U.S. patent application number 09/990255 was filed with the patent office on 2004-01-15 for method for the identification and use of substances that modulate pod function and/or structure.
This patent application is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Doucas, Vassilis, Evans, Ronald M., Trono, Didier.
Application Number | 20040009892 09/990255 |
Document ID | / |
Family ID | 25522854 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009892 |
Kind Code |
A1 |
Doucas, Vassilis ; et
al. |
January 15, 2004 |
Method for the identification and use of substances that modulate
POD function and/or structure
Abstract
The invention disclosed herein comprises assay methods for
identifying substances useful for treating pathogenic disorders.
The assay methods disclosed herein are based on the discovery that
POD function and structure are key elements in normal
transcriptional processes. Disruption of POD function and/or
structure contributes to the creation and/or maintenance of a
variety of pathogenic disorders.
Inventors: |
Doucas, Vassilis; (Geneva,
CH) ; Evans, Ronald M.; (La Jolla, CA) ;
Trono, Didier; (Collonge, CH) |
Correspondence
Address: |
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
The Salk Institute for Biological
Studies
|
Family ID: |
25522854 |
Appl. No.: |
09/990255 |
Filed: |
November 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09990255 |
Nov 20, 2001 |
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08975272 |
Nov 21, 1997 |
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6319663 |
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Current U.S.
Class: |
514/44R ;
514/19.3; 514/19.6; 514/3.7 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/6875 20130101; C12Q 1/6897 20130101; G01N 33/5008
20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/16 |
Claims
What is claimed:
1. A method of treating a pathogenic disorder, said method
comprising administering to a patient in need thereof an effective
amount of a substance that modulates POD structure and/or
function.
2. The method according to claim 1, wherein the disorder is a viral
infection.
3. The method according to claim 2, wherein the viral infection is
hepatitis virus infection, herpes virus infection, human T-cell
leukemia virus infection, human immunodeficiency virus infection,
varicella virus infection, papilloma virus infection or adenovirus
infection.
4. The method according to claim 1, wherein the disorder is a
cancer.
5. The method according to claim 4, wherein the cancer is a
leukemia.
6. The method according to claim 1, wherein the disorder is an
inflammatory disorder.
7. The method according to claim 1, wherein the disorder is an
autoimmune disorder.
8. A method of treating a pathogenic disorder, said method
comprising administering to a patient in need thereof an effective
amount of a substance that increases the level of a POD-localized
protein.
9. The method according to claim 8, wherein the disorder is a viral
infection.
10. The method according to claim 9, wherein the viral infection is
hepatitis virus infection, herpes virus infection, human T-cell
leukemia virus infection, human immunodeficiency virus infection,
varicella virus infection, papilloma virus infection or adenovirus
infection.
11. The method according to claim 8, wherein the disorder is a
cancer.
12. The method according to claim 11, wherein the cancer is a
leukemia.
13. The method according to claim 8, wherein the disorder is an
inflammatory disorder.
14. The method according to claim 8, wherein the disorder is an
autoimmune disorder.
15. A method of treating a viral infection, said method comprising
administering to a patient in need thereof an effective amount of a
substance that disrupts the interaction of a viral protein with a
POD-localized protein.
16. The method according to claim 15, wherein the viral infection
is hepatitis virus infection, herpes virus infection, human T-cell
leukemia virus infection, human immunodeficiency virus infection,
varicella virus infection, papilloma virus infection or adenovirus
infection.
17. A method of testing viral vectors to eliminate candidate
vectors that are not useful for gene therapy, said method
comprising: (a) transfecting a test cell with a test viral vector
or infecting a test cell with a virus; and (b) assaying for the
ability of the vector or virus to modulate POD function and/or
structure, wherein a vector or virus that modulates POD function
and/or structure is not useful for gene therapy.
18. A method of treating a pathogenic disorder, said method
comprising administering to a patient in need thereof a nucleic
acid construct comprising an exogenous nucleic acid encoding a
therapeutic polypeptide and a nucleic acid fragment encoding a
POD-localized protein or fragment thereof.
19. The method according to claim 18, wherein the POD-localized
protein or fragment thereof is selected from the group consisting
of PML, Sp-100, CBP, PIF13, PIF31, ND52, ND55, Isp20, and DipA.
20. A method of treating a pathogenic disorder, said method
comprising administering to a patient in need thereof a composition
comprising a therapeutic polypeptide and a POD-localized protein or
fragment thereof.
21. The method according to claim 20, wherein the POD-localized
protein or fragment thereof is selected from the group consisting
of PML, Sp-100, CBP, PIF13, PIF31, ND52, ND55, Isp20, and DipA.
22. A method of delivering an exogenous nucleic acid encoding a
therapeutic polypeptide to a subject in need thereof, said method
comprising administering to said subject a nucleic acid construct
comprising said exogenous nucleic acid and a nucleic acid fragment
encoding a POD-localized protein or fragment thereof.
23. The method according to claim 22, wherein the POD-localized
protein or fragment thereof is selected from the group consisting
of PML, Sp-100, CBP, PIF13, PIF31, ND52, ND55, Isp20, and DipA.
24. A method of delivering a therapeutic polypeptide to a subject
in need thereof, said method comprising administering to said
subject a composition comprising said polypeptide and a
POD-localized protein or fragment thereof.
25. The method according to claim 24, wherein the POD-localized
protein or fragment thereof is selected from the group consisting
of PML, Sp-100, CBP, PIF13, PIF31, ND52, ND55, Isp20, and DipA.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application Ser.
No. 08/975,272, filed Nov. 21, 1997, now pending, which is hereby
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to assay methods that can be used to
identify substances valuable in the treatment of pathogenic
disorders. In a particular aspect, the invention relates to assay
methods for identification of substances useful for treatment of
disorders related to transcription, translation and replication
such as viral infections, cancer, inflammatory disorders and in
particular disorders involving nuclear receptor functioning. In
another aspect, the invention relates to methods for using
substances, identified employing assay methods of the invention,
for the treatment of pathogenic disorders. An additional aspect of
the invention relates to methods for evaluating viral vectors for
their usefulness in gene therapy.
BACKGROUND OF THE INVENTION
[0003] Some of the processes by which a cell utilizes its genetic
material to direct its growth, differentiation, and other functions
include transcription, translation, and replication. Transcription
is the process by which DNA is converted into RNA for subsequent
translation into a protein. Replication is the process by which DNA
sequences are duplicated prior to cell division. These processes
represent the principle activities localized in the cell
nucleus.
