerative diseases including
neurodegeneration, myodegeneration and osteodegeneration disorders.
Pharmaceutical Compositions
This invention also provides compositions containing any of the
above-mentioned proteins, muteins, fragments, antibodies, nucleic acid
molecules encoding such proteins, muteins, antibodies or fragments thereof,
as well as vectors and host cells that express such nucleic acid molecules,
and an acceptable solid or liquid, carrier buffer, or diluent. An effective
amount of one or more active ingredient is used which sufficient to
accomplish the desired regulatory effect on a bone-forming activity or
apoptosis activity. An effective amount can be determined by conventional
dose-response curves for the desired activity. When the compositions are
used pharmaceutically, they are combined with a "pharmaceutically
acceptable carrier" for diagnostic and therapeutic use. The formulation of
such compositions is well known to persons skilled in this field.
Pharmaceutical compositions of the invention may comprise one or more
additional active components and, preferably, include a pharmaceutically
acceptable carrier. The additional active component may be provided to
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work in combination with an active based on a one or more SFRPs, as
described above. In alternative embodiments, the additional active is added
since it works on the same disease or disorder as SFRPs but by a different
mode of action from those actives based on SFRPs, or the additional active
may work on other diseases or disorders present in a human or animal.
Suitable pharmaceutically acceptable carriers and/or diluents include any and
all conventional solvents, dispersion media, fillers, solid carriers, aqueous
solutions, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like. The term "pharmaceutically
acceptable carrier" refers to a carrier that does not cause an allergic
reaction
or other untoward effect in patients to whom it is administered. Suitable
pharmaceutically acceptable carriers include, for example, one or more of
water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the
like, as well as combinations thereof. Pharmaceutically acceptable carriers
may further comprise minor amounts of auxiliary substances such as wetting
or emulsifying agents, preservatives or buffers, which enhance the shelf life
or effectiveness of one or more of the active components of the
composition. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any conventional
media or agent is incompatible with the active ingredient, use thereof in
immunogenic compositions of the present invention is contemplated.
These compositions also can be used for the preparation of
medicaments for the diagnosis and treatment of pathologies associated
neurodegenerative (i.e., Huntington's disease, Alzheimer's disease, spinal
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cord injuriesl, myodegerative (i.e., muscular dsytrophy, myasthenia gravis,
myotonic myopathies) and osteodegenerative disorders /i.e., osteoporosis).
In certain embodiments, antibodies that binds all or a portion of an
SFRP protein are employed in the composition to treat of any of the above
diseases or disorders. Polyclonal and monclonal antibodies can be prepared
by conventional methods. Generally, an antibody is raised against an amino
acid sequence (a) that is specific to a SFRP protein (or proteins) and (b)
that
is also more likely to be antigenic. One can select a sequence specific for an
SFRP protein by performing sequence analysis and using any conventional
programs for sequence alignment and sequence comparisons. An amino acid
sequence that is hydrophilic at one or more ends, preferably at both ends, is
generally preferred for raising antibodies. In addition to employing amino
acids that are hydrophilic, in preferred embodiments the hydrophilic amino
acids are also basic (non-acidicl. One can also employ any amino acid that
increases antigenicity. For example, often prolines are employed in the
center portion of the sequence. Antigenicity can be measured by an
increase in decrease in the amount of antibody that is produced when
generating antibodies against an initial test sequence, which is specific an
SFRP protein(sl. In certain - embodiments of the present invention, the
antibody is raised against a sequence comprising at least 8 consecutive
amino acids of an SFRP protein(sl, and preferably a sequence comprising at
least 10 consecutive amino acids of an SFRP protein(sl. In further preferred
embodiments, the antibody is raised against amino acid sequence comprising
about 15 to about 30 amino acids. In preferred embodiments, the antibody
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is raised against a sequence comprising at amino acids 217-231 of an SFRP
protein of SEQ ID NO 2 or sequence variations thereof.
The compositions of the present invention can be administered to an
individual in need of facilitated neural, muscle cartilage and bone growth by
numerous routes, including but not limited to intravenous, subcutaneous,
intramuscular, intrathecal, intracranial and topical. The composition may be
administered directly to an organ or to organ cells by in vivo or ex vivo
methods.
These compositions may be in soluble or microparticular form, or may
be incorporated into microspheres or microvesicles, including micelles and
liposomes.
Industrial Applicability
The compositions described above provide the components for an
assay to screen for agents and pharmaceutical compounds that are agonists
or antagonists of a Wnt receptor in a suitable cell.
It is also anticipated that the SFRP polynucleotides of the invention
will have utility as diagnostic agents or detecting genetic abnormalities
associated with genes encoding SFRP or with one or more genes involved in
the Wnt signaling pathway. Such genetic abnormalities include point
mutations, deletions, or insertions of nucleotides. Any of several genetic
screening procedures may be adapted for use with probes enabled by the
present invention, including restriction fragment length polymorphism (RFLP)
analysis, ligase chain reaction, or PCR. Mutations in this gene indicate
increased risk of developmental abnormalities.
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As provided in more detail below, the proteins and fragments thereof
are useful in a cell-free and cellular in vitro assay system to screen for
agents and pharmaceutical compounds which either inhibit or augment the
Wnt-receptor pathway and apoptosis and to test possible therapies for
S disorders associated with this pathway, e.g., bone formation diseases,
carcinogenesis, and cardiovascular diseases. Embryogenesis also can be
modulated.
Drug screening assays can be used to identify activators or inhibitors
of the SFRP protein. For example, an increase in cartilage growth in the
presence of a drug compared to SFRP alone may indicate activation of SFRP,
while a decrease may indicate inhibition of the SFRP activity.
A variety of compounds may be screened using methods of the
present invention. They include peptides, macromolecules, small molecules,
chemicals and biological mixtures. Such compounds may be biological,
synthetic, organic, or inorganic compounds.
In the present invention suitable cells are used for preparing
diagnostic assays, for the expression of SFPRs or for preparing nucleotide-
based diagnostic kits. The cells may be made or derived from yeast,
bacteria fungi, or viruses. In preferred embodiments, the cells are hOB cells,
in particular a novel immortalized pre-osteocytic cell line referred to as hOB-
01-C 1-PS-09 cells (which are deposited with American Type Culture
Collection in Manassas, Va. with the designation PTA-7851, and osteoblast
cells having the identifying characteristics of hOB-01-C 1-PS-09 cells as well
as osteoblast cells made therefrom, e.g. progeny. Immortalized refers to a
substantially continuous and permanently established cell culture with
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substantially unlimited cell division potential. That is, the cells can be
cultured substantially indefinitely, i.e., for at least about 6 months under
rapid conditions of growth, preferably much longer under slower growth
conditions, and can be propagated rapidly and continually using routine cell
culture techniques. Alternatively stated, the cells of the present invention
can be cultured for at least about 100, 150 or 200 population doublings.
