U.S. patent application number 10/155567 was filed with the patent office on 2003-11-27 for calbindin-d28k protection against glucocorticoid induced cell death.
This patent application is currently assigned to University of Medicine & Dentistry of New Jersey. Invention is credited to Christakos, Sylvia.
Application Number | 20030219421 10/155567 |
Document ID | / |
Family ID | 29549102 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219421 |
Kind Code |
A1 |
Christakos, Sylvia |
November 27, 2003 |
Calbindin-D28k protection against glucocorticoid induced cell
death
Abstract
The present invention provides novel compositions containing a
calbindin-D.sub.28k therapeutic element, which is involved in the
regulation of apoptosis, and may be administered for the prevention
of an abnormal apoptosis response in cells. In particular the
compositions and methods of the present invention may be used for
the prevention or induction of apoptosis in such cells types as
osteoblasts and osteocytes. Specifically, the compositions and
methods of the present invention are useful for the prevention of
diseases associated with glucocorticoid induced cell death.
Specifically, the compositions and methods of the present invention
may be useful in the prevention of glucocorticoid induced cell
death in osteoblasts and the treatment of such conditions as
glucocorticoid induced osteoporosis.
Inventors: |
Christakos, Sylvia;
(Mendham, NJ) |
Correspondence
Address: |
PERKINS COIE LLP
POST OFFICE BOX 1208
SEATTLE
WA
98111-1208
US
|
Assignee: |
University of Medicine &
Dentistry of New Jersey
|
Family ID: |
29549102 |
Appl. No.: |
10/155567 |
Filed: |
May 23, 2002 |
Current U.S.
Class: |
424/93.21 ;
435/320.1; 435/368; 435/456; 536/23.5 |
Current CPC
Class: |
C07K 14/4747 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
424/93.21 ;
435/320.1; 435/368; 536/23.5; 435/456 |
International
Class: |
A61K 048/00; C07H
021/04; C12P 021/02; C12N 015/86; C12N 005/08 |
Goverment Interests
[0001] This invention was made with government support by the
following National Institute of Health Grant DK38961. The
government may own certain rights in the present invention
Claims
What is claimed is:
1. A vector suitable for use in a human comprising a
polynucleotide, wherein said polynucleotide encodes a
calbindin-D.sub.28k polypeptide.
2. The vector of claim 1, wherein said polynucleotide sequence
encoding the CALBINDIN-D.sub.28k polypeptide is SEQ ID NO:1.
3. The vector of claim 1, wherein the polynucleotide sequence has
at least 70% identity to SEQ ID NO:1, said identity being
calculated over the entire length of SEQ ID NO:1.
4. The vector of claim 1, wherein the polynucleotide sequence is
identical to SEQ ID NO:1.
5. The vector of claim 1, wherein the CALBINDIN-D.sub.28k
polypeptide comprises an amino acid sequence of SEQ ID NO:2.
6. The vector of claim 5, wherein the CALBINDIN-D.sub.28k
polypeptide comprises an amino acid sequence that has at least 70%
identity to SEQ ID NO:2, said identity being calculated over the
entire length of SEQ ID NO:2.
7. The vector of claim 1, wherein said polynucleotide encodes a
calbindin-D.sub.28k antisense sequence to a nucleotide sequence
encoding a CALBINDIN-D.sub.28k polypeptide.
8. The vector of claim 7, wherein the polynucleotide sequence has
at least 70% identity to the antisense polynucleotide sequence of
claim 7, said identity being calculated over the entire length of
the sequence.
9. A vector comprising the polynucleotide sequence of claim 7,
wherein said vector is capable of inhibiting the expression of a
CALBINDIN-D.sub.28k polypeptide when said vector is present in a
compatible host cell.
10. A pharmaceutical composition suitable for use in a human
comprising a biologically effective amount of a CALBINDIN-D.sub.28k
polynucleotide and an acceptable carrier.
11. The composition of claim 10, wherein the CALBINDIN-D.sub.28k
polynucleotide sequence is substantially similar to SEQ ID NO:
1.
12. The composition of claim 10, wherein the CALBINDIN-D.sub.28k
polynucleotide sequence is an antisense sequence to SEQ ID NO:
1
13. A pharmaceutical composition suitable for use in a human
comprising a biologically effective amount of a CALBINDIN-D.sub.28k
polypeptide and an acceptable carrier.
14. The composition of claim 13, wherein the CALBINDIN-D.sub.28k
polypeptide sequence is substantially similar to SEQ ID NO: 2.
15. A method of treating a disease associated with abnormal
glucocorticoid induced cell death comprising the administration of
a pharmaceutical composition comprising a biologically effective
amount of a CALBINDIN-D.sub.28k polynucleotide and an acceptable
carrier.
16. The method of claim 15, wherein the disease comprises
glucocorticoid induced osteoporosis.
17. A method of treating a disease associated with abnormal
glucocorticoid induced cell death comprising the administration of
a pharmaceutical composition comprising a biologically effective
amount of a CALBINDIN-D.sub.28k polypeptide and an acceptable
carrier.
18. The method of claim 17, wherein the disease comprises
glucocorticoid induced osteoporosis.
19. A method of treating a disease associated with a lack of normal
cell death comprising the administration of a pharmaceutical
composition comprising a biologically effective amount of a
polynucleotide coding for the antisense sequence to SEQ. ID. No. 1,
and an acceptable carrier.
20. The method of claim 19, wherein the disease is selected from
the group consisting of osteoblastic cancer, osteocytic cancer,
prostrate cancer, lymphocytic cancer, leukemia and lymphoma.
21. A vector for the delivery of a calbindin-D.sub.28k therapeutic
element to a human for the treatment of diseases associated with
glucocorticoid induced cell death or an abnormal decrease in cell
death, wherein the vector comprises an expression cassette encoding
the calbindin-D.sub.28k therapeutic.
22. The vector of claim 21, wherein the calbindin-D.sub.28k
therapeutic is selected from the group consisting of an
CALBINDIN-D.sub.28k polynucleotide, a CALBINDIN-D.sub.28k
polynucleotide antisense sequence, a CALBINDIN-D.sub.28k protein,
and a CALBINDIN-D.sub.28k protein fragment.
23. The vector of claim 21, wherein the expression cassette
comprises one or more elements selected from the group consisting
of a host cell origin of replication, suitable promoter operably
linked to a heterologous genetic element, internal ribosome entry
site, splice donor site, splice acceptor site, suitable enhancer,
PPT track, heterologous genetic element, a reporter gene, and an
appropriate termination sequence.
24. The vector of claim 34 wherein the vector is selected from the
group consisting of: retrovirus, lentivirus, adenovirus, herpes
simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated
virus (AAV).
25. A method for introducing a CALBINDIN-D.sub.28k therapeutic into
a human for the treatment of a disease associated with
glucocorticoid induced cell death, comprising transducing the cell
with the vector of claim 21.
26. The method of claim 25, wherein the transduction occurs in
vivo.
27. The method of claim 25, wherein the transduction occurs ex
vivo.
28. The method of claim 25, wherein the cell is selected from the
group consisting of osteoblasts and osteocytes.
29. The cell of claim 28, wherein the cell comprises a neural
cell.
30. The method of claim 25, wherein the disease associated with
glucocorticoid induced cell death is osteoporosis.
31. A method for introducing a CALBINDIN-D.sub.28k therapeutic into
a human for the treatment of a disease associated with
glucocorticoid induced cell death, comprising transfecting the cell
with a plasmid comprising an expression cassette encoding the
CALBINDIN-D.sub.28k therapeutic.
32. The method of claim 31, wherein the CALBINDIN-D.sub.28k
therapeutic is selected from the group consisting of a
CALBINDIN-D.sub.28k polynucleotide, a CALBINDIN-D.sub.28k
polynucleotide antisense sequence, a CALBINDIN-D.sub.28k protein
and a CALBINDIN-D.sub.28k protein fragment.
33. The methods of claim 31, wherein said transfection is carried
out by a procedure selected from the group consisting of calcium
phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, scrape loading, ballistic introduction or
infection, use of a gene gun, lyposome and lipofectamine
transfection
34. The method of claim 31, wherein the transfection occurs in
vivo.
35. The method of claim 31, wherein the transfection occurs in
vitro.
36. The method of claim 31, wherein the cell is selected from the
group consisting of osteoblasts and osteocytes.
37. The method of claim 31, wherein the disease associated with
glucocorticoid induced cell death is osteoporosis.
38. The cell of claim 31, wherein the cell comprises a neural
cell.
39. The method of claim 31, wherein the transfection takes place as
part of an ex vivo procedure.
40. The method of claim 17, wherein the disease comprises
glucocorticoid induced neuronal cell death.
41. The method of claim 40, wherein the neuronal cell comprises a
hippocampal cell.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to the fields of molecular
biology, gene therapy, and the regulation of apoptosis. In
particular, this invention provides a novel mechanism for
regulating cell death. Specifically, the present invention is based
on the determination that a novel composition containing
calbindin-D.sub.28k can be administered to a subject to inhibit
glucocorticoid induced apoptosis in osteoblastic cells. Thus, the
compositions and methods of the present invention are useful for
the treatment of such diseases as Glucocorticoid induced
osteoporosis, which is the third most prevalent form of
osteoporosis.
BACKGROUND OF THE INVENTION
[0003] Various publications or patents are referred to throughout
this application or at the end of this specification to describe
the state of the art to which the invention pertains. Each of these
publications or patents is incorporated by reference herein.
Citations of scientific publications are set forth in the text or
at the end of the specification.
[0004] Control of cell numbers in mammals is determined, in part,
by a balance between cell proliferation and cell death. Cell death
involves processes that are equal in complexity and regulation to
those involved in cell proliferation. There are two forms of cell
death. One form of cell death is referred to as necrotic cell
death. It is typically characterized as a pathologic form of death
resulting from cellular trauma or injury. Necrosis is a process
that involves loss of membrane integrity and uncontrolled release
of cellular contents, giving rise to inflammatory responses. In
necrotic cell death, the cell has a passive role in initiating the
process of death, it is a response to pathologic changes initiated
outside of the cell that results in a change in the plasma membrane
permeability that results in cellular edema and the osmotic lysis
of the cell. In contrast, the other form of cell death, referred to
as apoptosis, usually proceeds in an orderly or controlled manner
wherein the cell undergoes an energy-dependent process of cellular
death initiated by specific signals in an otherwise normal
microenvironment (see, e.g., Barr, et al., Bio/Technology,
12:487-493 (1994); Steller, et al., Science, 267:1445-1449
(1995)).
[0005] Apoptosis, or programmed cell death, is a natural
"physiologic" process that occurs during growth and development,
and it is an important regulator of tissue homeostasis and aging.
It is the process, whereby organisms eliminate unwanted cells to
prevent uncontrolled cell proliferation and or disease. Early on in
development apoptosis plays a central role in sculpting the fetal
animal, precisely managing cell number in tissues and controlling
the formation of organs. In homeostasis, apoptosis regulates cell
number, facilitates morphogenesis, removes harmful or otherwise
abnormal cells, eliminates cells that have already performed their
function, and hence, plays a crucial role in the development and
maintenance of multicellular organisms by eliminating superfluous
or unwanted cells. It further serves as a defense mechanism to
remove potentially dangerous cells, including virus-infected cells,
self-reactive lymphocytes in autoimmune diseases, or malignant
cells and may minimize the risk of developing potentially cancerous
cells in tissues frequently exposed to mutagenic chemicals,
carcinogens, or UV radiation.
[0006] For instance, recent evidence has demonstrated that the rate
of bone formation is regulated not only by the rate of osteoblast
formation but also by the rate of osteoblast apoptosis. Thus,
increased osteoblast apoptosis is at least partially responsible
for the reduced bone formation in glucocorticoid excess-induced
osteopenia (Weinstein, R. S., Jilka, R. L., Parfitt, A. M., and
Manolagas, S. C. (1998) J. Clin. Invest. 102, 274-282). Conversely,
inhibition of osteoblast apoptosis is a likely mechanism of the
anabolic effect of intermittent administration of parathyroid
hormone (Jilka, R. L., Weinstein, R. S., Bellido, T., Roberson, P.
K., Parfitt, A. M., and Manolagas, S. C. (1999) J. Clin. Invest.
104, 439-446).
[0007] The programmed cell death process is often associated with
characteristic morphological and biochemical changes. Once
committed to apoptosis, cells undergo new rounds of protein
synthesis and various morphological and physiological changes. The
morphological characteristics of apoptosis include organelle
re-localization and compaction, cell shrinkage (condensation of
nuclear chromatin, nucleoplasm and cytoplasm), the appearance of
membrane ruffling, plasma and nuclear membrane blebbing and
formation of apoptotic bodies (membrane enclosed particles
containing intracellular material), and loss of cell-cell contact
followed by fragmentation. Signals for apoptosis promote the
activation of specific calcium- and magnesium-dependent
endonucleoases that cleave the double stranded DNA at linker
regions between nucleosomes. At the end of the process, the
phospholipid phosphatidylserine, which is normally hidden within
the plasma membrane, is exposed on the cell's surface and bound by
neighboring epitheliel cells, macrophages and dendritic cells that
engulf and phagocytose the fragments from the apoptotic cell before
lysis. In this way, dead cells are removed in an orderly manner
without any leakage of their noxious and potentially
proinflammatory contents. Because of this clearance mechanism,
inflammation is not induced despite the clearance of great numbers
of cells. By contrast, during the pathological form of cell death
referred to above as necrosis, the mitochondria within the cell
swell, lose their function and are rapidly lysed, thereby releasing
cytoplasmic contents that invariably trigger an inflammatory
response.
[0008] In general, the apoptosis cycle can be divided into three
phases: an initiation phase in which the various death stimuli take
so-called "private" pathways to converge on a common effector phase
involving the caspase family of proteins, which leads finally to
the degradation phase characterized by the typical biochemical
symptoms of cell death. In phase one, the cell undergoes genetic
reprogramming in which certain genes that were previously expressed
are now repressed, while other genes that were previously repressed
are now expressed. These genetic changes result in the activation
of double-stranded DNA fragmentation during the next phase.