[0004] Transcription is a complex process that controls gene
expression and largely determines the actions and properties of
cells. Most eukaryotic genes are regulated by multiple
transcription-control elements, specific DNA sequences lying
outside of the coding region of the gene. Transcription-control
elements serve as specific binding sites for a variety of protein
transcription factors, both activators and repressors.
[0005] In general, transcription activators are believed to control
the assembly of the transcription initiation complex and the rate
at which the poised RNA polymerase in this complex initiates
transcription. The common feature among transcription activators is
that each binds to one or several specific DNA regulatory elements,
then participates in moderating the activity of the transcriptional
apparatus. Generally transcriptional activators act in response to
a cascade of intracellular reactions initiated by an extracellular
signal. Although capable of acting alone, transcription activators
are also known to interact with additional protein components,
called coactivators, which can modify their activities. Well known
transcription activators include the nuclear receptors,
NF-.kappa.B, cAMP-response element binding protein (CREB), and the
STATs.
[0006] The nuclear receptors, also known as steroid/retinoid
hormone receptors, are transcription factors that exist in an
inactive form either in the cytoplasm or nucleus. Upon binding
their respective hormonal ligands, the receptors undergo activation
or "transformation," and the activated receptor can then
effectively bind to DNA and activate transcription of a cis-linked
gene. Well known examples of nuclear receptors include the
glucocorticoid receptor (GR), the retinoid receptors (RAR and RXR),
the estrogen receptor (ER), the mineralocorticoid receptor (MR),
the androgen receptor (AR), the progesterone receptor (PR),
peroxisome proliferator-activated receptors (PPAR), vitamin D
receptor (VDR), thyroid hormone receptor (THR) and the like.
Reviews discussing the structure and function of nuclear receptors
are found in Wahli and Martinez, FASEB J. 5:2243-2249, 1991; Evans,
Science 240:889-895, 1988; and Tsai and O'Malley, Ann. Rev.
Biochem. 63:451-486, 1994, all of which are hereby incorporated by
reference in their entirety. Nuclear receptor activity effects such
diverse cellular functions as differentiation, proliferation and
inflammatory response.
[0007] Nuclear factor-kappa B (NF-.kappa.B) encompasses a family of
inducible transcriptional activators critical in the regulation of
gene expression in the lymphocytic response to injury and
inflammatory stimuli. In the cell, NF-.kappa.B exists as a homo- or
heterodimer with distinct DNA binding specificities. Cellular
stimulation with inflammatory cytokines, such as the interleukins,
and other inflammatory stimuli results in the rapid translocation
of NF-.kappa.B to the nucleus where it binds to specific .kappa.B
elements. Many genes involved in the inflammatory response are
induced by NF-.kappa.B including pro-inflammatory cytokines,
chemotactic proteins and adhesion molecules. Steroids and retinoids
are widely reported to exert anti-proliferative and
anti-inflammatory effects in T lymphocytes in part by repressing
the activities of NF-.kappa.B (and AP-1). In turn, NF-.kappa.B
activity represses nuclear receptor function.
[0008] Elevation in the level of cytosolic cAMP, such as in
response to G-coupled protein receptor activity, induces the
expression of many genes. These genes are under the control of a
cAMP-response element (CRE), essential for their induction, which
is bound by the transcription activator CREB (cAMP-response element
binding protein). CREB activity is promoted by its phosphorylation,
which recruits the coactivator CREB-binding protein (CBP/p300). CBP
can act as a coactivator for a number of other transcription
activators including c-Jun, c-Myb, c-Fos, NF-.kappa.B and nuclear
receptors, among others. CBP/p300 interacts with the transcription
factor and tumor suppressor p53 and modulates its activity
(Avantaggiati et al., Cell 89:1175-1184, 1997). In addition to its
activity as a transcription factor, CBP enzymatically acetylates
histones and other proteins, such as p53. This acetylation activity
promotes CBP-dependent regulation of chromatin functioning,
(Ogryzko et al., Cell 87:953-959, 1996). Thus, CBP may perform an
important role in the integration of diverse signaling pathways
that result in changes in gene expression.
[0009] The cytokine-receptor superfamily includes receptors for
protein ligands such as the interferons, erthryropoetin, and the
like. Transcriptional activation in response to these receptors
involves the phosphorylation and subsequent dimerization of a
family of transcriptional activators called the STATs (Signal
Transducers and Activators of Transcription). STATs can form homo-
or heterodimers and bind to a number of DNA sequences including the
interferon-stimulated response-element (ISRE) and the
serum-inducible response element (SRE). STAT activity can also be
induced by the activity of some tyrosine kinase receptors, such as
the epidermal growth factor receptor. STAT activity regulates the
expression of therapeutically important genes. In response to the
binding of interferon, for example, cells are induced to express a
set of proteins that make them resistant to viral infection.
[0010] Recent research has uncovered new evidence for the
localization of several nuclear activities in substructures that
may contribute a topological function to these processes. The
nuclei of cells contain a variety of substructures among which are
several morphologically distinct types, called nuclear bodies,
including sphere organelles and coiled bodies. A third type of
nuclear body having a distinct nuclear topology has recently been
described, and is variously referred to as the PML Oncogenic Domain
(POD), Nuclear Domain (ND) 10, or Kr body. PODs and POD-localized
proteins have been proposed as playing a role in transcription and
may be involved in translation, replication and nuclear transport
as well. PODs have been found in all the cell types examined to
date, suggesting that they are a fundamental structure common to
all cells.
[0011] PODs are comprised of a number of cellular proteins, all of
unknown function, arranged in a vesicle-like structure around an
electron dense core. Among these proteins are PML, Sp-100, PIF13,
PIF31, ND52, ND55, and Isg20 (Doucas and Evans, Biochim. Biophys.
Acta 1288:M25-M29, 1996; Gongora et al., J. Biol. Chem.
272(31):19457-19463, 1997). Several of these proteins (e.g., PML,
Sp-100) are the targets of antibodies present in the serum of
patients with autoimmune disorders such as primary bilary cirosis
and systemic lupus erythramatosis (Andre et al., Exp. Cell Res.
229:253-260, 1996).
[0012] A chief component of PODs is the protein PML. PML was
originally identified by its role in Acute Promyelocytic Leukemia
(APL), a disorder characterized by a chromosomal translocation
(15;17) which fuses PML to the retinoic acid receptor resulting in
a PML:RAR fusion product that retains most of the functional
domains of its parental proteins.