These cells produce a complement of proteins characteristic of normal
human osteoblastic cells and are capable of osteoblastic differentiation. They
can be used in cell culture studies of osteoblastic cell sensitivity to
various
agents, such as hormones, cytokines, and growth factors, or in tissue
therapy. These cells are a post-senescent subclone of hOB-01-C1 cell line,
as previously disclosed by Bodine et al 1996 Endocrinology 137:4592-4604.
Since, as we report herein, SFRPs are new drug targets for
osteoporosis, certain embodiments relate to the expression of genes or
nucleic acids that encode all or portion of at least one SFRP protein.
Expression of such nucleic acids or genes in human osteoblast (hOB) cell
lines correlates with accelerated cell death and apoptosis. The hOB-
01 C1 PS-09 cells of this invention are particularly useful over other hOB
cells
since the present"-09" cells are adult osteoblast cells. In addition, these
cells are osteocytic (i.e. mature cells) in comparison to other hOB cells
which
are often osteoblastic. Furthermore, the hOB-01-C 1-PS-09 cells express
very low levels of FRP-1/SARP-2 message. Consequently, the hOB-01-C1-
PS-09 cell line will be a unique in vitro model to study the effects of FRP-
1 /SARP-2 reintroduction and over-expression. Another important feature of
the hOB-01-C1-PS-09 cells is that they can be used for both transient and
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stable transfection studies. Some of the many advantages of this cell over
the parental hOB-01-C 1 cells are as follows: the hOB-01-C 1-PS-09 cells are
truly immortal, they divide 2- to 3-times faster at 34°C, and yet they
retain
many of the pre-osteocytic characteristics of the parental cells.
The hOB-01-C1-PS-09 cells will be useful for establishing stable cell
lines that over-express potential osteoporotic drug targets. Such stable cell
lines will then be valuable for characterizing these drug targets, as well as
for developing high throughput screens and assays to identify compounds
that regulate them.
Examples
The present invention is further described by the following examples.
The examples are provided solely to illustrate the invention by reference to
specific embodiments. These exemplifications, while illustrating certain
specific aspects of the invention do not portray the limitations or
circumscribe the scope of the invention.
Example A: Generation and Analysis of hOB cells
The hOB-01-C1 cells are a conditionally-transformed cell line derived
from adult human bone that faithfully exhibit a pre-osteocytic phenotype.
These cells were transformed with a temperature-sensitive large T-antigen
(tsA 2091 and proliferate at the permissive temperature of 34°C when
the T-
antigen mutant is active; however, the cells stop dividing at the non-
permissive temperature ( > 37°C) when the T-antigen mutant is inactive.
Although the hOB-01-C1 cells are the first osteocyte cell line to be
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established and are suitable for exploratory research, they have some
disadvantages for drug discovery. Like other SV-40 large T-antigen
transformed human cell lines, the hOB-01-C1 cells undergo crisis and
senesce after 15-20 passages in culture. Thus, although often referred to as
"immortal", such cell lines are actually only "extended-life". The hOB-01-C1
cells also proliferate slowly in culture at 34°C with a doubling time
of about
once every 5 to 6 days. In order to overcome some of these draw-backs,
the hOB-01-C1-PS-09 cell line was established.
The hOB-01-C1-PS-09 cells were developed by passaging the parental
hOB-01-C1 cell line beyond the crisis point /i.e., passages 15-20) until
proliferation resumed (passages 20-251. The post-senescent cells were then
expanded in culture and sub-cloned. Clones were characterized using
reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to
measure the levels of expression of parathyroid hormone (PTHI-1 receptor
mRNA. The hOB-01-C1-PS-09 cells were chosen for further
characterization, since this clone expressed the highest level of PTH-1
receptor message at 39°C. This cell line went through approximately 20-
25
population doublings during the cloning and expansion procedure, and has
subsequently been passaged over 50-times. These cell line can be passaged
hundreds of times. Thus, the hOB-01-C1-PS-09 cells are truly an immortal
cell line.
Like the parental hOB-01-C1 cell line, the hOB-01-C1-PS-09 cells fail
to form monolayer cultures and leave spaces on the tissue culture dishes.
The cells also appear to form cell-to-cell contacts through long cellular
processes. From analysis by electron microscopy, the cells possess finger-
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like cellular projections that are reminiscent of pre-osteocytes, and these
processes form gap junctions when they contact adjacent cells. As with the
parental cell line, the hOB-01-C 1-PS-09 cells also express the tsA 209 large
T-antigen as determined by immunocytochemistry.
Unlike the parental hOB-01-C1 cell line, the hOB-01-C1-PS-09 cells
proliferate 2- to 3-times faster at the permissive temperature with a doubling
time of once every 2-3 days (Figure 22A1. However, like the parental cells,
the hOB-01-C1-PS-09 cells stop dividing at the non-permissive temperature
(Figure 22A1. This observation indicates that although the cells have passed
through the crisis point and become immortal, they still require an active T-
antigen for proliferation. Moreover, as with the parental cell line, the hOB-
01-C1-PS-09 cells require inactivation of the T-antigen in order to exhibit an
enhanced osteocytic phenotype. Alkaline phosphatase and osteocalcin are
two important markers of the osteocytic lineage. As shown in Figure 22B,
the ability of vitamin D3-treatment to up-regulate alkaline phosphatase
activity is enhanced about 4-fold when the cells are incubated at 39°C.
Furthermore, as depicted in Figure 22C, vitamin D3 is unable to induce
osteocalcin secretion from the cells at 34°C.
However, the secosteroid up-regulates production of this bone-
specific matrix protein 11-fold when the cells are incubated at 39°C.
It
should be noted that the basal levels of alkaline phosphatase expression and
osteocalcin secretion by the hOB-01-C1-PS-09 cells are similar to the
parental cell line. Thus, both morphologically and biochemically, the hOB-
01-C1-PS-09 cells resemble the parental hOB-01-C1 cells and are therefore a
reliable in vitro model to study human pre-osteocyte biology.