[0009] During phase two, the effector phase, the nuclear morphology
changes (i.e., nuclear condensation of chromatin), while the plasma
and lysosomal membranes remain intact and the mitochondria continue
to function. This phase is regulated by the mitochondrial
permeability transition pore since its open or closed conformations
determine the fate of the cells. It participates in regulating the
level of calcium, the pH and the transmembrane potential in the
mitochondria. It has been demonstrated that opening of the pore,
regulated by Bcl-2, is a critical event in the process leading to
apoptosis as it allows dissipation of the transmembrane potential,
disrupting the integrity of the outer membrane and leading to the
release of mitochondrial intermembrane proteins, such as cytochrome
c (Kroemer et al. 1997).
[0010] Subsequently in phase three, proteases are activated, like
proteases that hydrolyzes poly(ADP-ribose) polymerase, the lamins
in the nuclear membrane are degraded and the nucleus itself
undergoes fragmentation. Plasma membrane blebbing and eventual
cellular fragmentation into clusters of membrane-bound apoptotic
bodies then occurs. Once formed, these apoptotic bodies are rapidly
recognized, phagocytized, and digested by macrophages or by
adjacent epithelial cells.
[0011] Once initiated apoptosis leads to a cascade of biochemical
and morphological events that result in irreversible degradation of
the genomic DNA and fragmentation of the cell. Entry into this
programmed cell death pathway is regulated by a careful balancing
act between those specific gene products that promote and those
that inhibit apoptosis. A cell activates its internally encoded
suicide program as a result of either internal or external signals.
In a healthy cell, the protein Bcl-2 is expressed on the outer
membrane surface of mitochondria and bound to an Apoptosis
Activation Factor-1 (Apaf-1) protein. When a cell undergoes
internal damage, Bcl-2 is caused to release Apaf-1 resulting in the
disruption of the mitochondria membrane and the leakage of both
Apaf-1 and cytochrome c out of the mitochondria. Once released
cytochrome c and Apaf-1 bind to a caspase 9 protease to form a
complex called an apoptosome, which aggregates in the cytosol.
Caspase 9 is thus activated, which in turn activates other members
of the caspase family of proteases which results in the digestion
of the structural proteins of the cytoplasm, the degradation of
chromosomal DNA and ultimately in the phagocytosis of the cell.
[0012] Endogenous activation of apoptosis occurs due to the
positive presence of a tissue-specific external signal (such as
TNF-.alpha. or glucocorticoids) that induces cells to
self-destruct. One characteristic of the tumor necrosis factor
(TNF) family is the ability of many family members to induce
programmed cell death in a variety of cells, both normal and of
tumor origin (Wiley et al, Immunity 3:673-682, 1995, and references
therein). TNF is a cytokine that has been implicated in cell death.
There are two forms of TNF, they are the .alpha. and .beta. forms.
TNF-.alpha. is a soluble homotrimer of 17 kD protein subunits. A
membrane-bound 26 kD precursor form of TNF-.alpha. also exists. TNF
.alpha. and .beta. are produced from various cells, including, for
example, T cells, monocytes, macrophages, and natural killer cells,
by induction with prophlogistic agents such as bacteria, viruses,
various mitogens or the like.
[0013] TNF elicits a broad range of biological effects through two
distinct membrane receptors, TNF R1 and TNF R2, which are expressed
at low levels on most cell types. Endotoxins strongly activate
monocyte/macrophage production and secretion of TNF. It is a
mediator of the metabolic and neurohormonal responses to
endotoxins. TNF causes the proinflammatory actions that result from
tissue injury, increases the adherence of neutrophils and
lymphocytes, and stimulates the release of platelet activating
factor from macrophages, neutrophils and vascular endothelial
cells. To this extent, TNF is a key component in biological
activities such as inducing hemorrhagic necrosis in tumors,
apoptosis in cancer cells, production of prostaglandin's and
collagenase, expression of adhesion molecules (ICAM-1, ELAM-1) and
HLA class II molecules, production of inflammatory cytokines (e.g.,
IL-1, IL-6) and chemokines (IL-8, RANTES), and enhancement of
absorption of bone and cartilage.
[0014] One of the most striking features of TNF compared to other
cytokines is its ability to elicit programmed cell death. Apoptosis
induced by TNF is mediated primarily through TNF R1. The
intracellular domain of TNF R1 contains a "death domain" of
approximately 80 amino acids that is responsible for signaling cell
death by the receptor. When TNF .alpha. binds to its integral
membrane receptor (TNF R1) a signal is transmitted to the cytoplasm
activating caspase 8, which initiates an expanding cascade of
sequential caspase family activation and proteolytic activity that
results in the eventual phagocytosis of the cell.
[0015] Along those lines, it was determined that the inflammatory
cytokine TNF, which inhibits bone formation, collagen synthesis,
and alkaline phosphatase (Stashenko, P., Obernesser, M. S., and
Dewhirst, F. E. (1989) Immunol. Invest. 18, 239-249; and Centrella,
M., McCarthy, T. L., and Canalis, E. (1988) Endocrinology 123,
1442-1448), also induces apoptosis of osteoblastic cells (Jilka, R.
L., Weinstein, R. S., Bellido, T., Parfitt, A. M., and Manolagas,
S. C. (1998) J. Bone Miner. Res. 13, 793-802; Hill, P. A., Tumber,
A., and Meikle, M. C. (1997) Endocrinology 138, 3849-3858; and
Kitajima, I., Nakajima, T., Imamura, T., Takasaki, I., Kawahara,
K., Okano, T., Tokioka, T., Soejima, Y., Abeyama, K., and Maruyama,
I. (1996) J. Bone Miner. Res. 11, 200-210). However, it was further
determined that growth factors such as insulin-like growth factor
I, basic fibroblast growth factor, interleukin-6 type cytokines,
and transforming growth factor inhibit osteoblastic cell apoptosis
induced by TNF, serum deprivation, or activation of Fas (Jilka, R.
L., Weinstein, R. S., Bellido, T., Parfitt, A. M., and Manolagas,
S. C. (1998) J. Bone Miner. Res. 13, 793-802; Hill, P. A., Tumber,
A., and Meikle, M. C. (1997) Endocrinology 138, 3849-3858; and
Kitajima, I., Nakajima, T., Imamura, T., Takasaki, I., Kawahara,
K., Okano, T., Tokioka, T., Soejima, Y., Abeyama, K., and Maruyama,
I. (1996) J. Bone Miner. Res. 11, 200-210).
[0016] Interestingly, insulin growth factor I and fibroblast growth
factor induce the calcium binding protein calbindin-D.sub.28k
expression in neurons and promote neuronal survival (Collazo, D.,
Takahashi, H., and McKay, R. D. (1992) Neuron 9, 643-656;
Nieto-Bona, M. P., Busiguina, S., and Torres-Aleman, I. (1995) J.
Neurosci. Res 42, 371-376; Mattson, M. P., Murrain, M., Guthrie, P.
B., and Kater, S. B. (1989) J. Neurosci. 9, 3728-3740; and Cheng,
B., and Mattson, M. P. (1992) J. Neurosci. 12, 1558-1566). Recent
evidence suggests the involvement of apoptosis in the regulation of
osteoblastic bone formation and osteoclastic bone resorption during
adult bone remodeling (Hughes, D. E., and Boyce, B. F. (1997) J.
Clin. Pathol. (Lond.) 50, 132-137; and Manolagas, S. C. (1999)
Endocrinology 140, 4377-4381). Because of these facts the inventor
set out to determine whether expression levels of
calbindin-D.sub.28k had an effect on apoptosis of osteoblastic
cells. The results showed TNF induced nuclear fragmentation of
MC3T3-E1 cells transfected with an empty vector, but that this
pro-apoptotic effect of TNF was significantly attenuated in cells
transfected with calbindin-D.sub.28k cDNA. Hence, it was determined
that calbindin-D.sub.28k transfectants were resistant to
TNF-induced apoptosis.
[0017] Since some evidence indicated a role for calcium in the
initiation as well as in the degradation phase of apoptosis, it was
suggested that the anti-apoptotic effect of calbindin-D.sub.28k
could be due to its ability to chelate calcium. It was also
suggested that calbindin-D.sub.28k could inhibit the release of
cytochrome c from the mitochondria, which is needed for the
activation of caspase-3, by preventing calcium mediated apoptotic
damage of mitochondrial electron transport (Guo, Q., Christakos,
S., Robinson, N., and Mattson, M. P. (1998) Proc. Natl. Acad. Sci.
U.S.A. 95, 3227-3232). Because recent evidence indicates that the
caspase family of proteins play a role in inducing TNF evoked
apoptosis it was suggested that calbindin-D.sub.28k may interact
with one or more of the caspase family of proteases to inhibit
apoptosis.
[0018] The caspases, are a family of cysteine proteases that share
the characteristic feature of a conserved QAC(R/Q)G motif, in which
the Cys residue is part of the active site and is essential for
caspase-mediated apoptosis. These proteases are primarily
responsible for the degradation of cellular proteins that lead to
the morphological changes seen in cells undergoing apoptosis. It
has been shown that many members of the family are capable of
inducing apoptosis when overexpressed in mammalian cells (Henkart,
1996 and Miura et al., Cell 75:653 [1993]). For instance, it is
known that caspase 1 (interleukin 1.beta. converting enzyme or ICE)
is responsible for the activation of interleukin-1.beta.
(IL-1.beta.) and is necessary for apoptosis. It is a
substrate-specific cysteine protease that cleaves the inactive
prointerleukin-1 to produce the mature IL-1. IL-1 is a cytokine
involved in mediating a wide range of biological responses
including inflammation, septic shock, wound healing, hematopoiesis
and growth of certain leukemias and apoptosis. When caspase 1 is
overexpressed cell apoptosis can be induced. Over expression of
caspases 2 and 3 in fibroblasts and neuroblastoma cells also
results in cell death by apoptos.
[0019] Caspase 3 is a protein also known to be intimately involved
with a cell's ability to induce apoptosis in normal nuclei. Caspase
3 normally exists in the cytosolic fraction of cells as a 32 kDa
inactive precursor that is converted proteolytically to a 20 kDa
and a 10 kDa active heterodimer when cells are signaled to undergo
apoptosis (Schlegel, et al., Biol. Chem. 271:1841-1844, (1996);
Wang, et al., EMBO J. 15:1012-1020, (1996)). And it is known that
Bcl-2, prevents the activation of caspase-3 by blocking the
mitochondria from releasing cytochrome c, a necessary co-factor for
caspase-3 activation (Liu, et al., Cell 86:147-157, (1996); Yang,
et al., Science 275:1129-1132, (1997); Kluck, et al., Science
275:1132-1136, (1997)).
[0020] Another extrinsic signal that can trigger apoptosis comes
from glucocorticoid hormones. Glucocorticoids exert several effects
in tissues that have receptors for them. By binding to their
receptors, glucocorticoids regulate the expression of several genes
either positively or negatively, in a direct or indirect manner,
and are known to arrest cell growth and can induce cell death.
[0021] The human glucocorticoid receptor is made up of 777 amino
acids and is predominantly cytoplasmic in its unactivated, non-DNA
binding form. When activated, it translocates to the nucleus. There
are four major functional domains of the glucocorticoid receptor.
The first, from the amino terminal, is the tau 1 domain, which
spans amino acid positions 77-262 and regulates gene activation.
The second is the DNA binding domain, which spans amino acid
positions 421-486 and contains nine cysteine residues, eight of
which form two different zinc fingers. The DNA binding domain binds
to the regulatory sequences of genes that are induced (or
deinduced) by glucocorticoids. The tau 2 domain runs from amino
acids 532-555 and it is also important for transcriptional
activation. Towards the carboxyl terminal end, from amino acids 555
to 777, is the steroid binding domain, which binds glucocorticoid
to activate the receptor. This region of the receptor also has the
nuclear localization signal.
[0022] The glucocorticoid receptor is expressed in the cytoplasm of
a cell. When a glucocorticoid enters a cell and binds its receptor,
the receptor goes through a conformational change wherein it is
activated, forms a heterodimer with another glucocorticoid bound
receptor complex and is transported via a transport protein to the
nucleus of the cell. This heterodimer complex interacts with the
cell's DNA upregulating the production of the pro-apoptotic signal:
bax. The bax protein is transported to the surface membrane of a
mitochondria wherein it forms a pore, causing the release of
cytochrome c, which then binds to the Apaf-1 protein resulting in
the activation of the caspase cascade and phosphatidyl serine being
displayed, which leads to the engulfment and degradation of the
cell by neighboring epithelial and macrophage cells.
[0023] As stated above, signals that initiate apoptosis trigger the
so-called private pathways of death, which are specific for
particular groups of stimuli and lead to the conversion of
procaspases to active the caspases. Although both TNF .alpha. and
the glucocorticoid hormones can induce apoptosis by binding to
their respective receptors and activating the caspase family
cascade, little is known about the actual signaling events that
occur once the caspases are activated, nor what caspases are
activated and by which inducer, nor which lead to the common cell
death pathway. The end result of this cascade, however, is
chromatin condensation, nuclear fragmentation, increase in cell
membrane permeability, and ultimately cell death (Thornberry, N.
A., and Lazebnik, Y. (1998) Science 281, 1312-1316; and Green, D.
R. (1998) Cell 94, 695-698).
[0024] The inventor's previous work establishes a relationship
between calbindin-D.sub.28k and caspase-3 in that it was determined
that calbindin-D.sub.28k could prevent TNF induced apoptosis by
interacting with caspase-3 via an unknown mechanism. (See Bellido
et al. (2000) Journal of Biol. Chem. 275, 26326-26332.) The present
work of the inventor, and the subject matter of the present
invention establishes that calbindin-D.sub.28k can also prevent
glucocorticoid induced cell death. Given that apoptosis is tightly
regulated and has been linked to pathways that are dysregulated in
a variety of diseases including cancer, the present invention is
important because it further elucidates a mechanism by which to
control this process, especially as it relates to glucocorticoid
induced cell death. For instance, the compositions and methods of
the present invention are useful in the prevention and treatment of
glucocorticoid induced osteoporosis, which is the third most
prevalent form of osteoporosis after postmenopause and senile
osteoporosis.