[0013] PML contains several different protein:protein interaction
domains including a cysteine-rich domain containing a RING finger
motif and a coiled-coil or alpha-helical region. The presence of
protein:protein interaction domains predicts that PML possesses a
self-polymerizing feature that permits efficient packaging and
transfer of bound target molecules into a round vesicle-like
structure. In fact, PML:RAR forms homodimers and PML:RAR-PML
heterodimers in vitro, which results in disruption of normal POD
structure and may contribute to the oncogenic state in APL
patients. Treatment with retinoic acid results both in restoration
of POD structure and cellular differentiation with subsequent
clinical remission.
[0014] POD structure has also been linked with viral replication.
Doucas et al have shown that infection with adenovirus disrupts POD
structure (Doucas et al., Genes & Devel. 10:196-207, 1996), as
is also the case when cells are genetically engineered to express
certain adenovirus proteins. POD disruption can be reduced,
however, by an increase in the expression of PML, which also
reduces viral replication. These results suggest that adenovirus
replication may utilize POD components. It has not been
demonstrated, however, that POD-associated proteins directly
contribute to transcriptional/translational/replication control nor
has a role for PODs been shown using clinically relevant
viruses.
[0015] A variety of serious pathogenic disorders involve
transcription and translation at some level. For example, the
process of inflammation requires the transcription and translation
of a variety of proteins such as inflammatory cytokines and
chemokines, and the like. Current anti-inflammatory treatments
strive to ameliorate the undesirable effects of these proteins with
varying degrees of success, often producing severe side effects. A
substance capable of regulating the transcription and/or
translation of inflammatory proteins could modulate the
inflammatory process without the side effects of current
treatments.
[0016] Cancer is another pathogenic disorder that involves
transcriptional and translational regulation. Disruption of normal
transcription can result in an inability of cells to differentiate,
for example, a hallmark of the cancerous state. Restoration of
transcription would allow these cancer cells to resume a normal
differentiation pathway, ultimately ending in apoptosis. To date,
however, many, if not most treatments for cancer involve the use of
cytotoxic agents that target and kill any rapidly dividing cell,
including those that are perfectly normal. Therefore, a need still
exists for new methods of treating cancer that are more selective
and have fewer side effects.
[0017] Viral infections, such as with the human immunodeficiency
virus, can have a severe debilitating effect on the infected host.
Viruses themselves are simple organisms lacking the full range of
transcriptional machinery necessary for their efficient propagation
in the cell. Most viruses rely on their ability to co-opt the
transcriptional/translational apparatus of their hosts in order to
multiply. The anti-viral therapies currently in use often target
viral specific proteins, such as viral proteases. The rapid
replication and mutation rate of most viruses, however, often
allows new strains to emerge that are resistant to these
therapies.
[0018] Thus a need exists for new substances that can be used to
treat these and other pathogenic disorders. The invention disclosed
herein addresses that need.
BRIEF DESCRIPTIONS OF THE INVENTION
[0019] The invention disclosed herein comprises assay methods for
identifying substances useful for treating pathogenic disorders.
The assay methods disclosed herein are based on the discovery that
POD function and structure are key elements in the coordination of
regulatory signals needed for normal transcription, translation,
replication, nuclear import/export and RNA processing. Disruption
of POD function and/or structure contributes to the creation and/or
maintenance of a variety of pathogenic disorders.
[0020] In accordance with another aspect of the present invention,
it has further been discovered that restoration of POD function
and/or structure has a beneficial treatment effect and can
contribute to restoration of normal function in the organism so
effected.
[0021] In accordance with another aspect of the invention there is
provided a method for designing viral vectors useful for gene
therapy. Many methods of delivering exogenous genetic material to
cells for therapeutic purposes involve the use of viral vectors.
The discovery that certain viral proteins significantly disrupt
normal cellular transcription and translation suggests that gene
therapy delivery vehicles based on viral vectors should avoid the
presence of these viral proteins in order to avoid the potential
for deleterious effects on normal cellular function.
[0022] Thus the present invention provides methods for identifying
useful therapeutic substances, including those useful in treating
viral and inflammatory disorders and in gene therapy, and methods
for their use.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention disclosed herein comprises assay methods for
identifying substances useful for treating pathogenic disorders.
Invention methods comprise: a) contacting a test cell with a test
substance and b) assaying for the ability of the substance to
modulate POD function and/or structure. By "modulate" it is meant
that the substance can either inhibit, enhance or restore POD
function and/or structure.
[0024] In one aspect of the present invention, a test substance can
be identified as a potentially useful therapeutic agent by its
ability to enhance or restore POD function and/or structure by
increasing levels of a POD-localized protein. The inventors have
discovered that an increase in the levels of POD-localized proteins
has a beneficial therapeutic effect on cells infected with viruses
that cause serious disease in humans, such as the human
T-lymphotropic virus (HTLV-1) which is associated with adult T-cell
leukemia (ATLL) and inflammatory neurological diseases such as
tropical spastic paraparesis (TSP). As seen in the Examples below,
increasing the expression of the POD-localized protein PML reverses
the pathogenic effects of the HTLV-1 protein Tax.
[0025] Of particular interest is the relationship between POD
function and structure and viruses such as HTLVs 1 and 2 and HIV.
This group of viruses is unique in that their genome consists of a
single strand of RNA that encodes, among other things, a reverse
transcriptase enzyme that transcribes the viral RNA into DNA in the
host cell. This is in contrast to viruses whose genomic material is
DNA that can be readily integrated in to the host genome. The
profound effects of the Tax protein of HTLV-1 on POD function and
structure are striking in light of the distinctive nature of these
types of viruses.
[0026] Increase in POD-localized protein levels can be identified
by measuring for upregulation of mRNA encoding a POD-localized
protein, or of the promoter of a POD-localized protein or by direct
measurement of the protein itself. By "POD-localized protein" it is
meant a protein naturally occurring in a cell, said protein further
being naturally localized to a POD structure, either permanently or
in response to some cellular signal. Examples of POD-localized
proteins include PML, Sp-100, CBP, PIF13, PIF31, ND52, ND55, Isp20,
DipA, and the like.
[0027] A variety of methods are known to those skilled in the art
that can be employed to measure upregulation of mRNA, proteins or
promoter activity. Levels of mRNA, for example, can be measured by
isolating total RNA from treated test cells, transferring it to a
solid support, such as a nylon membrane, and quantitating the mRNA
so bound using a labeled complementary probe specific for mRNA
encoding a POD-localized protein. Alternatively, after isolation,
the RNA can be subjected to quantitative polymerase chain reaction
(PCR) analysis using a POD-localized protein specific primer.