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As mentioned above, the hOB-01-C 1-PS-09 cell line was selected for
further characterization based on its high-level of PTH-1 receptor mRNA
expression. As shown in Figure 23, incubation of the cells for 48 hr at
39°C
increases the steady-state message levels for the PTH-1 receptor by 7-fold
when compared to cells maintained at 34°C. Since PTH-1 receptor
expression is a marker of osteoblast/osteocyte differentiation, this is
another
indication that the cells exhibit a more pronounced osteocytic phenotype at
the non-permissive temperature. Consistent with this enhanced PTH-1
receptor expression, preincubation of the hOB-01-C1-PS-09 cells for 48 hr at
39°C followed by treatment with increasing concentrations of human PTH
1-
34 (hPTH 1-34) for 10 min at 37°C generates a dose-dependent 5- to 6-
fold
increase in intracellular cyclic-adenosine monophosphate (CAMP) levels
(Figure 24). In contrast, preincubation of the cells at 34°C does not
result in
a subsequent increase in CAMP concentrations after hPTH 1-34 treatment.
Thus, both PTH-1 receptor expression and responsiveness are enhanced
following inactivation of the tsA-209 T-antigen. One potential utility for
this
cAMP assay would be the ability to characterize the activities of PTH-
analogs or -mimetics in an important target cell under conditions where
levels of PTH-1 receptor expression are dramatically altered.
In addition to vitamin D3 and PTH, the hOB-01-C1-PS-09 cells also
respond to additional bone-active agents: for example, glucocorticoids and
transforming growth factor (TGF)-f31. Treatment of the cells with the
synthetic glucocorticoid, dexamethasone, up-regulates alkaline phosphatase
activity approximately 2-fold at 34°C (Figure 25), and this effect is
once
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again enhanced when the cells are incubated at 39°C. Likewise,
treatment
of the cells with recombinant human (rh) TGF-fit at 39°C results in a
dose-
dependent decrease in hepatocyte growth factor (HGF) secretion. HGF has
been shown to act as a chemotactic factor for osteoclasts, and may
therefore play a role in regulating bone resorption.
An important property of osteocytes is the ability to respond to
mechanosensory stimulation, such as that which occurs during weight-
bearing exercise. One method to simulate this stimulatory effect in vitro is
through the use of a Flexercell Strain Unit (Flexcell International,
Hillsborough, NC1. For this experiment depicted in Figure 26, the hOB-01-
C1-PS-09 cells were seeded onto BioFlex type I collagen coated 6-well tissue
culture dishes and incubated at 34°C for 24 hr. The cells were then pre-
incubated in serum-free medium at either 34°C or 39°C for an
additional 24
hr, and then subjected to a physiologically-relevant strain (3400 ~E, 2 Hz,
7200 cycles) at 37°C using an FX-3000 Flexercell Strain Unit. After the
strain-treatment, the cells were incubated for 4.5 hr, at which time the
conditioned medium was collected and analyzed for the presence of nitric
oxide (NO). It has been previously reported that in vitro mechanosensory
stimulation or shear-stress of rodent and chick osteoblasts and osteocytes
stimulates NO production. As shown in Figure 26, mechanosensory
stimulation (i.e., "Flex") of the hOB-01-C1-PS-09 cells enhances the
production of NO by 10- to 18-fold. Consequently, these data suggest that
this cell line will be a useful in vitro model to study the molecular
mechanisms of mechanosensory stimulation.
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Additional experiments establish that the hOB-01-C 1-PS-09 cells can
be used for both transient and stable transfection studies. This cell line can
be transfected using the Tfx-20 lipofection reagent (Promega, Madison, WI).
In such an experiment, the cells are seeded into 24-well tissue culture dishes
at varying densities and then transfected with 0.25 pg/well of (3-
galactosidase and luciferase expression plasmids (total DNA = 0.5 ~g/well).
After a 48 hr incubation at either 34°C or 39°C, cell lysates
are assayed for
~3-galactosidase and luciferase activity. From such experiments, results
establish that the levels of either (3-galactosidase or luciferase expression
increase with increasing cell number. Moreover, when the luciferase
expression is normalized to (3-galactosidase expression in order to control
for
transfection efficiency, the level of luciferase expression is 2- to 3-fold
higher when the cells are incubated at 34°C. Since luciferase
expression is
under the control of the SV-40 promoter, this observation is consistent with
the tsA 209 T-antigen being inactivated at 39°C. Consequently, since
these
cells are both immortal and capable of being transfected, they can be used
to develop stable over-expressing cell lines in a human pre-osteocytic
background.
Example 1: Isolation of SFRP
The hOB SFRP gene fragment was identified using RADE (rapid
analysis of differential expression) technology as described by Shiue 1997
Drug Develop. Res. 41: 142-159, the whole of which is incorporated herein.
Three hOB cell lines (hOB-03-C5, hOB-03-CE6 and hOB-01-C1 ), representing
three distinct stages of differentiation (proliferative, mature and
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preosteocytic, respectively) were used to isolate and identify SFRP. The
hOB cell lines were established and cultured as previously described (Bodine
et al. 1996 J. Bone Miner. Res. 11: 806-819; Bodine et al. 1996
Endocrinolocw 137: 4592-4604; Bodine et al. 1997 J. Cell. Biochem. 65:
368-387). These cell lines were immortalized with a temperature-sensitive
simian virus (SV) 40 large T-antigen and exhibited a transformed phenotype
at the permissive temperature (34°C) when the T-antigen mutant was
active.
However, in contrast to osteosarcoma cells (Stein and Lian 1993 Endocrine
Rev. 14: 424-442), the hOB cell lines were faithful to the
proliferation/differentiation relationship at the nonpermissive temperature
( > 37°C) when the T-antigen mutant is inactivated. The cell lines were
seeded into 150 mm dishes at -40,000 cells/cm2 with growth medium [D-
MEM/F-12 containing 10% (v/v) heat-inactivated fetal bovine serum (FBS),
1 % (v/v) Penicillin-Streptomycin and 2 mM GIutaMAX-1 ] and incubated
overnight at 34°C. The next day, the medium was removed, the cells were
rinsed with phosphate-buffered saline (PBS/, 20 ml of serum-free medium
was added to the dishes [phenol red-free D-MEM/F-12 (Gibco/BRL)
containing 0.25% (w/v) bovine serum albumin (BSA, Pentex crystallized,
Bayer), 1 % (v/v) Penicillin-Streptomycin, 2 mM GIutaMAX-1, 50 mM
ascorbate-2-phosphate (Wakol, and 10 nM menadione sodium bisulfite
(vitamin K3)], and the dishes were incubated at 39°C for 24 hr. The
next
day, the medium was removed and the cells were treated at 39°C for an
additional 24 hr with 20 ml of fresh serum-free medium containing either
vehicle (Control), 8 nM human parathyroid hormone 1-34 (PTH), 100 nM
prostaglandin Ez (PGEZ) or 0.1 nM human transforming growth factor-(31
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(TGF-(31 ). PTH, PGEz and TGF-(31 are known osteogenic agents (Whitfield
and Morley 1995 Trends Pharmaceut. Sci. 16:382-386; Jee and Ma 1997
Bone 21:297-304; Centrella et al. 1994 Endocrine Rev. 15:27-39). After
the treatment period, the dishes were rinsed with PBS and total cellular RNA
was isolated from the nontreated and treated cells using TRlzol according to
the manufacturers instructions (GibcoBRL). RADE was then performed with
the isolated RNA samples as above; the regulated gene fragments were
identified, cloned and sequenced. These experiments identified a total of 82
differentially expressed genes. A BLAST (basic local alignment search tool)
search of the public data bases was performed on the RADE-obtained gene
fragments; one of the gene fragments was highly homologous to mouse
SFRP-1. This gene fragment was identified during BADE using the following
primer pair: 5'-AAGCTTTTTTTTTTTA-3' (HT1 1 A13' end (reverse primer) and
5'-AAGCTTGATTGCC-3' (H-AP1 ) 5' end (forward primer), which
sequences are SEQ ID NO 11 and SEQ ID NO 12, respectively. The
expression and regulation of the gene fragment having homology to mouse
cDNA SFRP-1 was confirmed by Northern blot analysis.