SUMMARY OF THE INVENTION
[0025] The present invention provides novel compositions containing
a calbindin-D.sub.28k therapeutic element, which is involved in the
regulation of apoptosis, and may be administered for the prevention
of an abnormal apoptosis response in cells. In particular the
compositions and methods of the present invention may be used for
the prevention or induction of apoptosis in such cells types as
osteoblasts and osteocytes. Specifically, the compositions and
methods of the present invention may be useful in the prevention of
glucocorticoid induced cell death in osteoblasts and the treatment
of such conditions as glucocorticoid induced osteoporosis.
[0026] According to one aspect, this invention depends on an
isolated polynucleotide encoding a calbindin-D.sub.28k protein.
Preferably, the polynucleotide comprises the sequence of: SEQ ID
NO:1, with up to 30% conservative substitutions; an allelic variant
of SEQ ID NO: 1; a sequence hybridizing with SEQ ID NO:1 or its
complement under moderate hybridization and washing conditions, or
an antisense sequence to SEQ ID NO:1. Further, in another aspect,
this invention depends on an isolated polypeptide sequence having
an amino acid sequence of SEQ ID NO:2 with up to 30% conservative
substitutions.
[0027] Specifically, one aspect of the invention features a
recombinant calbindin-D.sub.28k DNA or RNA molecule comprising a
vector having an insert that includes part or all of a
calbindin-D.sub.28k polynucleotide, or its antisense polynucleotide
sequence. The invention also features a vector containing a
calbindin-D.sub.28k polypeptide. Pharmaceutical compositions
containing a biologically effective amount of the
calbindin-D.sub.28k polynucleotide, antisense sequence, protein
and/or protein fragments with acceptable carriers are also
provided.
[0028] Hence, in another, particular embodiment, the present
invention may be implicated in the treatment of diseases and
conditions such as increased cell death in such cells as
osteoblasts and osteocytes. Therefore, the invention relates to
compositions and methods for treating diseased conditions
associated with an abnormal increase in apoptosis, by administering
a calbindin-D.sub.28k gene or protein to decrease programmed cell
death. Specifically, a method of treating a disease associated with
abnormal glucocorticoid induced cell death involving the
administration of a pharmaceutical composition containing a
biologically effective amount of a calbindin-D.sub.28k
polynucleotide or polypeptide along with an acceptable carrier, is
provided.
[0029] Further, in another particular embodiment, the present
invention may be implicated in the treatment of diseases and
conditions associated with decreased apoptosis such as various bone
osteoblastic cancers. Therefore, the invention relates to
compositions and methods for treating diseased conditions
associated with an abnormal decrease in apoptosis, by administering
a pharmaceutical composition containing a biologically effective
amount of a calbindin-D.sub.28k antisense sequence along with an
acceptable carrier, downregulating the endogenous production of the
calbindin-D.sub.28k protein, especially in osteoblast or osteocyte
cells with an abnormal growth rate, and thereby increasing the
likelihood of apoptosis and thus the killing off of the cell
without evoking an immune response. This mechanism may also be
useful in other cells wherein endogenous calbindin-D.sub.28k is
produced, such as various cells of the prostrate.
[0030] Specifically, in a particular embodiment, the invention
relates to the administration of vectors for the delivery of a
calbindin-D.sub.28k therapeutic element to a cell for the treatment
of diseases associated with an abnormal increase or decrease in
cell death, wherein the vector contains an expression cassette
encoding the calbindin-D.sub.28k therapeutic. The
calbindin-D.sub.28k therapeutic can be a calbindin-D.sub.28k
polynucleotide, a calbindin-D.sub.28k polynucleotide antisense
sequence, a calbindin-D.sub.28k protein, or a calbindin-D.sub.28k
protein fragment. The expression cassette may contain one or more
of the following elements: a host cell origin of replication, a
suitable promoter operably linked to a heterologous genetic
element, an internal ribosome entry site, splice donor site, splice
acceptor site, a suitable enhancer, PPT track, heterologous genetic
element, a reporter gene, and/or an appropriate termination
sequence. One or more of these vectors, containing a
calbindin-D.sub.28k therapeutic, may be introduced into an
appropriate cell by a variety of means, including in vivo, in vitro
or ex vivo transduction or transfection using an appropriate
expression system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The invention is best understood from the following detailed
description when read in connection with the accompanying drawings,
in which:
[0032] FIG. 1 shows calbindin-D.sub.28k protection against
dexamethasone induced cell death of MLOY4 osteocytic cells.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Introduction
[0034] In light of the recent work by the Applicant
calbindin-D.sub.28k has been identified as a protein that can
regulate glucocorticoid induced cell death in various cell types,
but particularly in osteoblast and osteocyte cells. By binding
caspase 3, calbindin-D.sub.28k inhibits the ability of caspase 3 to
proteolytically cleave, thereby inhibiting its function and
preventing glucocorticoid induced cell death and therefore,
osteoporosis.
[0035] Although specific embodiments of the present invention will
now be described, it should be understood that such embodiments are
by way of example only and merely illustrative of but a small
number of the many possible specific embodiments that can represent
applications of the principles of the present invention. Various
changes and modifications obvious to one skilled in the art to
which the present invention pertains are deemed to be within the
spirit, scope and contemplation of the present invention as further
defined in the appended claims.
[0036] Definitions
[0037] Various terms relating to the biological molecules of the
present invention are used throughout the specification and
claims.
[0038] "Calbindin-D.sub.28k" refers generally to a
calbindin-D.sub.28k polypeptide that has been found to regulate
glucocorticoid induced cell death by inhibiting the ability of
caspase 3 to proteolytically cleave, and thereby preventing
glucocorticoid induced osteoporosis, in accordance with the present
invention, which is described in detail herein above and throughout
the specification.
[0039] "Calbindin-D.sub.28k activity or calbindin-D.sub.28k
polypeptide activity" or "biological activity of the
calbindin-D.sub.28k protein or calbindin-D.sub.28k polypeptide"
refers to the metabolic or physiologic function of said
calbindin-D.sub.28k including similar activities or improved
activities or these activities with decreased undesirable side
effects. In particular, the calbindin-D.sub.28k polynucleotide
encodes a protein that interacts with caspase-3 in such a way as to
inhibit glucocorticoid induced cell death.
[0040] "Calbindin-D.sub.28k gene" refers to a polynucleotide in
accordance with the present invention, which encodes a
calbindin-D.sub.28k polypeptide.
[0041] A "calbindin-D.sub.28k therapeutic" refers to a
therapeutically effective amount of a calbindin-D.sub.28k related
genetic sequence such as, but not limited to polynucleotide,
polynucleotide antisense sequence, and calbindin-D.sub.28k peptide,
protein or protein fragment.
[0042] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the
term is employed herein.
[0043] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and
DNA.
[0044] The term polynucleotide also includes DNAs or RNAs
containing one or more modified bases and DNAs or RNAs with
backbones modified for stability or for other reasons. "Modified"
bases include, for example, tritylated bases and unusual bases such
as inosine. A variety of modifications has been made to DNA and
RNA; thus, "polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0045] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques that are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs; as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods.
[0046] Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of various moiety
groups, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation,
racernization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. See, for instance, Proteins--Structure And
Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York, 1993 and Wold, F., "Posttranslational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in
"Posttranslational Covalent Modification Of Proteins", B, C.
Johnson, Ed., Academic Press, New York, 1983; Seifter et al.,
"Analysis for protein modifications and nonprotein cofactors", Meth
Enzymol (1990)
[0047] 182:626-646 and Rattan et al., "Protein Synthesis:
Posttranslational Modifications and Aging", Ann AIY Acad Sci (1992)
663:48-62.
[0048] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical.
[0049] A variant and reference polypeptide may differ in amino acid
sequence by one or more substitutions, additions, and deletions in
any combination. A substituted or inserted amino acid residue may
or may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a variant that is not known to
occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis. For instance, a conservative
amino acid substitution may be made with respect to the amino acid
sequence encoding the polypeptide.
[0050] A "conservative amino acid substitution", as used herein, is
one in which one amino acid residue is replaced with another amino
acid residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art,
including basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine).
[0051] The term "substantially the same" refers to nucleic acid or
amino acid sequences having sequence variation that do not
materially affect the nature of the protein (i.e. the structure,
stability characteristics, substrate specificity and/or biological
activity of the protein). With particular reference to nucleic acid
sequences, the term "substantially the same" is intended to refer
to the coding region and to conserved sequences governing
expression, and refers primarily to degenerate codons encoding the
same amino acid, or alternate codons encoding conservative
substitute amino acids in the encoded polypeptide. With reference
to amino acid sequences, the term "substantially the same" refers
generally to conservative substitutions and/or variations in
regions of the polypeptide not involved in determination of
structure or function.
[0052] With respect to single-stranded nucleic acid molecules, the
term "specifically hybridizing" refers to the association between
two single-stranded nucleic acid molecules of sufficiently
complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes
termed "substantially complementary"). In particular, the term
refers to hybridization of an oligonucleotide with a substantially
complementary sequence contained within a single-stranded DNA or
RNA molecule, to the substantial exclusion of hybridization of the
oligonucleotide with single-stranded nucleic acids of
non-complementary sequence.
[0053] With respect to oligonucleotide constructs, but not limited
thereto, the term "specifically hybridizing" refers to the
association between two single-stranded nucleotide molecules of
sufficiently complementary sequence to permit such hybridization
under pre-determined conditions generally used in the art
(sometimes termed "substantially complementary"). In particular,
the term refers to hybridization of an oligonucleotide construct
with a substantially complementary sequence contained within a
single-stranded DNA or RNA molecule of the invention, to the
substantial exclusion of hybridization of the oligonucleotide with
single-stranded nucleic acids of non-complementary sequence.
[0054] The term "substantially pure" refers to a "preparation
comprising at least 50-60% by weight the compound of interest
(e.g., nucleic acid, oligonucleotide, protein, etc.). More
preferably, the preparation comprises at least 75% by weight, and
most preferably 90-99% by weight, the compound of interest. Purity
is measured by methods appropriate to the compound of interest
(e.g. chromatographic methods, agarose or polyacrylamide gel
electrophoresis, HPLC analysis, and the like).
[0055] The term "expression cassette" refers to a nucleotide
sequence that contains at least one coding sequence along with
sequence elements that direct the initiation and termination of
transcription. An expression cassette may include additional
sequences, including, but not limited to promoters, enhancers,
sequences involved in post-transcriptional or post-translational
processes, as well as appropriate terminator sequences.
[0056] A "coding sequence" or "coding region" refers to a nucleic
acid molecule having sequence information necessary to produce a
gene product, when the sequence is expressed.
[0057] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement of other transcription control
elements (e.g., enhancers and regulators) in an expression
vector.
[0058] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0059] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3' side of the coding
region, or within the coding region, or within introns. Typically,
a promoter is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The typical 5' promoter sequence is
bounded at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence is a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
[0060] The term "nucleic acid construct" or "DNA construct" is
sometimes used to refer to a coding sequence or sequences operably
linked to appropriate regulatory sequences and inserted into a
vector for transforming a cell; in vitro or in vivo. This term may
be used interchangeably with the term "transforming DNA". Such a
nucleic acid construct may contain a coding sequence for a gene
product of interest, along with a selectable marker gene and/or a
reporter gene.
[0061] A "heterologous" region of a nucleic acid construct is an
identifiable segment (or segments) of the nucleic acid molecule
within a larger molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region
encodes a mammalian gene, the gene will usually be flanked by DNA
that does not flank the mammalian genomic DNA in the genome of the
source organism. In another example, a heterologous region is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native
gene). Allelic variations or naturally-occurring mutational events
do not give rise to a heterologous region of DNA as defined
herein.
[0062] The term "DNA construct", as defined above, is also used to
refer to a heterologous region, particularly one constructed for
use in transformation of a cell. A cell has been "transformed" or
"transfected" or "transduced" by exogenous or heterologous DNA when
such DNA has been introduced inside the cell. The transforming DNA
may or may not be integrated (covalently linked) into the genome of
the cell. In prokaryotes, yeast, and mammalian cells for example,
the transforming DNA may be maintained on an episomal element such
as a plasmid. With respect to eukaryotic cells, a stably
transformed cell is one in which the transforming DNA has become
integrated into a chromosome so that it is inherited by daughter
cells through chromosome replication. This stability is
demonstrated by the ability of the eukaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the transforming DNA.
[0063] The term "in vivo delivery" involves the use of any gene
delivery system, such as viral- and liposome-mediated
transformation for the delivery and introduction of a therapeutic
agent to the cells of a subject while they remain in the subject.
Such therapeutic elements may include, for example,
calbindin-D.sub.28k DNA, cDNA, RNA, and antisense polynucleotide
sequences.
[0064] As used herein, the term "transduction," is used to describe
the delivery of DNA to eukaryotic cells using viral mediated
delivery systems, such as, adenoviral, AAV, retroviral, or plasmid
delivery gene transfer methods. Preferably the viral mediated
delivery system is targeted specifically to the cell, wherein
delivery is sought. The production of targeted delivery systems is
well known and practiced in the recombinant arts. A number of
methods for delivering therapeutic formulations, including DNA
expression constructs (as described further below), into eukaryotic
cells are known to those skilled in the art. In light of the
present disclosure, the skilled artisan will be able to deliver the
therapeutic elements of the present invention to cells in many
different but effective ways. Naturally, different viral host
ranges will dictate the virus chosen for gene transfer.
[0065] "In vitro gene delivery" refers to a variety of methods for
introducing exogenous DNA into a cell that has been removed form
its host environment.
[0066] As used herein the term, "transfection" is used to describe
the delivery and introduction of a therapeutic agent to a cell
using non-viral mediated means, these methods include, e.g.,
calcium phosphate- or dextran sulfate-mediated transfection;
electroporation; glass projectile targeting; and the like. These
methods are known to those of skill in the art, with the exact
compositions and execution being apparent in light of the present
disclosure.
[0067] "Ex vivo gene delivery" refers to the procedure wherein
appropriate cells are removed form the host organism, transformed,
transduced or transfected in accordance with the teachings of the
present invention, and replaced back into the host organism, for
the purpose of therapeutic restoration and/or prevention.