[0028] Upregulation of POD-localized protein promoter activity is
likewise measurable by techniques known to those skilled in the
art. For example, test cells can be transfected with a genetically
engineered plasmid encoding a POD-localized protein promoter
operably linked to a detectable gene product prior to incubation
with a test substance. Increase in the quantity of detectable gene
product would indicate upregulation of POD-localized protein
promoter activity. This method is illustrated in the Examples below
using the PML promoter and a gene encoding luciferase as the
detectable gene product.
[0029] Alternatively, one may directly measure levels of a
POD-localized protein to determine if its synthesis has been
upregulated. Detection methods include the use of POD-localized
protein-specific antibodies (monoclonal or polyclonal) in in situ
hybridization or immunoprecipitation. The POD-localized
protein-specific antibodies can be themselves conjugated to a
detectable label, such as a fluorescent label or enzymatic label,
and the like. Additionally, the POD-localized protein-specific
antibodies can be unlabeled and detected with a secondary antibody,
such as an anti-IgG, anti-IgM, and the like, that is labeled with a
detectable label.
[0030] An increase in POD-localized protein levels can also be
measured by isolation of the protein from test cells using protein
fractionation methods, such as column chromatography, gel
filtration, and the like, and subsequent determination of the
amount of protein so obtained, such as by using the method of Lowry
et al., (Lowry et al., J. Biol. Chem. 193:265, 1951). Methods for
protein isolation and quantitative estimation are well known to
those skilled in the art.
[0031] In another aspect of the invention, an additional assay step
can be included, i.e. causing the test cell to express one or more
viral proteins prior to assaying for the ability of the test
substance to modulate POD function and/or structure. Test cells
can, for example, simply be infected with a virus using methods
well known to those skilled in the art. Alternatively, test cells
can be genetically engineered to express selected viral proteins.
In some cases, such as with Tax, the viral protein can simply be
added to the media in which the test cells are grown. Any virus of
interest can be used, however particularly preferred viruses
include adenovirus, herpes viruses, human immunodeficiency virus
(HIV), HTLV viruses 1 and 2, hepatitis viruses, papilloma virus,
varicella virus, and the like. Particularly preferred viral
proteins for genetically engineered expression include E4-ORF3 and
E4-ORF6 of adenovirus, Tax from HTLV-1, and the like.
[0032] The methods described above for assessing the activity of a
test substance can be used in assays comprising this additional
step. Alternatively, the activity of test substances can be
evaluated by their ability to 1) modulate the disruption of POD
morphology, or 2) modulate the degradation of POD-localized
proteins.
[0033] The ability of a test substance to maintain or restore
normal POD morphology would be an indication of potential
therapeutic activity. PODs are normally found in the nucleus
arranged in a punctate-type structure. In several disorders, such
as cancer or viral infection, however, the punctate morphology is
often disrupted. This disruption can be detected using in situ
hybridization techniques with a POD-localized protein-specific
antibody (polyclonal or monoclonal) labeled with a detectable label
as described above.
[0034] Additional methods of assessing the potential of a test
substance to act as an anti-viral agent is by its ability to
disrupt viral-protein:POD-localized-protein interactions or by its
ability to prevent virus-induced POD-localized protein
degradation/disassociation. Both can be evaluated using, for
example, labeled antibodies, in the former case one specific for
the viral protein and one specific for the POD-localized protein,
and the techniques discussed above.
[0035] The inventors have shown that PODs and POD-localized
proteins play an important role in the function of nuclear
receptors. Thus, in yet another aspect of the invention, the
therapeutic potential of a test substance is evaluated by its
ability to modulate nuclear receptor-mediated transcription in
cells expressing one or more viral proteins. This method comprises
the steps of the assays disclosed above and includes the additional
steps of 1) genetically engineering the test cell to express a
nuclear receptor and reporter gene (described in more detail below)
and 2) contacting the genetically engineered test cell with an
agent known to activate the nuclear receptor. This assay method is
disclosed in more detail in Example 6 below. A particularly useful
viral protein employed for expression in a test cell in this aspect
of the invention is the HTLV-1 Tax protein.
[0036] It is known that the oncoprotein Tax, a key protein in the
replication of HTLV-1 and HIV-1, activates the transcriptional
coactivator NF-.kappa.B. It is also known that NF-.kappa.B inhibits
nuclear receptor function. In accordance with the present
invention, it has been discovered that Tax expression also inhibits
nuclear receptor function, showing a synergistic effect with
NF-.kappa.B, and additionally disrupts normal POD morphology. These
effects can be overcome by increased expression of a POD-localized
protein, for example PML. Furthermore, in accordance with the
present invention, it has been discovered that the transcription
factor CBP, an essential coactivator of nuclear receptor activity,
localizes to the POD through its binding to the POD-localized
protein PML. This work strongly suggests that a modulator of POD
function and/or structure would have a beneficial effect in
treating disorders dependent on nuclear receptor function.
[0037] Suitable nuclear receptors useful in this aspect of the
invention include the gucocorticoid receptor (GR), the retinoid
receptors (RXRs, RARs), prostaglandin receptors (PPARs), and the
like. Suitable reporter genes include luciferase, chloramphenicol
acetyl transferase, and the like, under the control of the various
promoters such as MMTV (mouse mammary tumor virus), apolipoprotein
A1 (Apol4tk), AP2 promoter, thymidine kinase promoter, collagenase
promoter, synthetic promoters containing regulatory elements, such
as DR1-5 and the .beta.-response element, synthetic promoters
containing regulatory elements for PPAR.alpha., .beta., and
.gamma., natural promoters regulated by nuclear receptors, and the
like.
[0038] In test cells expressing a nuclear receptor, a reporter
construct and Tax, a test substance showing therapeutic potential
will show increased transcriptional activity when treated with a
stimulator of nuclear receptor activity (such as dexamethasone)
compared to test cells not exposed to the test substance. Since it
has also been discovered, in accordance with the invention, that
Tax synergizes with NF-.kappa.B, this assay may be slightly
modified such that the test cells are additionally genetically
engineered to express NF-.kappa.B.