Example 2: Characterization of hOB SFRP.
Figure 1 depicts a summary of the RADE results. The hOB SFRP
gene fragment (indicated by the arrows) was strongly up-regulated by PGEZ
in the proliferative-stage (hOB-03-C5) and maturation-stage (hOB-03-CE6)
hOB cell lines and down-regulated by TGF-(31 in the pre-osteocytic (hOB-01-
C1 ) cell line. Moreover, basal expression of this gene was dramatically
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increased in the pre-osteocytic cells, suggesting that hOB SFRP gene
expression is linked to the osteoblast differentiation process.
Example 3: Seguence Analysis of the hOB SFRP Gene Fragment
The hOB SFRP gene fragment identified in Example 1, above,
containing 276 base pair (bp) was cloned, sequenced, and a further
subjected to a BLAST search of the public databases. This search revealed
that this gene was homologous to two other previously identified cDNAs.
Sequence alignment indicated that the hOB SFRP gene fragment shared 77%
sequence identity to the 3'-end of mouse SFRP-1 gene (GeneBankT""
Accession #U88566; Rattner et al. 1997 Proc. Natl. Acad. Sci. USA 94:
2859-2863). The hOB SFRP gene fragment also exhibited significant
homology (87%) to the 3'-end of a related bovine cDNA called frizzled-
related protein A (FrzA, GeneBankT"" Accession #U859451. Additionally,
the hOB SFRP gene fragment was very homologous to at least three
expressed sequence tags (ESTs), human clone TM010 (GeneBankT""
Accession #U54715), human CA11 tumor suppresser (GeneBankT""
Accession #U69122) and a human infant brain EST (GeneBankT"" Accession
#H 16753, H 168611.
Example 4: Regulation of the hOB SFRP by Osteogenic Agents
In order to confirm the regulation of hOB SFRP gene expression by
different osteogenic agents (i.e., the DNA fragment identified in Example 1,
abovel, the hOB cell lines were treated with PTH, PGEZ, and TGF-X31; RNA
was then isolated for Northern hybridizations. The results are shown in
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Figures 2 through 5. The experiments were performed as follows. The hOB
cell lines were seeded into 150 mm dishes and treated as described in
Example 1, except that polyA + RNA was isolated from total cellular RNA
using Oligotex mRNA maxi kits as described by the manufacturer (Qiagen).
Northern blot analysis was performed using either the excised RADE hOB
SFRP gene fragment, the cloned hOB SFRP gene fragment or the cloned full-
length hOB SFRP cDNA as a 32P-labeled probe (as described in Bodine et al.
1996 J. Bone Miner. Res. 1 1: 806-819, which is incorporated herein). Each
of these probes detected a 4.4-4.6 kb message in the hOB cells. Expression
of the hOB SFRP mRNA was normalized to either glyceraldehyde phosphate
dehydrogenase (GAPDH) mRNA or (3-actin mRNA using the corresponding
a2P-DNA probes. Treatment of the proliferative-stage hOB-03-C5 cells with
100 nM PGEZ for 24 hr completely up-regulated the expression of a --4.6
kilobase pair (kb) message (Figure 2), confirming the regulation of this gene
by PGE2. When the cloned hOB SRFP gene fragment was used as a probe, a
predominant mRNA of -4.4 kb was observed in cells treated with PGE2,
confirming that this message is indeed the SFRP gene (Figure 3). This
mRNA corresponds in size to the transcript for the human FRP-1 /SARP-2
gene (Finch et al. 1997 Proc. Natl. Acad. Sci. USA 94: 6770-6775;
Melkonyan et al. 1997 Proc. Natl. Acad. Sci. USA 94: 13636-13641 ).
Northern blot analysis also confirmed the up-regulation of hOB SFRP mRNA
expression in maturation-stage hOB-03-CE6 cells treated with PGE2 (Figure
4). In addition, basal expression of this gene was elevated in pre-osteocytic
hOB-01-C1 cells. This basal level expression was suppressed by 35%
following treatment with 8 nM PTH. Furthermore, PGEz treatment of the
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maturation-stage hOB-03-CE6 cells elevated SFRP expression to the level
that was expressed basely by the pre-osteocytic cells, implying that up-
regulation of SFRP by PGE2 in the osteoblastic cells is related to the
enhancement of cellular differentiation. Lastly, treatment of the pre-
S osteocytic hOB-01-C1 cells with 100 pM TGF-(31 for 24 hr suppressed hOB
SFRP mRNA levels by 80% (Figure 51.
To confirm that hOB SFRP message levels change with increasing
cellular differentiation, total RNA was isolated from the pre-osteoblastic hOB-
03-C5 cells, the mature osteblastic hOB-03-CE6 cells, the pre-osteocytic
hOB-01-C1 cells and the mature osteocytic hOB-05-T1 cells. Basal SFRP
mRNA levels were then measured by TaqMan quantitative RT-PCR. When
compared to the hOB-03-C5 cells, basal SFRP message levels increased
about 4-fold in the hOB-03-CE6 cells and about 23-fold in the hOB-01-C1
cells. On the other hand, SFRP mRNA levels declined to about 0.5-fold in
the hOB-05-T1 cells. Thus, of the cells in the osteoblast lineage, the pre-
osteocyte appears to express the highest levels of hOB SFRP message.