[0068] "Delivery of a therapeutic element or agent" may be carried
out through a variety of means, such as by using parenteral
delivery methods such as intravenous and subcutaneous injection,
and the like. Such methods are known to those of skill in the art
of drug delivery, and are further described herein in the sections
regarding pharmaceutical preparations and treatment. Compositions,
include pharmaceutical formulations, comprising a
calbindin-D.sub.28k gene, protein, or antisense polynucleotide
sequence that may be delivered. In such compositions, the
calbindin-D.sub.28k may be in the form a DNA segment, plasmid,
recombinant vector or recombinant virus that is capable of
expressing a calbindin-D.sub.28k protein in a cell; specifically,
an osteoblast cell. These compositions, including those comprising
a recombinant viral gene delivery system, such as an adenovirus
particle, may be formulated for in vivo administration by
dispersion in a pharmacologically acceptable solution or buffer.
Preferred pharmacologically acceptable solutions include neutral
saline solutions buffered with phosphate, lactate, Tris and the
like.
[0069] The term "contacted" when applied to a cell is used herein
to describe the process by which a calbindin-D.sub.28k genetic
element, such as a gene, protein or antisense sequence is delivered
to a target cell or is placed in direct proximity with the target
cell. This delivery may be in vitro or in vivo and may involve the
use of a recombinant vector system. Any method may be used to
contact a cell with the calbindin-D.sub.28k associated protein or
nucleotide sequence, so long as the method results in either
increased or decreased levels of functional calbindin-D.sub.28k
protein within the cell. This includes both the direct delivery of
a calbindin-D.sub.28k protein to the cell and the delivery of a
gene or DNA segment that encodes calbindin-D.sub.28k, or its
antisense polynucleotide sequence, which gene or antisense sequence
will direct or inhibit, respectfully, the expression and production
of calbindin-D.sub.28k within the cell. Since protein delivery is
subject to drawbacks, such as degradation and low cellular uptake,
it is contemplated that the use of a recombinant vector that
expresses a calbindin-D.sub.28k protein, or encodes for a
calbindin-D.sub.28k polynucleotide antisense sequence, will be of
particular advantage for delivery.
[0070] With respect to "therapeutically effective amount" is an
amount of the polynucleotide, antisense polynucleotide or protein
of calbindin-D.sub.28k, or fragment thereof, that when administered
to a subject is effective to bring about a desired effect (e.g., an
increase or decrease of apoptosis) within the subject.
[0071] Polynucleotides
[0072] The present invention provides a novel composition
containing a therapeutic calbindin-D.sub.28k genetic element, such
as a gene or protein, which acts as a mediator of glucocorticoid
induced apoptosis. The calbindin-D.sub.28k gene and protein of the
present invention, when introduced to a cell have been found to
inhibit glucocorticoid induced cell death. The summary of the
invention described above is non-limiting and other features and
advantages of the invention will be apparent from the following
detailed description.
[0073] The present invention concerns compositions and methods for
treating various diseases associated with either an unhealthy
increase or decrease in programmed cell death. The invention is
based firstly on the inventor's discovery that calbindin-D.sub.28k
protects against dexamethasone, a glucocorticoid, induced cell
death. To determine if calbindin-D.sub.28k could protect against
glucocorticoid induced cell death, Applicant transfected MLO-Y4
osteocytic cells with either a pREP4 calbindin-D.sub.28k or
BSR.alpha. calbindin-D.sub.28k expression vector construct and then
incubated the cells and dexamethasone. After 6, 12 and 24 hours the
cells were assayed to determine characteristic apoptotic
morphological changes due to the presence of dexamethasone, and
MLO-Y4 cells transfected with the calbindin-D.sub.28k containing
vector were observed to have been protected against glucocorticoid
induced cell death over those cells transfected with the naked
vector alone. Thus, the inventor discovered that
calbindin-D.sub.28k plays a role in regulating glucocorticoid
induced cell death in bone cells, and may be useful in the
treatment of the various forms of glucocorticoid induced
osteoporosis.
[0074] As described in detail in Example 1, the calbindin-D.sub.28k
gene was cloned into two different expression vector constructs,
pREP4 and BSR.alpha. calbindin-D.sub.28k. The human
calbindin-D.sub.28k gene is set out in SEQ ID NO:1. The
calbindin-D.sub.28k polynucleotides of the present invention
include isolated polynucleotides encoding calbindin-D.sub.28k
proteins, polypeptides and/or fragments, and polynucleotides
closely related thereto. More specifically, calbindin-D.sub.28k
polynucleotides of the invention include a polynucleotide
comprising the human nucleotide sequences contained in SEQ ID NO:1
encoding a calbindin-D.sub.28k polypeptide of SEQ ID NO:2, and
polynucleotides having the particular sequence of SEQ ID NO: 1.
[0075] Calbindin-D.sub.28k polynucleotides further include a
polynucleotide comprising a nucleotide sequence that has at least
70% identity over its entire length to a nucleotide sequence
encoding the calbindin-D.sub.28k polypeptide of SEQ ID NO:2, and a
polynucleotide comprising a nucleotide sequence that is at least
70% identical to that of SEQ ID NO: 1, over its entire length. In
this regard, polynucleotides with at least 70% are preferred, more
preferably at least 80% even more preferably at least 90% identity,
yet more preferably at least 95% identity, 97% are highly preferred
and those with at least 98-99% are most highly preferred, with at
least 99% being the most preferred.
[0076] The present invention includes polynucleotides encoding
polypeptides which have at least 70% identity, preferably at least
80% identity, more preferably at least 90% identity, yet more
preferably at least 95% identity, even more preferably at least
97-99% identity, to the amino acid sequence of SEQ ID NO:2, over
the entire length of the recited amino acid sequences. The
nucleotide sequences encoding the calbindin-D.sub.28k polypeptide
of SEQ ID NO:2 may be identical to the polypeptide encoding
sequence contained in SEQ ID NO: 1, or it may be a sequence, which
as a result of the redundancy (degeneracy) of the genetic code,
also encodes the polypeptide of SEQ ID NO:2. Also included under
calbindin-D.sub.28k polynucleotides are nucleotide sequences that
code for polynucleotides that are complementary to such
calbindin-D.sub.28k polynucleotides, such as anti-sense
calbindin-D.sub.28k polynucleotide sequences.
[0077] Calbindin-D.sub.28k polynucleotides of the present invention
may be prepared by two general methods: (1) they may be synthesized
from appropriate nucleotide triphosphates, or (2) they may be
isolated from biological sources. Both methods utilize protocols
well known in the art. The availability of nucleotide sequence
information, such as the cDNA having SEQ ID NO: 1, enables
preparation of an isolated nucleic acid molecule of the invention
by oligonucleotide synthesis.
[0078] Synthetic oligonucleotides may be prepared by the
phosphoramadite method employed in the Applied Biosystems 38A DNA
Synthesizer or similar devices. The resultant construct may be
purified according to methods known in the art, such as high
performance liquid chromatography (HPLC). Long, double-stranded
polynucleotides, must be synthesized in stages, due to the size
limitations inherent in current oligonucleotide synthetic methods.
Thus, for example, a long double-stranded molecule may be
synthesized as several smaller segments of appropriate
complementarity. Complementary segments thus produced may be
annealed such that each segment possesses appropriate cohesive
termini for attachment of an adjacent segment. Adjacent segments
may be ligated by annealing cohesive termini in the presence of DNA
ligase to construct an entire long double-stranded molecule. A
synthetic DNA molecule so constructed may then be cloned and
amplified in an appropriate vector. In this respect, the
polynucleotide may also contain non-coding 5' and 3' sequences,
such as transcribed, non-translated sequences, splicing and
polyadenylation signals, ribosome binding sites and sequences that
stabilize mRNA.
[0079] Calbindin-D.sub.28k genes may be isolated from appropriate
biological sources using methods known in the art. In the exemplary
embodiment of the invention, calbindin-D.sub.28k may be isolated
from genomic libraries of rat kidneys as described in Panalni et
al., (1984) J. Biol. Chem. 259, 9735-9741. A preferred means for
isolating calbindin-D.sub.28k genes is PCR amplification using
genomic or cDNA templates and calbindin-D.sub.28k specific primers.
Genomic and cDNA libraries are commercially available, such as
those sold by Sigma, and can also be made by procedures well known
in the art. In positions of degeneracy where more than one nucleic
acid residue could be used to encode the appropriate amino acid
residue, all the appropriate nucleic acid residues may be
incorporated to create a mixed oligonucleotide population, or a
neutral base such as inosine may be used. The strategy of
oligonucleotide design is well known in the art. Nucleic acids of
the present invention may be maintained as DNA in any convenient
cloning vector. In a preferred embodiment, clones are maintained in
plasmid cloning/expression vector, such as pBluescript (Stratagene,
La Jolla, Calif.), that is propagated in a suitable E. coli host
cell.
[0080] Hence, in one particular aspect, the present invention
provides novel compositions containing a calbindin-D.sub.28k
therapeutic element, which is involved in the regulation of
apoptosis, and may be administered for the prevention of an
abnormal apoptosis response in cells. More particularly, the
invention pertains to novel compositions containing a
calbindin-D.sub.28k polynucleotide sequence that may be used for
the prevention or induction of apoptosis in such cells types as
osteoblasts and osteocytes. More particularly still, the
polynucleotide compositions and methods of the present invention
may be useful in the prevention of glucocorticoid induced cell
death in osteoblasts and osteocytes and the treatment of such
conditions as glucocorticoid induced osteoporosis.
[0081] Specifically, according to one aspect, this invention
depends on an isolated polynucleotide encoding a
calbindin-D.sub.28k protein. Preferably, the polynucleotide
comprises the sequence of: SEQ ID NO:1, with up to 30% conservative
substitutions; an allelic variant of SEQ ID NO:1; a sequence
hybridizing with SEQ ID NO:1 or its complement under moderate
hybridization and washing conditions, or an antisense sequence to
SEQ ID NO:1. In a particular preferred embodiment, a pharmaceutical
composition containing a therapeutically effective amount of a
calbindin-D.sub.28k polynucleotide along with a biologically
acceptable carrier is provided, where by the administration of said
composition will be useful for the prevention and/or treatment of
glucocorticoid induced osteoporosis.
[0082] Polypeptides
[0083] In one aspect, the present invention relates to
calbindin-D.sub.28k polypeptides, calbindin-D.sub.28k proteins, or
therapeutically active fragments thereof. The calbindin-D.sub.28k
polypeptides include the polypeptide of SEQ ID NO:2; as well as
polypeptides comprising the amino acid sequence of SEQ ID NO:2; and
polypeptides comprising the amino acid sequences that have at least
70% identity to that of SEQ ID NO:2, over its entire length.
Preferably calbindin-D.sub.28k polypeptides or proteins exhibit at
least one biological activity of calbindin-D.sub.28k, specifically
the ability to interact with the caspase family of proteins to
inhibit apoptosis. The present invention further provides for a
polypeptide that comprises an amino acid sequence that has at least
80% identity, more preferably at least 90% identity, yet more
preferably at least 95% identity, most preferably at least 97-99%
identity, to that of SEQ ID NO:2 over the entire length of SEQ ID
NO:2.
[0084] The calbindin-D.sub.28k polypeptides may be in the form of
the "mature" protein or may be a part of a larger protein, such as
a fusion protein. It is often advantageous to include an additional
amino acid sequence which contains secretory or leader sequences,
pre/pro-sequences, sequences which aid in purification such as
multiple histidine residues, or an additional sequence for
stability during recombinant production.
[0085] Fragments of the calbindin-D.sub.28k proteins are also
included in the invention. A fragment is a polypeptide having an
amino acid sequence that entirely is the same as part, but not all,
of the amino acid sequence of the aforementioned
calbindin-D.sub.28k polypeptides. Preferred fragments include, for
example, truncation polypeptides having the amino acid sequence of
calbindin-D.sub.28k polypeptides, except for deletion of a
continuous series of residues that includes the amino terminus, or
a continuous series of residues that includes the carboxyl terminus
or deletion of two continuous series of residues, one including the
amino terminus and one including the carboxyl terminus, but having
the same functionality as the endogenous calbindin-D.sub.28k
protein, namely, the ability to interact with the caspase family of
proteins to inhibit or reduce apoptosis. Other preferred fragments
are biologically active fragments. Biologically active fragments
are those that mediate calbindin-D.sub.28k activity, including
those with a similar activity or an improved activity, or with a
decreased undesirable activity. Also included are those that are
antigenic or immunogenic in an animal, especially in a human.
Preferably, all of these polypeptide fragments retain the
biological activity of the calbindin-D.sub.28k. Variants of the
defined sequence and fragments also form part of the present
invention. Preferred variants are those that vary from the
referents by conservative amino acid substitutions.
[0086] The calbindin-D.sub.28k proteins and polypeptides of the
invention can be prepared in any suitable manner or purchased,
recombinantly, from commercial sources such as Swant Swiss
Antibodies. If produced in situ, the polypeptides may be purified
from appropriate sources, e.g., appropriate vertebrate cells e.g.,
mammalian cells, for instance cells from human, mouse, bovine or
rat. Alternatively, the availability of nucleic acid molecules
encoding the polypeptides enables production of the proteins using
in vitro expression methods well known in the art. For example, a
cDNA or gene may be cloned into an appropriate in vitro
transcription vector, for in vitro transcription, followed by
cell-free translation in a suitable cell-free translation system.
In vitro transcription and translation systems are commercially
available, e.g., from Promega Biotech, Madison, Wis., or BRL,
Rockville, Md. While in vitro transcription and translation is not
the method of choice for preparing large quantities of the protein,
it is ideal for preparing small amounts of native or mutant
proteins for research purposes, particularly since it allows the
incorporation of radioactive nucleotides.
[0087] Larger quantities of calbindin-D.sub.28k encoded polypeptide
may be produced by expression in a suitable prokaryotic or
eukaryotic system. For example, part or all of a DNA molecule, such
as the coding portion of SEQ ID NO: 1 may be inserted into a
plasmid vector adapted for expression in a bacterial cell (such as
E. coli) or a yeast cell (such as Saccharomyces cerevisiae). Such
vectors comprise the regulatory elements necessary for expression
of the DNA in the host cell, positioned in such a manner as to
permit expression of the DNA into the host cell. Such regulatory
elements required for expression include appropriate origins of
replication, promoter sequences, transcription initiation sequences
and optionally, enhancer or termination sequences. Secretion
signals may be used to facilitate purification of the resulting
protein. An appropriate secretion coding sequence for the secretion
of the peptide is operably linked to the 5' end of the coding
sequence for the protein, and this hybrid nucleic acid molecule is
inserted into a plasmid adapted to express the protein in the host
cell of choice. Plasmids specifically designed to express and
secrete foreign proteins are available from commercial sources. For
example, if expression and secretion is desired in E. coli,
commonly used plasmids include pTrcPPA (Pharmacia); pPROK-C and
pKK233-2 (Clontech); and pNH8a, pNH16a, pcDNAII and pAX
(Stratagene), among others.