[0039] Test cells suitable for use in the assays of the invention
include mammalian cells, insect cells, fungal cells, bacterial
cells, and the like. Preferred test cells are mammalian cells,
particularly those that have been adapted to cell culture (more
commonly called cell lines). Particularly preferred test cells
include CV-1 (ATCC CCL 70), Hep-2 (ATCC CCL 23), NB-4 (Duprez et
al., Leukemia 6:1281-1287, 1992), NIH3T3 L1 cells (Blondel et al.,
Oncogene 5:857-865, 1990), P4-2 cells (Naldini et al., Science
272:263-267, 1996) liposarcoma cell lines LS857, LS175, LS707
(Tontonoz et al., Proc. Natl. Acad. Sci. USA 94:237-241, 1997) and
the like.
[0040] Test cells can be grown under in vitro cell culture
conditions using techniques well known in the art. Alternatively
the test cells can be grown in vivo as part of an animal. Common
sources of suitable test cells include, for example, the American
Type Culture Collection, Bethesda, Md. (ATCC).
[0041] Suitable test substances for use in the assays of the
invention include proteins, peptides, lipids, carbohydrates,
extracts from fungal or plant sources, small organic molecules, and
the like. Sources of these test substances include conditioned cell
culture media, commercially available libraries of compounds (small
organic molecules, peptides), and the like.
[0042] In another aspect of the invention, substances are
identified that inhibit nuclear receptor function. Such substances
are useful, for example, in treating disorders such as diabetes or
obesity where a reduction in mature adipocytes is desired. The
inventors have discovered that Tax expression inhibits the nuclear
receptor driven differentiation of adipocytes. Substances mimicking
Tax activity could, therefore, be useful as therapeutics. Test
cells as described above are genetically engineered to express a
nuclear receptor and a reporter gene construct. Test substances are
then assessed for their ability to inhibit nuclear receptor
activity such as disrupting POD structure and/or function.
[0043] Also encompassed in the present application is a method of
treating pathogenic disorders with a substance capable of
modulating POD structure and/or function. Treatment methods in
accordance with the invention comprise administering the substance
in an amount sufficient to modulate POD structure and/or function.
The organism to be so treated is preferably a mammal, and most
preferably human.
[0044] The particular substance selected for use in treatment can
be administered to a patient either by itself or in a
pharmaceutical composition where it is mixed with suitable carriers
or excipient(s). In treating a patient, a therapeutically effective
dose of the substance is administered. A therapeutically effective
dose refers to that amount of the substance that results in
amelioration of symptoms or a prolongation of survival in a
patient.
[0045] Toxicity and therapeutic efficacy of such substances can be
determined by standard pharmaceutical procedures in cell culture or
experimental animals. Cell culture assays and animal studies can be
used for determining the LD.sub.50 (the dose lethal to 50% of a
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of a population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which can be
expressed as the ratio LD.sub.50/ED.sub.50. Substances that exhibit
large therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a range of dosages for use in humans. The dosage of
such substances lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon a
variety of factors, e.g., the dosage form employed, the route of
administration utilized, the condition of the subject, and the
like.
[0046] For any substance used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays by determining an IC.sub.50 (i.e., the concentration
of the test substance that achieves a half-maximal modulation of
POD structure and/or function). A dose can then be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by HPLC.
The exact formulation, route of administration and dosage can be
chosen by the individual physician in view of the patient's
condition. (See e.g. Fingl et al., 1975, in "The Pharmacological
Basis of Therapeutics", Ch. 1 p. 1).
[0047] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity, to organ dysfunctions, and the like. Conversely,
the attending physician would also know to adjust treatment to
higher levels if the clinical response were not adequate
(precluding toxicity). The magnitude of an administered dose in the
management of the oncogenic disorder of interest will vary with the
severity of the condition to be treated, with the route of
administration, and the like. The severity of the condition may,
for example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose frequency,
will also vary according to the age, body weight, and response of
the individual patient. A program comparable to that discussed
above may be used in veterinary medicine.
[0048] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
"Remington's Pharmaceutical Sciences," 1990, 18th ed., Mack
Publishing Co., Easton, Pa. Suitable routes may include oral,
rectal, transdermal, vaginal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
or intraocular injections, just to name a few.
[0049] For injection, the substances of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0050] Use of pharmaceutically acceptable carriers to formulate the
substances herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The substances can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the substances of the
invention to be formulated as tablets, pills, capsules, dragees,
liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated.
[0051] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external microenvironment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Small organic molecules may be directly
administered intracellularly due to their hydrophobicity.
[0052] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an amount effective to achieve its intended purpose.
Determination of an effective amount is well within the capability
of those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0053] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
excipients and auxiliaries that facilitate processing of the active
compounds into preparations which can be used pharmaceutically.
[0054] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, lyophilizing
processes, or the like.
[0055] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active substances may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, dextran, or the
like. Optionally, the suspension may also contain suitable
stabilizers or agents that increase the solubility of the compounds
to allow for the preparation of highly concentrated solutions.
[0056] Pharmaceutical preparations for oral use can be obtained by
combining the active substances with solid excipient, optionally
grinding the resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores.
[0057] Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, sorbitol, and the
like; cellulose preparations such as, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethylcellulose, sodium
carboxymethylcellulose, polyvinylpyrrolidone (PVP), and the like,
as well as mixtures of any two or more thereof. If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, alginic acid or a salt thereof such as sodium
alginate, and the like.
[0058] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions, suitable organic solvents or solvent mixtures, and the
like. Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0059] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
[0060] Gene therapy has the potential to treat a variety of
inherited and acquired disorders. The basic premise behind gene
therapy is the introduction of exogenous genetic material into the
cells of the organism to be treated. This has been most often
accomplished using gene delivery vehicles, or vectors, based on
recombinant versions of viruses, with retroviruses serving as
prototypes (Wilson, Clin. Exp. Immunol. 107(Suppl 1):31-32, 1997;
Miller and Whelan, Hum. Gene Ther. 8(7):803-815, 1997).
[0061] In accordance with the present invention, the identification
of the viral proteins that interfere with POD organization provides
important information in the design of viral vectors. The
techniques described above can be used to determine if the
introduction of a viral vector proposed for use in gene therapy
disrupts POD structure and/or function in such a way that the
therapeutic gene being introduced into the cells is rejected.
[0062] The invention will now be described in greater detail by
reference to the following non-limiting Examples.
EXAMPLES
Example 1
Assay for Identifying Therapeutic Substances by an Increase in
POD-Localized Proteins
[0063] The following example describes assays for the
identification of substances with the potential to be useful
therapeutics. The assays measure an increase in POD-localized
proteins in a test cell following contact with a test
substance.