Example 5: Kinetics of hOB SFRP Expression
Proliferative hOB-03-C5 cells were seeded into 150 mm dishes and
treated with increasing concentrations of PGEZ for 24 hr or with 100 nM
PGEZ for varying lengths of time as described in Example 1. PolyA+ RNA
Northern blot analysis was performed with the excised RADE hOB SFRP 276
by gene fragment or a cloned 1.1 kb hOB SRFP gene fragment as a 32P-
labeled probe. Treatment of the proliferative-stage hOB-03-C5 cells with
increasing concentrations of PGEZ up-regulated hOB SFRP mRNA expression
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in a dose-dependent manner with an ECSO of approximately 8 nM. Likewise,
treatment of the mature hOB-03-CE6 cells with increasing concentrations of
PGE2 also up-regulated hOB SFRP mRNA levels in a dose-dependent manner,
although the EC5° of PGEZ for this response was about 10-times higher
than
in the hOB-03-C5 cells.
Treatment of the hOB-03-C5 cells with 100 nM PGEZ for 2 to 24 hr
up-regulated hOB SFRP mRNA expression in a time-dependent manner. A
significant increase in SFRP gene expression was observed after 2 to 4 hr of
treatment, and the steady-state mRNA levels continued to increase up to 24
hr after the addition of PGE2 to the cell culture medium. These results
suggest that SFRP may be a late-response gene to PGEZ treatment of the
hOB cells, and implies that it may be under the secondary control of another
gene product. Similar results were also obtained with the mature hOB-03-
CE6 cells, although the fold-increase in expression of the hOB SFRP message
was not as great as it was in the hOB-03-C5.
In a similar manner, the hOB-01-C1 cells were seeded into 100 mm
dishes and treated for 24 hr with increasing concentrations of TGF-~i1. Total
RNA was then isolated from the cells, and hOB SFRP mRNA was measured
by TaqMan quantitative RT-PCR. TGF-(31 suppressed SFRP gene expression
by approximately 70% in a dose-dependent manner with an IC5° of about 4
pM.
In addition to PGE2, treatment of either the hOB-03-C5 cells or the
hOB-03-CE6 cells with interleukin (IL)-1 ~3, 9-cis-retinoic acid or all-trans-
retinoic acid also increases hOB SFRP mRNA levels. On the other hand, as
with TGF-(31, treatment of various hOB cell lines with PTH, bone
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morphogenetic protein (BMP)-2, insulin-like growth factor (IGF)-I, 17(3-
estradiol (17(3-Ezl, vitamin D3 (VD3), dexamethasone (Dex), or fetal bovine
serum (FBS) suppresses hOB SFRP message expression. Overall, there is a
good (albeit imperfect) correlation between the abilities of these various
agents to either increase or decrease hOB SFRP expression, and their ability
to either enhance or suppress osteoblast/osteocyte apoptosis (see for
example Manolagas 2000 Endocrine Reviews 21: 115-137).
Example 6: Tissue Distribution of hOB SFRP
In order to determine the tissue distribution for the expression of the
hOB SFRP gene, multiple human tissue polylA) + RNA Northern blots
(obtained from Clonetech) were probed with the excised RADE hOB SFRP
gene fragment as described in Example 4. When the RADE fragment was
used to probe the poly (A) + RNA Northern blots, a --4.4 kb transcript was
expressed by several tissues (Figure 6). When these results were normalized
to ~i-actin, SFRP expression was ranked as follows:
Kidney > heart > placenta > liver -
skeletal muscle = stomach = thyroid gland
> adrenal gland = testis = uterus = small
intestine = pancreas = brain > trachea =
spinal cord = prostate = colon > spleen >
lung = lymph node = bone marrow;
No expression was observed in thymus and peripheral blood lymphocytes.
This expression pattern is similar to the human FRP-1 /SARP-2 gene (Finch
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et al. 1997 Proc. Natl. Acad. Sci. USA 94: 6770-6775; Melkonyan et al.
1997 Proc. Natl. Acad. Sci. USA 94: 13636-13641 ).
Example 7: Distribution of SFRP in Osteoblast Cell Lines
In addition to the hOB cell lines from which SFRP was initially
identified, additional in vitro human osteoblast models were examined for the
presence of the gene. SaOS-2 human osteosarcoma osteoblast-like cells
were obtained from the American Type-Culture Collection (ATCC) and were
cultured at 37°C in McCoy's 5A Modified medium containing 10% FBS, 1
(v/v) Penicillin-Streptomycin and 2 mM GIutaMAX-1. Likewise, explant
cultures of normal human osteoblasts (hOBs) were established from
cancellous bone chips as previously described (Bodine et al. 1996 J. Bone
Miner. Res. 11: 806-819, which is incorporated herein by reference). The
cells were then seeded into 150 mm dishes and treated as described in
Examples 1 and 4, except that the cells were incubated at 37°C
instead of
39°C. PolyA + RNA Northern blot analysis was performed using the cloned
1.1 kb hOB SRFP gene fragment as a 32P-label). SaOS-2 cells expressed
relatively low basal levels of SFRP mRNA which was not regulated by
treatment with either PTH, PGEz or TGF-[31 (Figure 7). It was difficult to
quantify expression of this gene in these cells, since the level of expression
was low. In contrast, normal hOB cells expressed higher basal levels of
SFRP message, and treatment of the cells with 100 nM PGEZ for 24 hr
appeared to slightly up-regulate the steady-state levels of this mRNA (- 1.3-
fold). TaqMan quantitative RT-PCR analysis of these RNA samples indicated
that PGEz upregulated SFRP 10-fold in the hOB cells. Due to the low basal
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level expression in osteosarcoma cells, these cells may not be satisfactory in
vitro models to study the regulation of the SFRP gene. Since the gene is
expressed and regulated by PGEZ in cultures of normal human osteoblasts,
the use of the hOB cell lines described in the present invention is validated
for in vitro osteoblast models. Northern blot analysis and RT-PCR of total
RNA isolated from a human giant cell tumor of bone failed to detect
expression of the hOB SFRP mRNA in this tissue. These results suggest that
osteoclast-like cells may not express this gene.
Example 8: Isolation of Full Length hOB SFRP cDNA.
Since the cloned hOB SFRP gene fragment from RADE was identical
to several human ESTs, an analysis of the EST database was performed in
order to assemble the full-length cDNA for the hOB gene. This analysis
suggested that the hOB SFRP was in fact the known human gene, FRP-1
(also called the secreted apopotisis-related protein-2 or SARP-2). Based on
this analysis, and the observation that the mouse SFRP-1 gene is apparently
homologous to the human FRP-1 gene and the human SARP-2 gene (Rattner
et al. 1997 Proc. Natl. Acad. Sci. USA 94: 2859-2863; Finch et al. 1997
Proc. Natl. Acad. Sci. USA 94: 6770-6775; Melkonyan et al. 1997 Proc.