[0088] The calbindin-D.sub.28k proteins produced by in vitro
transcription and translation or by gene expression in a
recombinant prokaryotic or eukaryotic system may be purified
according to methods known in the art. Recombinant proteins can be
purified by affinity separation, such as by immunological
interaction with antibodies that bind specifically to the
recombinant protein or fusion proteins such as His tags. Such
methods are commonly used by skilled practitioners.
[0089] Using appropriate amino acid sequence information, synthetic
calbindin-D.sub.28k proteins of the present invention may be
prepared by various synthetic methods of peptide synthesis via
condensation of one or more amino acid residues, in accordance with
conventional peptide synthesis methods. Preferably, peptides are
synthesized according to standard solid-phase methodologies, such
as may be performed on an Applied Biosystems Model 430A peptide
synthesizer (Applied Biosystems, Foster City, Calif.), according to
manufacturer's instructions. Other methods of synthesizing peptides
or peptidomimetics, either by solid phase methodologies or in
liquid phase, are well known to those skilled in the art. The
protein produced may be purified from the cells and directly
injected to the BM tissue, infused to osteoblast cells, or
delivered in a lyophilized carrier.
[0090] Hence, in one particular embodiment, the present invention
provides novel compositions containing a calbindin-D.sub.28k
therapeutic element, specifically a calbindin-D.sub.28k
polypeptide, protein or protein fragment, which may act as a
regulator of apoptosis, and may be administered for the prevention
of an abnormal apoptosis response in cells, in particular
osteoblasts and osteoclasts. However, it has also been shown that
calbindin-D.sub.28k can protect against glucocorticoid induced
lymphocyte cell death (Dowd. D. P., et al., Stable expression of
calbindin-D.sub.28k complimentary DNA apoptotic pathway in
lymphocytes. Endocrinology 6:1843-1848, 1992) hence, it is possible
that the compositions and methods of the present invention may also
be useful for preventing premature or excessive lymphocytic death
caused by glucocorticoids.
[0091] In particular the calbindin-D.sub.28k protein compositions
and methods of the present invention may be used for the prevention
of apoptosis in such cell types as osteoblasts and osteocytes, as
well as lymphocytes. Specifically, the compositions and methods of
the present invention may be useful in the prevention of
glucocorticoid induced cell death in osteoblasts and the treatment
of such conditions as glucocorticoid induced osteoporosis
[0092] According to one specific aspect, this invention depends on
an isolated polypeptide encoding a calbindin-D.sub.28k protein.
Preferably, the polypeptide comprises the amino acid sequence of:
SEQ ID NO:2, with up to 30% conservative substitutions. In a
particular aspect, the invention relates to compositions and
methods for using such polypeptides for treating diseases
associated with abnormal or increased programmed cell death, by
administering a calbindin-D.sub.28k polypeptide, in a
pharmaceutically acceptable and appropriate delivery vehicle, to
decrease apoptosis.
[0093] Vectors, Host Cells, and Expression
[0094] In one particular embodiment the present invention relates
to vectors that comprise calbindin-D.sub.28k therapeutic related
genetic elements, such as a polynucleotide or polynucleotides of
the present invention and to the production of polypeptides and
proteins of the invention by recombinant techniques both in vitro
and in vivo, as well as ex vivo procedures. Cell-free translation
systems can also be employed to produce such proteins using RNAs
derived from the DNA constructs of the present invention.
[0095] Host cells can be genetically engineered to incorporate
expression systems or portions thereof for polynucleotides of the
present invention. In accordance with the methods of the present
invention, host cells, such as osteoblasts and/or osteocytes, may
also be obtained from the bone marrow of a subject by procedures
well known in the medical research arts. Introduction of
polynucleotides into host cells can then be effected by methods
described in many standard laboratory manuals, such as Davis et
al., Basic Methods In Molecular Biology (1986) and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). These methods
include calcium phosphate transfection, DEAE-dextran mediated
transfection, microinjection, cationic lipid-mediated transfection,
lipofectamine transfection, electroporation, transduction, scrape
loading, ballistic introduction or infection.
[0096] Representative examples of appropriate hosts for in vitro
procedures include bacterial cells, such as Streptococci,
Staphylococci, E. coli, Streptomyces, Lactobacillus, Bacillus
cells; fungal cells, such as non-pathogenic yeast cells and
Aspergiffits cells; and animal cells such as CHO, COS, HeLa, C127,
3T3, BHK, HEK 293 and Bowes melanoma cells. The selection of an
appropriate host is deemed to be within the scope of those skilled
in the art from the teachings herein.
[0097] More particularly, the present invention also includes
recombinant constructs comprising a calbindin-D.sub.28k DNA, cDNA
or RNA sequence as well as compliment nucleotide sequences, i.e.,
for triplexing duplex DNA, and antisense polynucleotide sequences.
The construct comprises a vector, such as a plasmid or viral
vector, into which the clone has been inserted, in a forward or
reverse orientation. In a preferred aspect of this embodiment, the
construct further comprises regulatory sequences, including, for
example, a promoter, operably linked to the genetic sequence, and
may include a suitable origin of replication or termination
sequence. Large numbers of suitable vectors and promoters are known
to those of skill in the art, and are commercially available. The
following vectors are provided by way of example; Bacterial: pQE70,
pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX 174,
pbluescript SK, pbsks, pNH8A, pNH 16a, pNH18A, pNH46A (Stratagene);
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic:
pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL (Pharmacia); of particular importance are the pREP4
calbindin-D.sub.28k and BSR.alpha. calbindin-D.sub.28k expression
vectors of the present invention. As further examples,
calbindin-D.sub.28k cDNA may be inserted in the pEF/myc/cyto vector
(from Invitrogen) and/or the pCMV-Tag3b vector (from Stratagene),
which can then be used with anti-Myc Ab, to transform Stem, HeLa,
Osteoblasts (or other) cells with the calbindin-D.sub.28k DNA.
However, any other plasmid or vector may be used as long as they
are replicable and viable in the host.
[0098] In addition, a complete mammalian transcription unit and a
selectable marker can be inserted into a prokaryotic plasmid for
use in in vivo procedures. The resulting vector is then amplified
in bacteria before being transfected into cultured mammalian cells,
i.e., osteoblasts or delivered directly to the subject with an
acceptable biological carrier as described below. Examples of
vectors of this type include pTK2, pHyg, pRSVneo, pREP4 or BSRA.
Hence, these plasmids, constructs and vectors may be used in both
in vivo and ex vivo procedures. Ex vivo procedures involve the
removal of a host cell, such as bone marrow, osteoblast or
osteoclast cells, from the subject, recombinant manipulation of the
cell (i.e., transformation, transduction or transfection with a
suitable calbindin-D.sub.28k expression system vector), and the
re-delivery of the cell back into its host environment.
[0099] A wide variety of recombinant plasmids and delivery methods
may be engineered to express the calbindin-D.sub.28k protein and
used for delivery of calbindin-D.sub.28k to a cell. These include
the use of naked DNA and calbindin-D.sub.28k plasmids to directly
transfer genetic material into a cell (Wolfe et al., 1990);
formulations of trapped liposomes encoding a therapeutic
calbindin-D.sub.28k genetic element (Ledley et. al., 1987) or in
proteoliposomes that contain other viral envelope receptor proteins
(Nicolau et al., 1983); and calbindin-encoding DNA, or antisense
sequence, coupled to a polysineglycoprotein carrier complex. Hence
methods for the delivery of nucleotide sequences to cells are well
known in the recombinant arts. Such methods for in vitro delivery,
further include, but are not limited to: microinjection, calcium
phosphatase, lyposomes, lipofectamine transfection and
electroporation.
[0100] Accordingly, one particular embodiment of the present
invention, a therapeutic calbindin-D.sub.28k genetic element such
as, a DNA, cDNA, RNA, may be directly injected to the bone marrow,
specifically osteoblasts or osteocytes, for the production of
calbindin-D.sub.28k endogenously. This may be useful for the
prevention of glucocorticoid induced cell death in various cell
types, in particular osteoblasts and osteocytes, and thereby
effective in preventing osteoporosis, but also in neuronal cells
such as hippocampal cells, thereby possibly being effective for the
prevention of glucocorticoid induced neural degeneration. On the
other hand, the genetic element containing a calbindin-D.sub.28k
DNA, cDNA, RNA or polynucleotide sequences coding for the antisense
sequence encoding the protein may also be delivered using other
appropriate means, including vectors, as herein described, and well
known in the recombinant arts. These may be useful in preventing
various forms of cancers such as prostate cancer and the various
forms of lymphocytic cancers such as leukemia and lymphoma.
[0101] Genetic material, such as the nucleotides of the present
invention, may be delivered to cells, in vivo, using various
different plasmid based delivery platforms, including but not
limited to recombinant ADV (such as that described in U.S. Pat. No.
6,069,134 incorporated by reference herein), AAV (such as those
described by U.S. Pat. No. 5,139,941 incorporated by reference
herein), MMLV, Herpes Simplex Virus (U.S. Pat. No. 5,288,641,
incorporated by reference herein), cytomegalovirus, lentiviral, and
overall, retroviral gene delivery systems, well known and practiced
with in the art.
[0102] Techniques for preparing replication defective, infective
viruses are well known in the art, as exemplified by
Ghosh-Choudhury & Graham (9187); McGory et al. (1988); and
Gluzman et al. (1982), each incorporated by reference herein. These
systems typically include a plasmid vector including a promoter
sequence (such as CMV early promoter) operably linked to the
nucleotide coding the gene of interest (inserted into an
appropriate gene insertion site; i.e., an IRES site), as well as a
terminating signal (such as a Poly-A tail i.e., BGH), and the
appropriate mutations so as to make the delivery vehicle
replication defective (e.g., Psi sequence deletions) and safe for
therapeutic uses. The construction of the appropriate elements in a
vector system containing the nucleotides of the present invention
is well within the skills of one versed in the recombinant
arts.
[0103] A great variety of vector and/or expression systems can be
used. Such systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia, viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate
calbindin-D.sub.28k therapeutic nucleotide sequence may be inserted
into an expression system by any of a variety of well-known and
routine techniques, such as, for example, those set forth in
Sambrook et al., Molecular Cloning, A Laboratory Manual
(supra).
[0104] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol acetyl transferase) vectors or other vectors
with selectable markers. Two appropriate vectors are pKK232-8 and
pCM7. Particular named bacterial promoters include lacI, lacZ, T3,
T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retrovirus, and mouse metallothionein-1. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. A particularly good promoter for in vivo
use would be to use a promoter specific to osteoblasts, for
instance the osteocalcin, calcium binding protein promoter, which
would only allow transcription in osteoblastic cells.
[0105] Further still, the therapeutic calbindin-D.sub.28k genetic
elements such as DNA, cDNA, RNA or polynucleotide sequences coding
for the antisense sequence encoding the protein, may be delivered
to the cells of the bone marrow, specifically to osteoblasts or
osteocytes, for the production or inhibition of calbindin-D.sub.28k
endogenously, by use of biologically compatible carriers or
excipients. This may be useful in inducing or inhibiting programmed
cell death. Pharmaceutically acceptable carriers for therapeutic
use are well known in the pharmaceutical arts, and are described,
for example, in Remington's Pharmaceutical Sciences (A. P. Gennaro,
ed.; Mack, 1985). For example, sterile saline or phosphate-buffered
saline at physiological pH may be used. Preservatives, stabilizers,
dyes, and even flavoring agents may be provided in the
pharmaceutical-composition. For example, sodium benzoate, sorbic
acid, and esters of p-hydroxybenzoic acid may be added as
preservatives. Antioxidants and suspending agents may also be
used.
[0106] The above-described constructs, plasmids and vectors are
useful in gene therapy procedures. Successful gene therapy
generally requires the integration of a gene capable of correcting
the genetic disorder into the host genome, where it would co-exist
and replicate with the host DNA and be expressed at a level to
compensate for the defective gene. Ideally, the disease would be
cured by one or a few treatments, with no serious side effects.
There are several approaches to gene therapy proposed.
[0107] As described above, basic transfection methods exist in
which DNA containing the gene of interest is introduced into cells
non-biologically, for example, by permeabilizing the cell membrane
physically or chemically. Liposomes or protein conjugates formed
with certain lipids and amphophilic peptides can be used for
transfection. (Stewart et al., 1992; Torchilin et al., 1992; Zhu et
al., 1993, incorporated herein by reference.) This approach is
particularly effective in ex vivo procedures involving osteocytes,
which can be temporarily removed from the body and can tolerate the
structural manipulation of the treatment.
[0108] A basic transduction approach capitalizes on the natural
ability of viruses to enter cells, bringing their own genetic
material with them. For example, retroviruses have promise as gene
delivery vectors due to their ability to integrate their genes into
the host genome, transferring a large amount of foreign genetic
material, infecting a broad spectrum of species and cell types and
of being packaged in special cell-lines (Miller, 1992, incorporated
herein by reference).
[0109] A third method uses other viruses, such as adenovirus,
herpes simplex virues (HSV), cytomegalovirus (CMV), and
adeno-associated virus (AAV), which are engineered to serve as
vectors for gene transfer. Although some viruses that can accept
foreign genetic material are limited in the number of nucleotides
they can accommodate and in the range of cells they infect, these
viruses have been demonstrated to successfully effect gene
expression. For example, adenovirus gene transfer systems may be
used. Such a system is based upon recombinant, engineered
adenovirus which is rendered replication-incompetent by deletion of
a portion of its genome, such as El, and yet still retains its
competency for infection. Relatively-large foreign proteins can be
expressed when additional deletions are made in the adenovirus
genome. For example, adenoviruses deleted in both E1 and E3 regions
are capable of carrying up to 10 Kb of foreign DNA and can be grown
to high titers in 293 cells (Stratford-Perricaudet and Perricaudet,
1991a). Surprisingly persistent expression of transgenes following
adenoviral infection has also been reported.