[0064] Test cells, such as Hep-2 cells, are placed in approximately
equal numbers in wells of a 96 well microtiter plate and grown in
standard growth media, such as, for example, Dulbecco's Modified
Growth Media (DMEM)(Gibco) plus 5% fetal calf serum. Test substance
is dissolved in a convenient solvent, water, phosphate buffered
saline (PBS) or dimethylsulfoxide (DMSO) for example, and added to
the wells containing the test cells. At various time points
post-treatment, for example 0, 2, 6, and 12 hours post-treatment,
the cell culture media is removed, and RNA isolated from the
treated test cells using standard techniques. Quantitative reverse
transcriptase-polymerase chain reaction assays are performed using
the isolated RNA and employing appropriate primers obtained from
cDNA encoding a POD-localized protein (such as PML or Sp-100). The
amount of POD-localized protein specific RNA in test substance
treated cells is compared to cells not exposed to a test substance.
An increase in POD-localized protein specific RNA indicates that
the test substance has the potential to be a useful therapeutic
agent.
[0065] In a variation on this assay, isolated RNA is transferred to
a solid support such as a nylon membrane, and the amount of
POD-localized protein specific RNA detected using a specific
nucleotide sequence that is capable of hybridizing to the
POD-localized protein specific RNA.
[0066] Potential therapeutic agents are also identified by
measuring amounts of POD-localized protein in treated cells. Test
cells, such as Hep-2 cells, are place in approximately equal
numbers in wells of a 96 well microtiter plate and grown in
standard growth media. Test substance dissolved in a convenient
solvent, water, phosphate buffered saline (PBS) or
dimethylsulfoxide (DMSO) for example, and added to the wells
containing the test cells. At various time points post-treatment,
for example 0, 2, 6, and 12 hours post-treatment, the cell culture
media is removed, and the cells fixed with freshly prepared
paraformaldehyde in PBS at room temperature. The fixed cells are
washed in PBS and permeabilized for 20 minutes on ice with 0.2%
Triton X-100 in PBS (v/v). Alternatively, cells can be fixes at
-20.degree. C. for 5 minutes on ice with fresh, cold acetone,
followed by fresh, cold absolute methanol.
[0067] The fixed cells are incubated with POD-localized
protein-specific antibody conjugated to a detectable label, such as
a fluorescent chromophore, under conditions that allow the antibody
to bind to the POD-localized protein, generally about 1 hour at
room temperature. The cells are washed to remove unbound antibody
and the amount of antibody bound determined using the detectable
label. The amount of POD-localized protein present in cells treated
with a test substance is compared to untreated cells. An increase
in POD-localized protein levels indicates that the test substance
may be a useful therapeutic.
[0068] Alternatively, the assay procedure can be performed as
above, however, rather that determining the amount of antibody
bound quantitatively, the cells are visualized and evaluated for
changes in POD morphology. Enlargement of the POD structure, for
example, is an indication of an increase in POD-localized
protein.
Example 2
Assay for Identifying Therapeutic Substances by Inhibiting Viral
Effects on PODs
[0069] The following example describes assays for the
identification of potential therapeutic substances by measuring the
ability of the substance to inhibit the negative effects of viral
proteins on POD structure and/or function.
[0070] Test cells, such as Hep-2 or CV-1 cells, are placed in
approximately equal numbers in wells of a 96 well microtiter plate
and grown in standard growth media, such as, for example, DMEM
(Gibco) plus 5% fetal calf serum. Test substance is dissolved in a
convenient solvent, water, phosphate buffered saline (PBS) or
dimethylsulfoxide (DMSO) for example, added to the wells containing
the test cells and allowed to incubate for approximately 16 hours.
The test cells are then transfected with an expression vector
expressing a viral protein, such as E4-ORF3 from adenovirus, using
standard transfection procedures (Doucas, et al, Genes &
Devel., supra). The test cells are provided with fresh growth
medium and allowed to grow for approximately 24 hours. The cells
are then fixed and treated with antibodies specific for a
POD-localized protein, such as PML, and the viral protein using the
procedures described in Example 1. Substances with therapeutic
potential will block the ability of the viral protein to
disorganize or degrade the POD-localized protein, which can be
detected visually.
Example 3
The Effects of Tax Expression are Overcome by PML
[0071] The following example demonstrates that expression of HTLV-1
Tax protein disrupts POD structure and function and that this
effect can be overcome by an increase in POD-localized protein
expression.
[0072] Hep-2 cells were transfected with pcTax1 expression vector
(expressing HTLV-1 Tax protein) and fixed 48 hours later as
described in Doucas et al, Genes & Devel. 10:196-207, 1996.
Antigen localization was determined with double incubation of fixed
cells with Tax specific antibodies (Ab6605 (rabbit), Ab1189 (goat))
and the PML-specific monoclonal antibody followed by incubation
with a secondary monoclonal antibody conjugated to fluorescein or
Texas red as previously described in Doucas, et al, supra.
Fluorescence images were analyzed using a Nikon Microshoft-SA
immunofluorescence microscope.
[0073] POD structure was significantly disrupted in cells
expressing Tax protein. Anti-PML antibody showed that PML had been
removed from its normal nuclear location and redistributed to
co-localize with Tax either perinuclearly or in the cytoplasm. In
the majority of experiments, the PML was partially distributed in
track-like structures.
[0074] A similar experiment was conducted to determine if an
increase in POD-localized protein expression could reverse the
disruptive effects of Tax. CV-1 cells were co-transfected with
pcTax1 and CMXHA-PML (a vector expressing PML, Doucas, et al,
supra). The cells were fixed 36-48 hours later and Tax and PML
proteins visualized with antibodies as described above. Increase in
PML expression reversed the Tax-induced disruption of the POD
structure and returned PML localization to the nucleus.
Example 4
Human Immunodeficiency Virus Localizes to PODs
[0075] The following example demonstrates that the human
Immunodeficiency virus localizes to the PODs after infection, much
as does HTLV-1. Furthermore, POD proteins appear to facilitate
transfer of HIV RNA to the nucleus after infection. Substances that
restore POD structure and/or function would be useful, therefore,
in treating HIV infection.
[0076] P4-2 cells (Naldini et al., Science 272:263-267, 1996) were
grown in round coverslips and maintained in DMEM supplemented with
10% fetal calf serum in a humidified atmosphere containing 5%
CO.sub.2. The cells were infected with two different concentrations
(100 ng and 1 .mu.g) of HIV virus, (HIV-1 (W13) wt.) or no virus
(empty). Cells were fixed at 3, 6, 9, 24, 32, hrs post-infection by
incubating at room temperature for 5 min. with freshly prepared 1%
paraformaldehyde in PBS, washed with PBS, and permeabilized for 20
min on ice with 0.2% Triton X-100 in PBS (v/v) (Sigma Chemical
Co.).