Natl. Acad. Sci. USA 94: 13636-13641 ), an RT-PCR-based strategy was
designed to obtain the full-length 1.1 kb hOB SFRP cDNA from both human
placenta RNA and PGEz-treated hOB-03-CE6 cell RNA. RT-PCR was
performed using 1 ~g of total RNA, primers that spanned the coding region
of hFRP-1 /SARP-2 (forward primer: 5'-GCTGGGGACTGCGCCTTTTGT-3'
SEQ ID NO 13; reverse primer: 5'-CCTGCCCCCGGGAGAATCACTTA-3'
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SEQ ID NO 14), 35 cycles of PCR, and the Advantage-GC PCR kit
(Clonetech) according to the manufacturer's instructions. In order to detect
expression of the mRNA, a Southern blot analysis was performed with the
RT-PCR products using a 3zP-oligonucleotide probe which specifically
hybridized to bases 501 to 530 of the hFRP-1 /hSARP-2 coding region (refer
to Bodine et al. 1997 J. Cell. Biochem. 65: 368-387 for experimental details
concerning RT-PCR and Southern hybridizationsl. Full-length 1.1 kb cDNA
for the hOB SFRP was isolated and was up-regulated by 100 nM PGEz-
treatment of the hOB-03-CE6 cells for 24 hr (Figure 8). Likewise, RT-PCR of
total RNA isolated from hOB-03-C5 cells treated with PGEZ identified a 2.2
kb cDNA which spanned from the 5'-region of the hFRP-1 /SARP-2 cDNA to
the 276 by RADE fragment at the 3'-end. These cDNA fragments were
cloned into either the pcDNA3.1 (Invitrogen) mammalian expression vector
(1.1 kb cDNA) or the TA (Invitrogen) cloning vector (2.2 kb cDNA) and
sequenced. Sequence analysis of the hOB SFRP 1.1 kb /SEQ. ID. NO.:
1land 2.2 kb cDNAs enabled the assembly of a 2.6 kb cDNA which included
the transcription start site at the 5'-end and the RADE fragment at the 3'-
end. A BLAST search of the public databases using the 1.1 kb cDNA
indicated that it essentially was identical to human FRP-1 /SARP-2. The
deduced amino acid sequence of the coding region of the SFRP cDNA is
shown in SEQ. ID. NO. 2. The sequence contains one amino acid difference
from the published sequence for human SARP-2: alanine 174 instead of
proline at this position (Melkonyan et al. 1997 Proc. Natl. Acad. Sci. USA
94: 13636-136411.
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Example 9: Characterization of the Apoptotic Activity of hOB
Since the cloned full-length hOB SFRP gene was identical to human
SFRP-1 /FRP-1 /SARP-2, the biological role of this gene in the hOB was
investigated to determine whether the gene product regulated hOB cell
viability and Wnt signaling (Figures 9-16).
As shown in Figure 9, hOB cells were seeded at 200,000 cells/well
into 6-well dishes and incubated at 34°C. The next day, one set of
plates
were rinsed with PBS, trypsinized, and baseline cell number (and mean cell
volume) was determined with a Coulter Multisizer as previously described in
Bodine et al. 1996 J. Bone Miner. Res. 11: 806-819, which is incorporated
herein by reference. The other set of plates was placed at the non-
permissive temperature of 39°C, and cell number was determined 6 days
later (the medium was changed on day 3). The hOB-03-C5 cells, which are
in the proliferative-stage of osteoblast differentiation, divided slowly at
39°C
and cell number increased by 60 to 80% after 6 days; this rate of cell
division was similar to explant cultures of normal hOB cells (Bodine et al.
1996 Endocrinology 137: 4592-4604). In contrast, the maturation-stage
hOB-03-CE6 cells stopped dividing at the non-permissive temperature and
cell number remained constant, while the pre-osteocytic hOB-01-C1 cells
slowly died at 39°C such that fewer than 40% of the cells remained
alive
after 6 days. As noted previously, overexpression of SARP-2 in MCF-7
breast cancer cells accelerated the rate of cell death. Consistent with this
observation, and as shown in Figures 1, 4 and 5, basal SFRP-1 /FRP-1 /SARP-
2 mRNA expression dramatically increased in the pre-osteocyte hOB-01-C1
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cells when compared to the proliferative hOB-03-C5 and mature hOB-03-CE6
cell lines.
The hypothesis that up-regulation of SFRP-1 /FRP-1 /SARP-2 gene
expression accelerates hOB cell death, while down-regulation of SFRP-1 /FRP-
1 /SARP-2 gene expression suppresses cell death, was next examined.
These results are depicted in Figure 10. hOB-03-C5, hOB-03-CE6 or hOB-
01-C1 cells were seeded with growth medium at about 200,000 cells per
well into 6-well plates and incubated at 34°C overnight. The next day,
the
6-well plates were rinsed with PBS, placed in BSA-medium, and treated at
39°C in the absence or presence of either 100 nM PGEZ (in order to up-
regulate SFRP-1 /FRP-1 /SARP-2 steady-state mRNA levels; panels A & B), or
0.01-1.0 nM TGF-(31 (in order to down-regulate SFRP-1 /FRP-1 /SARP-2
message levels; panel C). Incubating cells in serum-free medium is a
common method to induce apoptosis (Melkonyan et al. 1997 Proc. Natl.
Acad. Sci. USA 94: 13636-13641 ~, and the hOB-03-C5, hOB-03-CE6 and
hOB-01-C1 cell lines all stopped dividing and gradually died under these
conditions. However, the rate of cell death was significantly accelerated
when the hOB-03-C5 and hOB-03-CE6 cells were treated with PGE2, such
that over 40% fewer cells remained alive after 6-days of treatment. In
contrast, treatment of the hOB-01-C1 cells with TGF-(31 increased cell
viability about 2-fold in a dose-dependent manner. Treatment of the cells
with PGEZ not only accelerated cell death, but also significantly reduced the
mean cell volume by 10 to 20%. This observation was consistent with the
induction of apoptosis, which is known to result in cytoplasmic blebbing, the
loss of water and a decrease in cell volume (Mesner and Kaufmann 1997 in
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Advances in Pharmacology, vol. 41, pp. 57-88). Also consistent with the
induction of apoptosis, PGEZ-treatment of the hOB-03-C5 cells resulted in
the generation of histone-associated DNA fragments. Finally, treatment of
the hOB-03-C5 cells with PGEZ increased annexin V (a specific marker for
apoptosis) binding to the cell as measured by flow cytometry (Figure 1 1 ).