[0110] Hence, in one particular aspect the invention features a
recombinant calbindin-D.sub.28k DNA or RNA molecule comprising a
vector having an insert that includes part or all of a
calbindin-D.sub.28k polynucleotide, or its antisense polynucleotide
sequence. The invention also features a vector containing a
calbindin-D.sub.28k polypeptide. Pharmaceutical compositions
containing a biologically effective amount of the
calbindin-D.sub.28k polynucleotide, antisense sequence, protein
and/or protein fragments with acceptable carriers are also
provided.
[0111] Specifically, in a particular embodiment, the invention
relates to the administration of vectors for the delivery of a
calbindin-D.sub.28k therapeutic element to a cell for the treatment
of diseases associated with an abnormal increase or decrease in
cell death, wherein the vector contains an expression cassette
encoding the calbindin-D.sub.28k therapeutic. The
calbindin-D.sub.28k therapeutic can be a CALBINDIN-D.sub.28k
polynucleotide, a CALBINDIN-D.sub.28k polynucleotide antisense
sequence, a CALBINDIN-D.sub.28k protein, or a CALBINDIN-D.sub.28k
protein fragment. The expression cassette may contain one or more
of the following elements: a host cell origin of replication, a
suitable promoter operably linked to a heterologous genetic
element, an internal ribosome entry site, splice donor site, splice
acceptor site, a suitable enhancer, PPT track, heterologous genetic
element, a reporter gene, and/or an appropriate termination
sequence. One or more of these vectors, containing a
calbindin-D.sub.28k therapeutic, may be introduced into an
appropriate cell by a variety of means, including in vivo, in vitro
or ex vivo transduction or transfection using an appropriate
expression system.
[0112] Delivery/Administration of Calbindin-D.sub.28k
Therapeutics
[0113] The pharmaceutical compositions of the present invention may
be formulated and used as tablets, capsules, or elixirs for oral
administration; suppositories for rectal or vaginal administration;
sterile solutions and suspensions for parenteral administration;
creams, lotions, or gels for topical administration; aerosols or
insufflations for intratracheobronchial administration; and the
like. Preparations of such formulations are well known to those
skilled in the pharmaceutical arts. The dosage and method of
administration can be tailored to achieve optimal efficacy and will
depend on factors that those skilled in the medical arts will
recognize.
[0114] When administration is to be parenteral, such as intravenous
on a daily basis, injectable pharmaceuticals may be prepared in
conventional forms, either as liquid solutions or suspensions;
solid forms suitable for solution or suspension in liquid prior to
injection; or as emulsions. Suitable excipients are, for example,
water, saline, dextrose, mannitol, lactose, lecithin, albumin,
sodium glutamate, cysteine hydrochloride, or the like. In addition,
if desired, the injectable pharmaceutical compositions may contain
minor amounts of nontoxic auxiliary substances, such as wetting
agents, pH buffering agents, and the like. If desired, absorption
enhancing preparations (e.g. liposomes) may be utilized.
[0115] Hence, in another preferred embodiment the present invention
is directed to a novel pharmaceutical composition that includes a
biologically acceptable carrier along with an effective amount of a
therapeutic calbindin-D.sub.28k genetic element such as a
calbindin-D.sub.28k DNA, cDNA, RNA or protein for the treatment
and/or prevention of diseases associated with an abnormal or
increased rate of apoptosis. The pharmaceutical composition
includes a calbindin-D.sub.28k sequence substantially identical to
SEQ ID No. 1 and/or a protein encoded by an amino acid sequence
substantially identical to the sequence of SEQ ID No. 2. For the
treatment of and/or prevention of diseases associated with an
unhealthy decrease in regulated cell death, a pharmaceutical
composition that includes an effective amount of a nucleotide
sequence coding for the antisense sequence of SEQ. ID. NO:1, may be
administered. An example of such diseased state that may be treated
by the compositions of the present invention are osteoblastic or
osteocytic cancer, as well as hormone independent prostate
cancer.
[0116] The methods for the treatment of diseases associated with an
unhealthy increase of programmed cell death in a subject are also
provided. These methods involve administering to the subject a
pharmaceutical composition that includes an effective amount of a
therapeutic calbindin-D.sub.28k genetic element, which may include
a calbindin-D.sub.28k protein or a nucleotide sequence coding for
the calbindin-D.sub.28k protein or a nucleotide sequence that codes
for the antisense sequence of the nucleotide sequence coding for
the calbindin-D.sub.28k protein (wherein an increased rate of cell
death is sought). These may be delivered by suitable means, as
described above, including the use of vectors and or acceptable
biological carriers.
[0117] For administration, the therapeutic agent will generally be
mixed, prior to administration, with a non-toxic, pharmaceutically
acceptable carrier substance. Usually, this will be an aqueous
solution, such as normal saline or phosphate-buffered saline (PBS),
Ringer's solution, lactate-Ringer's solution, or any isotonic
physiologically acceptable solution for administration by the
chosen means. Preferably, the solution is sterile and pyrogen-free,
and is manufactured and packaged under current Good Manufacturing
Processes (GMP's), as approved by the FDA. The clinician of
ordinary skill is familiar with appropriate ranges for pH,
tonicity, and additives or preservatives when formulating
pharmaceutical compositions for administration by intravascular
injection, intrathecal injection, injection into the bone marrow,
direct injection into the osteoblast cell, or by other routes. In
addition to additives for adjusting pH or tonicity, the
therapeutics agent may be stabilized against aggregation and
polymerization with amino acids and non-ionic detergents,
polysorbate, and polyethylene glycol.
[0118] Optionally, additional stabilizers may include various
physiologically acceptable carbohydrates and salts. Also,
polyvinylpyrrolidone may be added in addition to the amino acid.
Suitable therapeutic immunoglobulin solutions, which are stabilized
for storage and administration to humans, are described in U.S.
Pat. No. 5,945,098, incorporated fully herein by reference. Other
agents, such as human serum albumin (HSA), may be added to the
therapeutic composition to stabilize the antibody conjugates. The
compositions of the invention may be administered using any
medically appropriate procedure, e.g., intravascular (intravenous,
intraarterial, intracapillary) administration, injection into the
bone marrow, intracavity or direct injection in the osteoblast
cell. Intravascular injection may be by intravenous or
intraarterial injection.
[0119] The effective amount of the therapeutic composition to be
given to a particular patient will depend on a variety of factors,
several of which will be different from patient to patient. A
competent clinician will be able to determine an effective amount
of a therapeutic composition to administer to a patient to retard
or promote apoptosis in target cells, such as osteoblasts. Dosage
of the therapeutic will depend on the type of treatment, route of
administration, the nature of the therapeutics, sensitivity of the
cell to the therapeutics, etc. Utilizing LD.sub.50 animal data, and
other information available for the administration of such
compositions, a clinician can determine the maximum safe dose for
an individual, depending on the route of administration. For
instance, an intravenously administered dose may be more than an
intrathecally administered dose, given the greater body of fluid
into which the therapeutic composition is being administered.
Similarly, compositions, which are rapidly cleared from the body,
may be administered at higher doses, or in repeated doses, in order
to maintain a therapeutic concentration. Utilizing ordinary skill,
the competent clinician will be able to optimize the dosage of a
particular therapeutic composition in the course of routine
clinical trials.
[0120] Typically the dosage will be 0.001 to 100 milligrams of
therapeutic per Kilogram subject body weight. Doses in the range of
0.01 to 1 mg per kilogram of patient body weight may be utilized
for a therapeutic composition that is administered. The therapeutic
can be administered to the subject in a series of more than one
administration. For therapeutic compositions, regular periodic
administration (e.g., every 2-3 days) will sometimes be required,
or may be desirable to reduce toxicity. For therapeutic
compositions that will be utilized in repeated-dose regimens,
moieties that do not provoke HAMA or other immune responses are
preferred.
[0121] The foregoing is intended to be illustrative of the
embodiments of the present invention, and are not intended to limit
the invention in any way. Although the invention has been described
with respect to specific modifications, the details thereof are not
to be construed as limitations, for it will be apparent that
various equivalents, changes and modifications may be resorted to
without departing from the spirit and scope thereof and it is
understood that such equivalent embodiments are to be included
herein. All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
EXAMPLES
[0122] The following description sets forth the general procedures
involved in practicing the present invention. To the extent that
specific materials are mentioned, it is merely for purposes of
illustration and is not intended to limit the invention. Unless
other-wise specified, general cloning procedures, such as those set
forth in Sambrook et al., Molecular Cloning, supra or Ausubel et
al. (eds) Current Protocols in Molecular Biology, John Wiley &
Sons (2000) are used.
Example I
[0123] A. Materials
[0124] Calbindin-D.sub.28k was purified from rat kidney, as
described previously (Pansini, A. R., and Christakos, S. (1984) J.
Biol. Chem. 259, 9735-9741). Purified rat bone osteocalcin was
purchased from Biomedical Technologies Inc. (Stoughton, Mass.).
Purified bovine calbindin-D.sub.9k, S100, calmodulin, murine
gelsolin, and antibody to -actin were purchased from Sigma. The
caspase-3 activity assay kit, recombinant and purified human
caspase-3, and the cell-permeable caspase-3 inhibitor
Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) were obtained from Biomol
Research Laboratories, Inc. (Plymouth Meeting, Pa.). The
glucocorticoid dexamethasone can be obtained from several
commerical sources. SuperFect, Lipofectin, and ECL reagents were
purchased from Qiagen (Santa Clarita, Calif.), Life Technologies,
Inc., and NEN Life Science Products, respectively. The enhanced
green fluorescent protein (Clontech Laboratories, Palo Alto,
Calif.) containing the SV40 large T antigen nuclear localization
sequence (Kalderon, D., Roberts, B. L., Richardson, W. D., and
Smith, A. E. (1984) Cell 39, 499-509) attached to the carboxyl
terminus was provided by Dr. Charles O'Brien (University of
Arkansas for Medical Sciences, Little Rock, Ark.).
[0125] B. Cell Culture Conditions
[0126] Murine osteocytic MLO-Y4 cells (provided by Dr. Lynda
Bonewald, University of Texas Health Center at San Antonio, Tex.),
and primary bone cells (isolated from neonatal murine calvaria)
were cultured as indicated previously (Kato, Y., Windle, J. J.,
Koop, B. A., Mundy, G. R., and Bonewald, L. F. (1997) J. Bone
Miner. Res. 12, 2014-2023) (Bellido, T., Borba, V. Z. C., Roberson,
P. K., and Manolagas, S. C. (1997) Endocrinology 138, 3666-3676).
All culture media contained 100 units/ml penicillin and 100
.mu.g/ml streptomycin. Cultures were kept in a humidified
atmosphere of 5% CO.sub.2 in air at 37.degree. C.
[0127] C. Transient and Stable Transfections of
Calbindin-D.sub.28k
[0128] Transient transfections of MLO-Y4 cells were carried out in
12-well culture plates using SuperFect. Cells
(0.1.times.10.sup.6/well) were transfected in triplicate, using
lipofectamine, with 2 .mu.g of the expression vectors pBSR (a gift
of Dr. Michael Olszowy, Washington University School of Medicine,
St. Louis, Mo.) or pREP4 (Invitrogen, Carlsbad, Calif.), alone or
containing the cDNA for calbindin-D.sub.28k. The
calbindin-D.sub.28k expression plasmids were prepared using the
full-length calbindin-D.sub.28k cDNA isolated by reverse
transcriptase-polymerase chain reaction from rat renal distal
tubular mRNA. For the establishment of MLO-Y4 cells expressing
different levels of calbindin-D.sub.28k, parent MLO-Y4 cells were
transfected with pREP4 or pREP4-calbindin-D.sub.28k as described in
Guo, Q., Christakos, S., Robinson, N., and Mattson, M. P. (1998)
Proc. Natl. Acad. Sci. U.S.A. 95, 3227-3232. MLO-Y4 cells were then
cultured in .alpha.MEM (phenol red free) supplemented with 5% FBS,
5% bovine calf serum, 100 U/ml penicillin and 100 .mu.g/ml
streptomycin. Cells were plated at 1.times.10.sup.4 to
2.times.10.sup.4 cells/cm.sup.2 on collagen I-coated plates. After
6, 12 and 24 h, apoptosis was induced by addition of 10.sup.-6M
dexamethasone and quantified as indicated below or cell lysates
collected for the analysis of calbindin-D.sub.28k by Western
blotting and radioimmunoassay (cite 65 of grant).
[0129] D. Apoptosis
[0130] Cell viability was monitored by trypan blue exclusion.
Nonadherent cells were combined with adherent cells released from
the culture dish using trypsin-EDTA, resuspended in medium
containing serum and collected by centrifugation. 0.04% trypan blue
was added and the percentage of cells exhibiting cytoplasmic and
nuclear staining was determined by a hemocytometer (at least 150
cells per condition are counted). DNA fragmentation was measured by
the TUNEL (transferase biotin-dUTP nick endlabeling). We have
previously demonstrated that the percentage of cells exhibiting
trypan blue staining correlates with the percentage of terminal
deoxynucleotidyl transferase-mediated nick end labeling-labeled
cells, indicating that apoptotic osteoblasts could be reliably
quantified by either method (Jilka, R. L., Weinstein, R. S.,
Bellido, T., Parfitt, A. M., and Manolagas, S. C. (1998) J. Bone
Miner. Res. 13, 793-802; Jilka, R. L., Weinstein, R. S., Bellido,
T., Roberson, P. K., Parfitt, A. M., and Manolagas, S. C. (1999) J.
Clin. Invest. 104, 439-446; Plotkin, L. I., Weinstein, R. S.,
Parfitt, A. M., Roberson, P. K., Manolagas, S. C., and Bellido, T.
(1999) J. Clin. Invest. 104, 1363-1374; and Bellido, T., O'Brien,
C. A., Roberson, P. K., and Manolagas, S. C. (1998) J. Biol. Chem.
273, 21137-21144).
[0131] After 6, 12 and 24 h in the presence or absence of
dexamethasone cells were fixed, permeabilized (0.1%
Triton.times.100 in 0.1% citrate), washed and labeled using
terminal deoxynucleotidyl transferase (TdT) which catalyzes the
addition of fluorescein dUTP at free 3'OH groups in single and
double stranded DNA. Cells were then washed and analyzed by
fluorescence microscopy (a negative control included all steps
without the addition of terminal transferase (In situ cell death
detection kit, Boehringer Mannheim). Cells were also examined
microscopically to assess morphological changes in the presence of
dexamethasone and in the presence or absence of calbindin.