[0077] For the detection of HIV-RNA in the infected cells, fixed
coverslips were incubated for 10 hrs at 42.degree. C. with 50 ng of
a HIV-1-specific 300 bp RNA probe labeled with fluorescent UTP, in
a solution containing 50% formamide. Cells were washed in a
PBS/SDS/NaCl solution for 90 min. To detect PML, the washed cells
were incubated with anti-PML antibodies. Cells were incubated with
the first Ab diluted in PBS--10% calf serum for two hrs at RT in a
humidified chamber. Coverslips were washed for 10 min in PBS--0.05%
Tween 20. The conjugated to Texas red second monoclonal Ab (Jackson
Lab.) was applied for 1 hr at RT in the humidified chamber. Cells
were analyzed in a light or confocal microscopy.
[0078] In cells infected with HIV, virus was detected in the
nucleus co-localized with PML. POD structure was additionally
reorganized with PML being detectable perinuclearly or in the
cytoplasm. Furthermore, the localization of PML and HIV over time
suggests that PML is moving into the cytoplasm and facilitating
import of HIV RNA into the nucleus.
Example 5
DipA is a POD-Localized Protein
[0079] The following example demonstrates that DipA is a
POD-localized protein. DipA is a mammalian counterpart to a protein
encoded by the hepatitis delta virus (HDV) (Brazas and Ganem,
Science 274:90-94, 1996). Previous research suggested that DipA
might be involved in HDV replication, although the mechanism was
unknown (Brazas, supra). The inventors have discovered that DipA is
a POD-localized protein, thus linking POD structure and function to
control of HDV replication.
[0080] Hep-2 cells were grown in round coverslips and maintained in
DMEM supplemented with 10% fetal calf serum in a humidified
atmosphere containing 5% CO.sub.2. Cells were treated with 5 .mu.g
of gamma interferon (Biosource) or mocked treated for 18 hrs before
fixation. Cells were fixed by one of two methods. In a first
method, the cells were fixed at room temperature for 5 min with
freshly prepared 1% paraformaldehyde in PBS, washed with PBS, and
permeabilized for 20 min on ice with 0.2% Triton X-100 in PBS (v/v)
(Sigma Chemical Co.). Antigen localization (PML and DipA) was
determined after incubation of permeabilized cells with rabbit
antiserum (DipA Ab) and a specific PML mab diluted in PBS for 1 hr
at RT. Cells were double labeled with the respective second
antibodies labeled either with FITC or Texas red, using FITC for
the structures with the lowest staining intensity.
[0081] Alternatively, cells were fixed at -20.degree. C. for 5 min
with fresh and cold acetone followed by fresh and cold absolute
methanol.
[0082] The cells were incubated with the first Ab diluted in
PBS-10% calf serum for two hrs at RT in a humidified chamber.
Coverslips were washed for 10 min in PBS 0.05% Tween 20. The
conjugated to fluorescein or Texas red second monoclonal Abs
(Jackson Lab.) were applied for 1 hr at RT in the humidified
chamber. For double immunofluorescence, permeabilized cells were
incubated with the two Abs under the same conditions. Cells were
then stained for DNA with 0.5 .mu.g/ml of bis-benzimide (Hoecht
33258; Sigma) in PBS or with 0.5 .mu.g/ml DAPI and mounted with
gelvatol or Fluoromount G (Fischer Scientific). The two fixation
methods gave similar results.
[0083] Fluorescence images were analyzed using either the Leica
confocal scanning microscope or a Nikon Microphot-SA
immunofluorescence microscope. Confocal images represent single
optical sections obtained either by simultaneous or sequential
scanning.
[0084] In these experiments, DipA protein localized in the POD
structure. It is of interest to note the increased co-localization
of PML and DipA in the POD structures in interferon gamma treated
cells, and the exclusive localization of DipA in specific
structures in the nucleoli.
Example 6
Tax-Mediated Inhibition of Nuclear Receptor Function is Reversed by
an Increase in PML Expression
[0085] The following example demonstrates that Tax synergizes with
NF-.kappa.B to inhibit nuclear receptor function and that this
inhibition can be reverse by an increase in PML. These results
suggest that substances that increase POD-localized proteins and/or
restore POD function will be useful as anti-inflammatory treatments
and in treating other disorders involving nuclear receptor
function.
[0086] a) Tax Expression Activates NF-.kappa.B and Inhibits Nuclear
Receptor Function
[0087] CV-1 cells were transfected with expression plasmids
expressing either Tax (pcTax), NF-.kappa.B (CMXp65) or both as
described in Doucas and Evans, supra in addition to a reporter
gene, Ig.kappa.-tk-Luc. The Ig.kappa.-tk-Luc reporter contains six
oligonucleotides corresponding to the .kappa.B-site of the
immunoglobulin .kappa.-light chain enhancer (Chiao et al., Proc.
Natl. Acad. Sci. USA 91:28-32, 1994) linked to the minimal
thymidine kinase promoter upstream of the coding sequence for the
luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725-737, 1987).
The CMXp65 and pcTax expression vectors were transfected at 0.12
.mu.g and 0.18 .mu.g, respectively. Transient expression of Tax or
NF-.kappa.B stimulated the transcription of an Ig.kappa.-tk-Luc
reporter gene by 6 and 12 fold, respectively.
[0088] A similar experiment was done to assess the effect of Tax
expression on nuclear receptor activity. Transient expression of
the glucocorticoid receptor (GR) in CV-1 cells activates the
transcription of the MMTV-Luc reporter by 450-fold in the presence
of 1 .mu.M dexamethasone (Doucas, Genes & Dev, supra). For this
experiment, 0.10 .mu.g of MMTV-Luc, 0.075 .mu.g of GR and 0.15
.mu.g of pcTaxI and pcTaxII expression vectors were trisected per
30,000-40,000 cells together with 0.075 .mu.g of a
beta-galactosidase expression vector as an internal control.
Co-transfect of either TaxI or TaxII repressed GR activity to a
level of less than 100 units.
[0089] CV-1 cells were transfected with MMTV-Luc reporter gene, GR
and 0.075 .mu.g of CMXp65 in a similar experiment. Transient
expression of p65 (NF-.kappa.B) repressed the activity of GR
8-fold. Co-transfection of Tax and p65 at 0.1 and 0.075 .mu.g
respectively repressed the transcriptional activity of GR
approximately 500-fold.