Example 10: Reversal of Cell Death Induction
Reversal of cell death using an antisense oligonucleotide to SFRP-
1 /FRP-1 /SARP-2 is demonstrated in Figure 12 using hOB-03-C5 and hOB-03-
CE6. These experiments were performed in a similar manner to the ones
depicted in Figure 10. However, for these experiments, the cells were co-
treated with either vehicle control (i.e., 0.1 % ethanol) or PGEZ in the
absence or presence of sense (control) or antisense initiation-site directed
phosphorothioate oligonucleotides to human SARP-2. The results are
presented as either the % relative to the day 0 control (i.e., --200,000 cells
per well of a 6 well plate) or as the % relative to the vehicle treated
control.
The results of this experiment indicate that co-treatment of the hOB cells
with the antisense oligonucleotide to SARP-2 reversed the ability of PGE2 to
accelerate the rate of cell death, while co-treatment with the sense (control)
oligonucleotide has no effect on this process. In addition, co-treatment of
the cells with PGE2 and an anti-peptide antibody to SARP-2 blocked the
ability of PGEZ to induce hOB cell death. The sequences for the sense and
antisense oligonucleotides to human SFRP-1 /SARP-2 are as follows:
Sense: 5'-GGCATGGGCATCGGGCGC-3' (SEQ ID NO. 15)
Antisense: 5'-GCGCCCGATGCCCATGCC-3' (SEQ ID NO. 16)
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Example 1 1: Overexpression of SFRP Accelerates hOB Cell Death
Figure 13 shows the results of a cell viability experiment with the
hOB-01-C1-PS-09 cells using the Coulter cell counter. For these
experiments, the cells were stably transfected with either an hOB SFRP
cDNA (i.e., SFRP-1 /FRP-1 /SARP-2) mammalian expression plasmid or the
empty vector (i.e., pcDNA3.1, which is obtained from Invitrogen of Carlsbad,
CA). Standard cloning technigues were used (See Ausubel et al. 1997 Short
Protocols in M < olecular Biology, 3'd edition, Wiley New York}). The results
are presented as the % of the day 0 control cells. The results of this
experiment indicated that overexpression of SFRP-1 /FRP-1 /SARP-2 by the
hOB cells accelerates the rate of cell death when compared to the empty
vector which has no effect on this process. Autoradiograms of a Northern
blot of total RNA isolated from the empty vector (v) or SFRP (S) expressing
cells demonstrated that the hOB cells overexpressed the SFRP gene as
expected. In addition, TaqMan quantitative Rt-PCR analysis indicated that
the SFRP overexpressing cells expressed 5-6 times more SFRP mRNA than
the empty-vector expressing cell.
In order to improve the rate of cell death that was induced by SFRP-
1 /FRP-1 /SARP-2, the overexpressing hOB-01-C 1-PS-09 were subcloned and
characterized (Figure 14). TaqMan quantitative RT-PCR analysis indicated
that one subclone (SARP-2 Clone #1 ) expressed 50-60-times more SFRP-
1 /FRP-1 /SARP-2 mRNA than the pcDNA3.1 (empty vector) expressing cells.
Likewise, the SARP-2 Clone #1 cells died at greatly accelerated rate (t"z =
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39.2 hr) in BSA-medium when compared to the empty vector expressing
cells (t"2 = ~ 120 hrl.
Example 12: Effect of hOB SFRP on Wnt Activity
In order to determine if the up-regulation of SFRP-1 /FRP-1 /SARP-2
gene expression results in an antagonism of the Wnt signaling pathway,
hOB-03-CE6 cells were treated with PGEZ and the resulting cells probed with
(3-catenin monoclonal antibody. Overexpression of Wnt proteins in cells up-
regulates a signaling protein known as (3-catenin (reviewed in Moon et al.
1997 Cell 88: 725-728; Barth et al. 1997 Curr. Opin. Cell Biol. 9: 683-690;
and Nusse 1997 Cell 89: 321-323). Moreover, overexpression of SFRP-
1 /FRP-1 /SARP-2 in MCF-7 cells down-regulated (3-catenin levels, which is
consistent with an antagonism of Wnt activity (Melkonyan et al. 1997 Proc.
Natl. Acad. Sci. USA 94: 13636-136411. Therefore, hOB-03-CE6 cells were
plated and treated with PGEZ as described in Example 1, except that total
cellular protein was extracted and a Western blot analysis for (3-catenin was
performed using a monoclonal antibody to the protein (Transduction
Laboratories) as previously described (Bodine et al. 1996 Endocrinology 137:
4592-4604; Melkonyan et al. 1997 Proc. Natl. Acad. Sci. USA 94: 13636-
13641 ). Consistent with up-regulation of SFRP-1 /FRP-1 /SARP-2 steady-
state mRNA levels, treatment of hOB-03-CE6 cells with 100 nM PGEZ for 24
hr down-regulated (3-catenin protein levels indicating an antagonism of Wnt
activity (Figure 151. In addition, cotransfecting SFRP-1 /FRP-1 /SARP-2 cDNA
into either hOB-01-C1-PS-09 or hOB-02-C1-PS-02 cells down-regulated TCF-
luciferase expression which is an authentic measurement of Wnt signaling
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and (3-catenin nuclear activity (Figure 16) (e.g., Bafico et al. 1999 J. Biol.
Chem. 274: 16180-16187). Both human and rat SFRP-1 /FRP-1 /SARP-2, as
well as human Frzb-1 /FrzB/Fritz, suppressed TCF-luciferase activity in the
hOB cells.
S All together, these observations suggest that a Wnt proteinls)
prolongs the life of human osteoblasts in vitro and that antagonism of Wnt
signaling by SFRP-1 /FRP-1 /SARP-2 promotes osteoblast cell death. Thus, an
inhibitor of SFRP-1 /FRP-1 /SARP-2 function may increase osteoblast/pre-
osteocyte survival and therefore enhance bone formation in vivo.
Using several methods to characterize Wnt expression in the hOB
cells (e.g., RT-PCR, GeneChip analysis and cDNA cloning), we have evidence
that these cell lines express, to varying degrees, Wnt-2B/13, -3, -4, -5A, and
-11. Anyone or all of these Wnts could be involved in prolonging hOB cell
life. Wnt-2B/13 is also known as Wnt-x.
Example 13: Use of hOB SFRP in a Screening Method for Anabolic A
A new screening paradigm for an anabolic bone agent using SFRP is
designed. As outlined in Figure 17, this screening paradigm uses the hOB
cells and SFRP-1 /FRP-1 /SARP-2 to identify compounds that are capable of
preventing or slowing osteoblast cell death. Such compounds act by
blocking the ability of SFRP-1 /FRP-1 /SARP-2 to accelerate hOB cell death.