[0132] E. Western Blot Analysis and Radioimmunoassay
[0133] Cell lysates, protein electrophoresis and Western blot
analysis were performed as described previously (Bellido, T.,
Borba, V. Z. C., Roberson, P. K., and Manolagas, S. C. (1997)
Endocrinology 138, 3666-3676). Membranes were incubated overnight
at 4.degree. C. with an antibody to either calbindin-D.sub.28k
(Sonnenberg, J., Pansini, A. R., and Christakos, S. (1984)
Endocrinology 115, 640-648) or -actin, followed by incubation for 1
h with the corresponding secondary antibody conjugated with
horseradish peroxidase. Blots were developed by ECL. Quantification
of the intensity of the bands in the autoradiograms was performed
by laser densitometry. Calbindin-D.sub.28k protein levels were
determined by radioimmunoassay using antiserum against rat renal
calbindin-D.sub.28k and purified rat renal calbindin-D.sub.28k as
standard, as described previously (Sonnenberg, J., Pansini, A. R.,
and Christakos, S. (1984) Endocrinology 115, 640-648).
[0134] F. In Vitro Digestion With Caspase-3
[0135] Purified gelsolin (3 .mu.g) or calbindin-D.sub.28k (3 .mu.g)
and caspase-3 (50 ng) were incubated at 37.degree. C. for 10 min in
6 mM Tris-HCl (pH 7.5), 1.2 mM CaCl.sub.2, 1.5 mM dithiothreitol,
1.5 mM MgCl.sub.2, and 1 mM KCl. Samples were analyzed by SDS-PAGE
followed by staining with Coomassie Blue.
[0136] G. Caspase-3 Activity
[0137] Since dexanethasone induced apoptosis in MLO-Y4 cells and in
primary osteoblasts has been shown to be accompanied by activation
of caspase 3 (cite 47 of grant) and since we predicted that
calbindin-D.sub.28k inhibits caspase 3 (cite 29 of grant), we
examined MLO-Y4 cells for an increase in caspase 3 in response to
dexamethasone and used calbindin-D.sub.28k transfected MLO-Y4 cells
to determine whether calbindin-D.sub.28k blocks dexamethasone
induced caspase 3 activity in these cells. Caspase 3 activity was
measured by the degradation of the colorimetric caspase 3 substrate
DEVD-paranitroanilide (DEVD-pNA) that contains the sequence of the
caspase 3 cleavage site in poly(ADP-ribose) polymerase (Schlegel,
J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A.,
Yamin, T. T., and Nicholson, D. W. (1996) J. Biol. Chem. 271,
1841-1844) (cite 110 of grant). Cell pellets obtained from MLO-Y-4
cells transfected with calbindin-D.sub.28k or vector and treated
with dexamethasone was resuspended in lysis buffer (50 mM Hepes pH
7.4, 0.1% CHAPS, 1 mM DDT, 0.1 mM EDTA, 0.1% Triton.times.100).
After centrifugation (20,000.times.g 10 min) 30-100 ug cell lysate
was added to the assay buffer (50 mM Hepes. 100 mM NaCl, 0.1%
CHAPS, 10% v/v glycerol, 1 mM EDTA and 10 mM DDT) containing 200
.mu.M DEVD-pNA (total volume=100 ul). Incubation was at 37.degree.
C. and plate absorbency was read at 405 nm every 10 min up to 120
min in a microtiter plate reader. As a positive control purified
caspase 3 (30U) was incubated with DEVD-pNA. As an additional
control the specific inhibitor of caspase 3 (DEVD-CHO) can be added
to cell lysates (0.1 M) to control for any non-specific hydrolysis
of DEVD-pNA. The caspase 3 assay (BIOMOL QuantiZyme Assay System)
may be used for these purposes.
[0138] H. Results of Analysis
[0139] Data were analyzed by one-way analysis of variance, and the
Student-Keuls-Newman method was used to estimate the level of
significance of differences between means. The effect of
dexamethasone treatment and calbindin-D.sub.28k overexpression on
the proportion of apoptotic cells in the experiment of FIG. 1 was
analyzed using exact procedures for logistic regression (otulsky,
H. (1995) Intuitive Biostatistics, Oxford University Press, New
York) (LogXact software; CYTEL Software Corp.; Cambridge, Mass.).
Following this, individual pair-wise comparisons of experimental
groups were conducted using exact tests, adjusting the p values
with a Bonferroni correction. All p values (unadjusted for logistic
regression; Bonferroni-adjusted for individual group comparisons)
were compared with a value of 0.05 to determine significance.
Findings shown in FIG. 1 show the protection by calbindin-D.sub.28k
against glucocorticoid induced apoptosis.
Example 2
[0140] A. Further Analysis
[0141] In addition to MLO-Y4 cells, it will also be possible to
determine the effect of dexamethasone in the presence or absence of
calbindin-D.sub.28k on apoptotic cell death and on caspase 3
activity (with the methodology outlined above) using primary
osteoblastic cells from calbindin-D.sub.28k KO and WT mice. Murine
osteoblasts will be isolated from pups 30-72 h old from our colony
of WT (C57BL6) or calbindin-D.sub.28k KO mice (cite 22 of grant,
cite 111 of grant) by controlled digestion with collagenase (cite
112 of grant). Briefly calvariae, trimmed of fibrous tissue and
divided in 2 are transferred to 15 ml conical sterile tubes, washed
with PBS (5 ml) 3.times. at 37.degree. C. (10 min/wash) in an
oscillating H.sub.2O bath. Calvariae are then subjected to a series
of collagenase digestions (200 .mu.g/Ml PBS collagenase CLS2 from
Worthington) in an oscillating 37.degree. C. H.sub.2O bath
(calvariae from 5-10 mice/approx. 5 ml collagenase buffer/10 min.
followed by low speed centrifugation and resuspension of the pellet
and repeat digestion). The first 2 digests are discarded and
digests from 3, 4 and 5 are neutralized with MEM, pooled, filtered
through sterile mesh. The filtrate is centrifuged for 6 min. at
1500 rpm, the supernatant removed and the cells resuspended in 3-5
ml MEM containing 100%FCS. After isolation cells are cultured in
10%FBS at 1.times.10.sup.4 to 2.times.10.sup.4 cells/cm2. In
preliminary studies using primary osteoblasts K. Gengaro found that
after 16 h dex. treatment (10.sup.-6M) the % of dead cells (assayed
by trypan blue uptake) was 23.2.+-.3 in the absence of
calbindin-D.sub.28k and 12.9.+-.2.4 in the presence of
calbindin-D.sub.28k (p<0.05). This experiment will be important
in determining that there is an increase in capsae 3 activity after
glucocorticoid treatment and establishing that calbindin-D.sub.28k
inhibits that increase in bone cells. The ability of
calbindin-D.sub.28k to protect against induction of other caspases
such as caspase 6, 7, 9 and 10 will also be examined in accordance
with the experiments set forth above.
Sequence CWU 1
1
4 1 3352 DNA RATTUS NORVEGICUS 1 gggctatgtc tcgtcccttt ggagtagcat
tgttgtttgg aggagttgat gagaaagggc 60 ccaactgttt cacatggacc
catctgggac cttcgtacag tgtgatgctc gagcaattgg 120 ttctgcgtac
gagggtgcca gagctcttgc aggaagttta ccacaagtct atgactctga 180
aggaggccat caagtcttca ctcatcatcc tcaagcaagt catggaggag aagctgaacg
240 caactaacat cgagctggcc acagtgcagc ctggtcagaa tttccacatg
ttcacaaagg 300 aagaactgga ggaggtgatc aaggacattt aaggaggggc
catcctcgaa cttctgtggg 360 acagtttcag ttctaatggc tcttagactt
tatttccaac tccacgtcgt gaaaatatcc 420 agtatatgta tgtgtgtttt
tttatgatgt ctgtacataa cagcaattct gaaataaaaa 480 aaatttacaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcac cgcggacagc 540
gccggccgcg ccgcgcccag ctcagcctgc tcagccctct cgtcccgagg ttcgcgctca
600 gcgctctctc taactagccg ctgcaccatg gcagaatccc acctgcagtc
atctctgatc 660 acagcctcac agttttttga gatctggctt catttcgacg
ctgatggaag tggttacctg 720 gaaggaaagg agctgcagaa cttgatccag
gagcttctgc aggcacgaaa gaaggctgga 780 ttggagctat cacctgagat
gaaaaccttt gtggatcaat atgggcagag agatgatggg 840 aaaataggaa
ttgtagagtt ggcccatgtc ttacccaccg aagagaattt cctgctgctc 900
tttcgatgcc agcaactgaa gtcctgcgag gaattcatga agacttggag aaagtatgac
960 actgaccaca gtggcttcat agaaacggag gaacttaaga actttcttaa
ggacctgcta 1020 gagaaagcaa acaagaccgt gcatgatacg aaacttgctg
agtacacaga cctcatgctg 1080 aagctgttcg actcaaataa tgatgggaag
ctggagctga cagagatggc caggttacta 1140 ccagtgcagg aaaatttcct
tcttaaattc cagggaatca aaatgtgtgg gaaagagttc 1200 aataaggctt
ttgagttata tgatcaggat ggcaacggat acatagatga aaatgagctg 1260
gatgccttac tgaaagacct gtgtgagaaa aacaaacagg aattggatat taacaatatt
1320 tctacataca agaagaacat aatggccttg tcggatggag ggaagctgta
ccgaacagat 1380 cttgccctta ttctctctgc tggggacaac tagagttggt
ggccacaacc acttgctagt 1440 gatacattgt atctaaaacc ataactgtgc
gctataaagg agtaggctgt attttctttt 1500 atatctgtaa attctactgc
atatagagaa ttatccagga tgtgtggcac attcttttct 1560 gcttgtttct
atactgtttg taatgtacag tttttgtaag catataattg aaaagaagaa 1620
agtctatgct taggccagtc agtataatcc attttcaaag atgaatctaa catgattctg
1680 ctttcataaa tacagatgaa cacttggatt tccctaaaac tctaccatct
caacaattct 1740 agtgtcagat gtgtaaatgc agctgtcagt gagtaaaaga
ataattcatg acaagccaag 1800 tgttttttaa tttaggcaat catagaactg
tcccacaaag cacttctgtg cgtttccatc 1860 tagtggaagg gatgtgcttc
tgcttgtgaa gcaccaaata tcaatagtta actatggctt 1920 tatcataaaa
cgatctccct agagatttaa tttactgatc agtggcatgt ctactgcttg 1980
aatagatacc acactgttgg ttcaagctgg cttggtggca agggaaggta gccagatgac
2040 acataaatct gtctgatact atgcctatat ttccaagaag tctattgcag
agagtatgac 2100 cttagcccat tttctaaatt attttcatgt gttccagatg
acaattattc tagtaaactg 2160 ctgttttgtg tcatattctg tgtgtactct
ctgattaaat tcaatgtact gagtaaaaaa 2220 aaaaaacggg gctgcggcat
tgcgcccagc agcggactcc gagatccata gacctcggcg 2280 aacccttgct
ctcgaccacc cacccacttt cggaaccatg gccgcagtgg cagcagcctc 2340
ggcagaactg ctcatcatcg gctggtacat cttccgcgtg ctgcagaagc ctggcgcaca
2400 cggtgtcccg gaccggcggc aggtgctggg ggagcgcagg caccgagccc
ccaactgagg 2460 ccccatctcc cagccctggg cggccgtgtc atcaggtgct
cctgtgcttc tcgaccagca 2520 tgggagccaa tgccgcgcag gaatgggggg
tcccctgtgc tccctcgtca gagagcactt 2580 gccaaggtca gtgaggggcc
ggtagtccag aaaagcagca ccggaccaat gatgaagaca 2640 tccagttcct
ttcccagccc cccccccctt tgcccctgtc ccatggccgg cgggtgggag 2700
aggatggggg aagaggggag caaccctcga gatatgggcg taggcaccac attctgatct
2760 ggaccaagtt ggaacagcag ccatctcagc cgcacaagat cctaccatgg
agagctaaca 2820 cccccaccaa ccagcagaat ggacattctg acatcaccag
ctgaaaccct gaatctcggt 2880 ggcagaagag aaagtgtcaa ctgcgtgcag
cactggggga gtggagggtg tgggtggtgg 2940 aggaagaggg ttaagaaaac
tagtggggcc ctcttgctgt ccctgtccta tggcacgcat 3000 attcctgcct
tgctccctca ctccccctct cccctgcctt ccaaagcccc accccccaaa 3060
aatgtgtcac ttgattcgga cctattcaac cagtaattgg atcccacctt taccaaaaca
3120 ccgtctctga cccccggccc ttcactgatc ttgcttatcc ctggtctcac
gcagcagttg 3180 tggttgctat tgtggtagtc gctaattgta ctagtttacg
tgtgcattag ttgtgtctcc 3240 ccggctagat tgtaagctcc tggagacagg
gaccacctcc acaaaaaata aaaaaacgga 3300 cctctcctgt cttgtagtgt
cctaggaccc tgcagggcag tgggggtgca cc 3352 2 261 PRT RATTUS
NORVEGICUS 2 Met Ala Glu Ser His Leu Gln Ser Ser Leu Ile Thr Ala
Ser Gln Phe 1 5 10 15 Phe Glu Ile Trp Leu His Phe Asp Ala Asp Gly
Ser Gly Tyr Leu Glu 20 25 30 Gly Lys Glu Leu Gln Asn Leu Ile Gln
Glu Leu Leu Gln Ala Arg Lys 35 40 45 Lys Ala Gly Leu Glu Leu Ser
Pro Glu Met Lys Thr Phe Val Asp Gln 50 55 60 Tyr Gly Gln Arg Asp
Asp Gly Lys Ile Gly Ile Val Glu Leu Ala His 65 70 75 80 Val Leu Pro
Thr Glu Glu Asn Phe Leu Leu Leu Phe Arg Cys Gln Gln 85 90 95 Leu
Lys Ser Cys Glu Glu Phe Met Lys Thr Trp Arg Lys Tyr Asp Thr 100 105
110 Asp His Ser Gly Phe Ile Glu Thr Glu Glu Leu Lys Asn Phe Leu Lys
115 120 125 Asp Leu Leu Glu Lys Ala Asn Lys Thr Val Asp Asp Thr Lys
Leu Ala 130 135 140 Glu Tyr Thr Asp Leu Met Leu Lys Leu Phe Asp Ser
Asn Asn Asp Gly 145 150 155 160 Lys Leu Glu Leu Thr Glu Met Ala Arg
Leu Leu Pro Val Gln Glu Asn 165 170 175 Phe Leu Leu Lys Phe Gln Gly
Ile Lys Met Cys Gly Lys Glu Phe Asn 180 185 190 Lys Ala Phe Glu Leu
Tyr Asp Gln Asp Gly Asn Gly Tyr Ile Asp Glu 195 200 205 Asn Glu Leu
Asp Ala Leu Leu Lys Asp Leu Cys Glu Lys Asn Lys Gln 210 215 220 Glu
Leu Asp Ile Asn Asn Ile Ser Thr Tyr Lys Lys Asn Ile Met Ala 225 230
235 240 Leu Ser Asp Gly Gly Lys Leu Tyr Arg Thr Asp Leu Ala Leu Ile
Leu 245 250 255 Ser Ala Gly Asp Asn 260 3 1440 PRT RATTUS
NORVEGICUS 3 Thr Gly Cys Thr Gly Gly Thr Gly Gly Gly Ala Thr Cys
Ala Ala Ala 1 5 10 15 Gly Cys Gly Cys Ala Gly Thr Gly Thr Cys Cys
Thr Gly Cys Gly Gly 20 25 30 Cys Gly Gly Gly Gly Ala Gly Cys Thr
Thr Gly Gly Ala Ala Cys Gly 35 40 45 Cys Thr Ala Ala Gly Ala Ala
Ala Ala Gly Thr Gly Ala Cys Cys Ala 50 55 60 Thr Gly Gly Ala Gly
Ala Ala Cys Ala Ala Cys Ala Ala Ala Ala Cys 65 70 75 80 Cys Thr Cys
Ala Gly Thr Gly Gly Ala Thr Thr Cys Ala Ala Ala Ala 85 90 95 Thr
Cys Cys Ala Thr Thr Ala Ala Thr Ala Ala Thr Thr Thr Thr Gly 100 105
110 Ala Ala Gly Thr Ala Ala Ala Gly Ala Cys Cys Ala Thr Ala Cys Ala
115 120 125 Thr Gly Gly Gly Ala Gly Cys Ala Ala Gly Thr Cys Ala Gly
Thr Gly 130 135 140 Gly Ala Cys Thr Cys Thr Gly Gly Gly Ala Thr Cys
Thr Ala Thr Cys 145 150 155 160 Thr Gly Gly Ala Cys Ala Gly Thr Ala
Gly Thr Thr Ala Cys Ala Ala 165 170 175 Ala Ala Thr Gly Gly Ala Thr
Thr Ala Thr Cys Cys Thr Gly Ala Ala 180 185 190 Ala Thr Gly Gly Gly
Cys Ala Thr Ala Thr Gly Cys Ala Thr Ala Ala 195 200 205 Thr Ala Ala
Thr Thr Ala Ala Thr Ala Ala Thr Ala Ala Gly Ala Ala 210 215 220 Cys
Thr Thr Cys Cys Ala Thr Ala Ala Gly Ala Gly Cys Ala Cys Thr 225 230
235 240 Gly Gly Ala Ala Thr Gly Thr Cys Ala Thr Cys Thr Cys Gly Cys
Thr 245 250 255 Cys Thr Gly Gly Thr Ala Cys Gly Gly Ala Thr Gly Thr
Gly Gly Ala 260 265 270 Cys Gly Cys Ala Gly Cys Cys Ala Ala Cys Cys
Thr Cys Ala Gly Ala 275 280 285 Gly Ala Gly Ala Cys Ala Thr Thr Cys
Ala Thr Gly Gly Gly Cys Cys 290 295 300 Thr Gly Ala Ala Ala Thr Ala
Cys Cys Ala Ala Gly Thr Cys Ala Gly 305 310 315 320 Gly Ala Ala Thr
Ala Ala Ala Ala Ala Thr Gly Ala Thr Cys Thr Thr 325 330 335 Ala Cys
Thr Cys Gly Thr Gly Ala Ala Gly Ala Cys Ala Thr Thr Thr 340 345 350
Thr Gly Gly Ala Ala Thr Thr Ala Ala Thr Gly Gly Ala Thr Ala Gly 355
360 365 Thr Gly Thr Thr Thr Cys Thr Ala Ala Gly Gly Ala Ala Gly Ala
Thr 370 375 380 Cys Ala Thr Ala Gly Cys Ala Ala Ala Ala Gly Gly Ala
Gly Cys Ala 385 390 395 400 Gly Cys Thr Thr Thr Gly Thr Gly Thr Gly
Thr Gly Thr Gly Ala Thr 405 410 415 Thr Cys Thr Ala Ala Gly Cys Cys
Ala Thr Gly Gly Thr Gly Ala Thr 420 425 430 Gly Ala Ala Gly Gly Gly
Gly Thr Cys Ala Thr Thr Thr Ala Thr Gly 435 440 445 Gly Gly Ala Cys
Ala Ala Ala Thr Gly Gly Gly Cys Cys Thr Gly Thr 450 455 460 Thr Gly
Ala Ala Cys Thr Gly Ala Ala Ala Ala Ala Gly Thr Thr Gly 465 470 475
480 Ala Cys Thr Ala Gly Cys Thr Thr Cys Thr Thr Cys Ala Gly Ala Gly
485 490 495 Gly Cys Gly Ala Cys Thr Ala Cys Thr Gly Cys Cys Gly Gly
Ala Gly 500 505 510 Thr Cys Thr Gly Ala Cys Thr Gly Gly Ala Ala Ala
Gly Cys Cys Gly 515 520 525 Ala Ala Ala Cys Thr Cys Thr Thr Cys Ala
Thr Cys Ala Thr Thr Cys 530 535 540 Ala Gly Gly Cys Cys Thr Gly Cys
Cys Gly Gly Gly Gly Thr Ala Cys 545 550 555 560 Gly Gly Ala Gly Cys
Thr Gly Gly Ala Cys Thr Gly Thr Gly Gly Cys 565 570 575 Ala Thr Thr
Gly Ala Gly Ala Cys Ala Gly Ala Cys Ala Gly Thr Gly 580 585 590 Gly
Gly Ala Cys Thr Gly Ala Thr Gly Ala Gly Gly Ala Gly Ala Thr 595 600
605 Gly Gly Cys Thr Thr Gly Cys Cys Ala Gly Ala Ala Gly Ala Thr Ala
610 615 620 Cys Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Thr Gly Ala
Cys Thr 625 630 635 640 Thr Cys Cys Thr Gly Thr Ala Thr Gly Cys Thr
Thr Ala Cys Thr Cys 645 650 655 Thr Ala Cys Ala Gly Cys Ala Cys Cys
Thr Gly Gly Thr Thr Ala Cys 660 665 670 Thr Ala Thr Thr Cys Cys Thr
Gly Gly Ala Gly Ala Ala Ala Thr Thr 675 680 685 Cys Ala Ala Ala Gly
Gly Ala Cys Gly Gly Gly Thr Cys Gly Thr Gly 690 695 700 Gly Thr Thr
Cys Ala Thr Cys Cys Ala Gly Thr Cys Cys Cys Thr Thr 705 710 715 720
Thr Gly Cys Ala Gly Cys Ala Thr Gly Cys Thr Gly Ala Ala Gly Cys 725
730 735 Thr Gly Thr Ala Cys Gly Cys Gly Cys Ala Cys Ala Ala Gly Cys
Thr 740 745 750 Ala Gly Ala Ala Thr Thr Thr Ala Thr Gly Cys Ala Cys
Ala Thr Thr 755 760 765 Cys Thr Cys Ala Cys Thr Cys Gly Cys Gly Thr
Thr Ala Ala Cys Ala 770 775 780 Gly Gly Ala Ala Gly Gly Thr Gly Gly
Cys Ala Ala Cys Gly Gly Ala 785 790 795 800 Ala Thr Thr Cys Gly Ala
Gly Thr Cys Cys Thr Thr Cys Thr Cys Cys 805 810 815 Cys Thr Gly Gly
Ala Cys Thr Cys Cys Ala Cys Thr Thr Thr Cys Cys 820 825 830 Ala Cys
Gly Cys Ala Ala Ala Gly Ala Ala Ala Cys Ala Gly Ala Thr 835 840 845
Cys Cys Cys Gly Thr Gly Thr Ala Thr Thr Gly Thr Gly Thr Cys Cys 850
855 860 Ala Thr Gly Cys Thr Cys Ala Cys Gly Ala Ala Ala Gly Ala Ala
Cys 865 870 875 880 Thr Gly Thr Ala Cys Thr Thr Thr Thr Ala Thr Cys
Ala Cys Thr Ala 885 890 895 Gly Ala Gly Gly Ala Ala Thr Gly Ala Thr
Thr Gly Gly Gly Gly Gly 900 905 910 Thr Gly Gly Gly Gly Gly Gly Gly
Gly Gly Cys Gly Thr Gly Thr Thr 915 920 925 Thr Cys Thr Gly Thr Thr
Thr Thr Gly Thr Thr Ala Thr Gly Cys Cys 930 935 940 Ala Ala Ala Thr
Gly Ala Gly Ala Ala Ala Gly Cys Thr Gly Thr Cys 945 950 955 960 Ala
Gly Gly Gly Ala Gly Ala Cys Thr Cys Thr Cys Ala Thr Thr Thr 965 970
975 Ala Ala Ala Thr Cys Thr Ala Ala Thr Cys Thr Gly Ala Cys Gly Gly
980 985 990 Thr Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Thr Thr Thr
Gly Thr 995 1000 1005 Ala Cys Gly Cys Thr Ala Cys Cys Ala Cys Thr
Gly Cys Cys Thr 1010 1015 1020 Ala Gly Cys Ala Ala Thr Gly Cys Ala
Gly Cys Cys Ala Gly Cys 1025 1030 1035 Cys Ala Cys Ala Gly Thr Gly
Cys Ala Gly Cys Thr Ala Cys Cys 1040 1045 1050 Thr Cys Ala Ala Cys
Thr Thr Cys Gly Ala Cys Ala Thr Cys Ala 1055 1060 1065 Gly Gly Thr
Ala Gly Thr Thr Gly Ala Ala Ala Thr Gly Ala Ala 1070 1075 1080 Ala
Thr Thr Thr Ala Ala Thr Thr Thr Ala Ala Thr Ala Ala Gly 1085 1090
1095 Gly Ala Gly Cys Ala Ala Gly Thr Ala Ala Cys Thr Gly Thr Cys
1100 1105 1110 Ala Ala Thr Gly Ala Thr Gly Gly Thr Ala Cys Thr Ala
Thr Cys 1115 1120 1125 Ala Thr Cys Cys Thr Ala Gly Ala Thr Gly Ala
Ala Ala Thr Thr 1130 1135 1140 Ala Cys Ala Ala Ala Gly Thr Thr Gly
Cys Cys Cys Thr Thr Thr 1145 1150 1155 Thr Ala Thr Ala Ala Thr Thr
Ala Gly Cys Ala Ala Gly Ala Thr 1160 1165 1170 Thr Thr Gly Gly Cys
Gly Ala Thr Ala Cys Thr Ala Thr Gly Ala 1175 1180 1185 Ala Thr Thr
Thr Thr Gly Ala Ala Gly Thr Cys Ala Thr Thr Thr 1190 1195 1200 Thr
Gly Ala Ala Gly Cys Ala Gly Thr Ala Cys Ala Gly Cys Thr 1205 1210
1215 Thr Thr Thr Cys Cys Ala Cys Thr Gly Ala Thr Gly Ala Thr Thr
1220 1225 1230 Thr Thr Ala Thr Ala Cys Thr Cys Cys Cys Cys Ala Cys
Thr Cys 1235 1240 1245 Ala Thr Gly Thr Thr Ala Ala Gly Ala Ala Thr
Gly Thr Thr Gly 1250 1255 1260 Thr Thr Cys Thr Ala Gly Thr Thr Thr
Thr Cys Gly Thr Thr Ala 1265 1270 1275 Ala Ala Cys Gly Thr Ala Gly
Ala Ala Ala Cys Ala Ala Thr Ala 1280 1285 1290 Ala Thr Gly Thr Cys
Ala Ala Ala Thr Gly Ala Thr Ala Ala Thr 1295 1300 1305 Gly Thr Cys
Thr Thr Ala Gly Ala Ala Cys Thr Thr Gly Ala Ala 1310 1315 1320 Thr
Cys Cys Ala Thr Gly Ala Gly Cys Ala Gly Ala Gly Thr Cys 1325 1330
1335 Ala Ala Ala Gly Gly Ala Thr Gly Gly Ala Ala Cys Cys Cys Thr
1340 1345 1350 Thr Gly Thr Thr Thr Thr Gly Gly Ala Cys Cys Thr Gly
Ala Thr 1355 1360 1365 Thr Thr Ala Thr Gly Gly Ala Ala Gly Thr Gly
Ala Ala Gly Ala 1370 1375 1380 Gly Thr Thr Gly Gly Ala Cys Cys Ala
Cys Cys Ala Thr Ala Gly 1385 1390 1395 Cys Ala Thr Gly Cys Ala Thr
Thr Ala Thr Ala Gly Cys Thr Ala 1400 1405 1410 Cys Thr Gly Gly Thr
Thr Thr Thr Gly Thr Gly Ala Cys Ala Gly 1415 1420 1425 Thr Thr Gly
Thr Cys Cys Ala Cys Ala Ala Cys Ala 1430 1435 1440 4 277 PRT
homosapiens 4 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile
Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met
Asp Ser Gly Met Ser 20 25 30 Trp Asp Thr Gly Tyr Lys Met Asp Tyr
Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe
His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val
Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr
Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val
Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105
110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe
115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe
Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys
Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp
Cys Gly Ile Glu Thr Asp Ser 165
170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Asp Ala
Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser
Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu
Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met
His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu
Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys
Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr
Phe Tyr His 275
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