These compounds bind to SFRP-1 /FRP-1 /SARP-2 and prevent it from binding
a Wnt protein, or they may bind to a Wnt and prevent it from binding to
SFRP-1 /FRP-1 /SARP-2. If SFRP-1 /FRP-1 /SARP-2 has activities that are
independent of Wnt-binding (e.g., binding to a cell surface receptor), then
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these compounds could also act by preventing this Wnt-independent
function as well.
For the initial assay of the screening paradigm outlined in Figure 17,
compounds are incubated with an hOB cell line and SFRP-1 /FRP-1 /SARP-2.
This assay could use purified or partially purified SFRP-1 /FRP-1 /SARP-2
protein, or conditioned-media or cell extracts that contained SFRP-1 /FRP-
1 /SARP-2. The hOB cell line could be one that naturally expressed high
basal levels of SFRP-1/FRP-1/SARP-2 /e.g., hOB-01-C1 cells), that
transiently or stably overexpressed SFRP-1/FRP-1/SARP-2 (e.g., hOB-01-C1-
PS-09 cellsl, or that stably or naturally expressed SFRP-1 /FRP-1 /SARP-2 in a
conditional manner (e.g., hOB-03-C5 cells treated with PGEz). As a
measurement of hOB cell death, assays quantify cell number (e.g., MTT or
MTS dye-conversion or CyQuant DNA flourescence) or apoptosis (e.g., DNA
fragmentation or annexin V binding) could be used. CyQuant kits were
purchased from Molecular Probes (Eugene, OR).
An example of a high-throughput screening assay (HTS) for SFRP-
1 /FRP-1 /SARP-2 inhibitors is depicted in Figure 18. For this assay, either
empty vector (pcDNA3.1 ) or SFRP-1 /FRP-1 /SARP-2 (SARP-2 #11 stable
overexpressing hOB-01-C1-PS-09 cells were seeded at 5000 cells per well
into 96-well plates using growth medium. After a brief 6 hr incubation at
34°C, the wells were rinsed with PBS and incubated in BSA-medium at
39°C
for 3 days. At the end of the incubation, the wells were rinsed again with
PBS and then assayed for DNA content using the CyQuant DNA
fluorescence assay (Molecular Probes). When compared to the empty vector
cells, the SARP-2 overexpressing cells died faster at 39°C such that
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days, only 20-30% of the cells were still alive. In contrast, 50-60% of the
empty vector cells survived the incubation. When the SARP-2
overexpressing cells were treated with an antipeptide antisera (AS)
generated to amino acids 217-231 of SFRP-1 /FRP-1 /SARP-2, 50-60% of the
cells were alive after 3 days. This indicates that inhibition of SFRP-1 /FRP-
1 /SARP-2 protein function prevents it from accelerating hOB cell death. As
controls, the pre-immune serum had no effect on the SARP-2 overexpressing
cells, and neither the pre-immune nor the immune sera affected the empty
vector expressing cells.
Compounds that blocked hOB cell death induced by SFRP-1 /FRP-
1 /SARP-2 would then move on to additional in vitro assays. These assays
measure the ability of these compounds to block hOB cell death in an SFRP-
1 /FRP-1 /SARP-2-dependent or independent manner, and they would also
determine the potency and efficacy of these compounds for these effects.
Additional assays are designed to determine the cell selectivity of these
compounds for these effects /e.g., by using MCF-7 or other cellsl, as well as
the specificity of these compounds for SFRP-1 /FRP-1 /SARP-2 versus another
member of the SFRP/SARP family (e.g., FrzB/Fritz, SARP-1, or SARP-3).
Additional assays could also be used to determine if these compounds
regulate down-stream signaling events involved in apoptosis /e.g., caspase
activity) or Wnt activity (e.g., ~i-catenin levels and function via the TCF-
luciferase assayl. Finally, compounds that exhibited appropriate activities in
these in vitro assays would then be used in a variety of animal models for
bone formation, osteopenia, or osteoporosis (e.g., ovariectomized rats or
micel. A compound that inhibited osteoblast/osteocyte apoptosis would
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conceivably be an anabolic bone agent by prolonging the lives of these cells
and thereby either increasing the amount of bone matrix that is synthesized
and mineralized and/or maintaining the integrity of the bone.
It is clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible in light of
the above teachings and therefore are within the scope of the appended
claims.
Examale 14: Development and Use of SFRP-1 Knock-Out Mice
As stated in Example 13, a compound that inhibited
osteoblast/osteocyte apoptosis would conceivably be an anabolic bone agent
by prolonging the lives of these cells and thereby either increasing the
amount of bone matrix that is synthesized and mineralized and/or
maintaining the integrity of the bone. In order to test this hypothesis and
determine if SFRP-1 /FRP-1 /SARP-2 affects the skeleton, to SFRP-1 "89 mice
were prepared (See Wattler et al. 1999. BioTechniques 26: 1150-11601.
Deleting the SFRP-1 /FRP-1 /SARP-2 gene from mice would be akin to
inhibiting its function with a drug, and this process allows us to validate
this
gene/protein as a potential drug target for osteoporosis.
As summarized in Figure 19, the SFRP-1 knock-out mice were
generated by substituting exon 1 of the mouse SFRP-1 gene with a (3-
galactosidase reporter gene/neomycin drug resistance gene expression
cassette. As shown in Figure 20, Northern blot analysis of poly A + RNA
isolated from either female of male kidneys (age 16-18 weeks) demonstrated
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high levels of SFRP-1 mRNA expression (4.4 kb) in the wild-type (WT)
control mice, but a complete absence of gene expression in the knock-out
(KO) mice.
As shown in Figure 21, micro computerized tomography (micro-CT)
was used to characterize the trabecular bone architecture of the distal
femurs from male and female wild-type control (+/+) and knock-out (-/-)
mice (for a review of this technique, see Genant et al. 1999 Bone 25: 149-
152 and Odgaard 1997 Bone 20: 315-3281. In the 20 week old males
(panel A), the -/- mice had 31 % more trabecular bone volume (BV/TV) and
an 8% increase in trabecular thickness (Tb. Th.) when compared to the +/+
control mice. In the 26-27 week old females (panel B), the -/- mice had a .
91 % increase in trabecular connectivity density (Conn. Den.), a 16%
increase in trabecular number (Tb. N.) and a 16% decrease in trabecular
spacing (Tb. Sp.) when compared to the +/+ control mice. Thus, in
support of our hypothesis, these results demonstrate that deletion of the
SFRP-1 gene in mice leads to increased parameters of trabecular bone
formation (P. J. Meunier 1995 Bone Histomorphometry, in Osteoporosis:
Etiology, Diagnosis, and Management, 2"d edition, B. L. Riggs & L. J. Melton
III, editors, Lippincott-Raven, Philadelphia, pages 299-3181.
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