[0090] b) Increase in PML Reverses Tax Mediated Inhibition of
Nuclear Receptors
[0091] CV-1 cells were transfected with expression plasmids
expressing GR, Tax, PML and the MMTV-Luc reporter gene in various
combinations as described previously. Transient expression of GR
(0.05 .mu.g) and reporter induced MMTV-Luc transcription
approximately 300-fold in the presence of 1 .mu.M dexamethasone. In
cells transiently transfected with GR, MMTV-Luc and Tax (0.18
.mu.g), Tax repressed GR activity by more than 50%. However, in
cells transiently transfected with GR, MMTV-Luc and PML (0.10
.mu.g), GR activity was stimulated 5-15 fold. Co-expression of TaxI
and PML in increasing molar ration blocked the Tax repression
effect on GR and GR regained more than a 300-fold activation on the
MMTV reporter plasmid.
Example 7
PODs are and Important Site of Nuclear Receptor and CBP
Activity
[0092] The following example demonstrates that the transcription
factor CBP is a POD-localized protein and that localization to and
the integrity of PODs is essential for nuclear receptor
function.
[0093] a) CBP and PML Co-Localize to Nuclear PODs
[0094] To investigate whether CBP localizes to PODs, a double
immunofluorescence experiment was undertaken in asynchronous CV-1
cells measuring endogenous proteins. CV-1 cells were maintained as
monolayers in Dulbecco's modified Eagle's minimal essential medium
(DMEM) supplemented with 10% bovine calf serum (Gibco) and 100 U/ml
penicillin-streptomycin. Cultures were maintained at 37 degrees C.
and in 7% CO.sub.2. For immunofluorescence, cells were grown on
round coverslips to 80-90% confluency (Corning Glass Inc.) in 6
well plates. The cells were fixed as previously described (Doucas
and Evans, Genes & Devel. 10:196-207, 1996). The fixed cells
were incubated with affinity purified polyclonal rabbit antiserum
against human PML (Ab PML 5311, Mab 5E10, PML mouse Ab (Santa Cruz
Biotechnology) (Doucas, supra)), or CBP (CBP A22, CBP C20 (Santa
Cruz Biotechnology)) diluted in PBS for 1 hour at room temperature.
Second antibodies conjugated to fluorescein or Texas red were
applied for 1 hour at room temperature in a humidified chamber.
Cells were then stained for DNA with 0.5 .mu.g/ml bis-benzimide
(Hoechst 33258, Sigma) in PBS and mounted with gelvatol or
Fluoromount G (Fisher Scientific). Fluorescence images were
analyzed with a Nikon Microsphot-SA immunofluorescence microscope
and with confocal microscopy.
[0095] PML localization was almost identical with the CBP
localization, both of which appeared in a nuclear speckled
pattern.
[0096] b) PML and CBP Interact Directly
[0097] Given that CBP and PML proteins co-localize in the nucleus,
the inventors investigated whether CBP and PML interact at the
protein level. CV-1 cells were grown to 50-80% confluency as
described above. The cells were transiently transfected with 1-3
.mu.g of expression vectors expressing PML (wild type--CMX-PML,
coiled-coil domain deleted--PML (216-331), (217-331)) and CBP
(CMX-Gal-CBP.wt) (Doucas et al., Proc. Natl. Acad. Sci. USA
90:9345-9349 (1993). Co-transfection of the wide-type PML increases
transactivation by Gal-CBP.wt by 10-12-fold, whereas
co-transfection with one of the deletion mutants not only abolished
transactivation but repressed the basal level of activity
2-3-fold.
[0098] These data indicate that PML is a novel co-factor for
nuclear receptor and CBP activity.
[0099] c) PML Functions as a Nuclear Receptor Co-Factor
[0100] CV-1 cells, grown as described above, were transiently
transfected with 1.2 .mu.g reporter construct (MMTV-Luc), 0.9 .mu.g
of CMX-beta-gal, 0.6 .mu.g RSV-GR and expression plasmids
containing PML (wild-type or deletion mutants) as previously
described. Luciferase and beta-galactosidase were assayed as
described in Hollenberg and Evans, Cell 55:899-906, 1988 using
10-fold serial dilutions of dexamethasone from 10.sup.-9 M to
10.sup.-6 M. Extracts were prepared 24-30 hours after transfection.
Transient transfection of GR induces MMTV-Luc transcription from 30
to 200 fold in a dexamethasone dependent manner. PML synergizes
with GR and activates MMTV transcription 600-fold at 10 mM of
dexamethasone.
[0101] In a similar experiment, CV-1 cells, grown as described
above, were transiently transfected with 1.2 .mu.g reporter
construct (Apo14-tk-Luc), 0.9 .mu.g of CMX-beta-gal, 1.2 .mu.g
CMX-RXRalpha and the expression vectors containing CBP, PML
(wild-type and deletion mutants) as previously described.
Apo-tk-Luc contains 4 synthetic oligonucleotides linked to the
minimal thymidine kinase promoter upstream of the coding sequence
for the luciferase gene. The oligonucleotides correspond to the A
site of the apolipoprotein AI promoter (Rottman et al., Mol. Cell.
Biol. 11:3814-3820, 1991). Transfected cells were treated with
9-cis retinoic-acid at 1 .mu.M for 8 hours before the assay.
Co-transfection of PML and RXR-alpha enhanced reporter gene
transcription 250-fold over cells mock transfected with empty
expression vector (CMX). Transfection with PML enhance reported
gene activity by 50-fold.
[0102] In a third experiment, CV-1 cells were transfected with
Gal-RXR alpha F (a Gal-4 DNA binding domain/RXR fusion), CMX-PML,
pSVP-PML delta 216-331, and CMX-CBP.m (each 1.2 .mu.g) and treated
with 1 .mu.M 9-cis retinoic-acid for 8 hours before performing the
reporter assay. (These expression plasmids are described in Doucas
et al., Proc. Natl. Acad. Sci. USA 90:9245-9349, 1993).)
Co-transfection of CBP, PML or the combination augments ligand
dependent transcription of the Gal reporter. PML augmented ligand
dependent transcription driven by the Gal-4/RXR fusion protein more
than 40-fold in a concentration dependent manner. In contrast,
expression of the PML deletion mutant failed to enhance
transcription and actually acted as a modest transcription
inhibitor.
[0103] PML expression markedly enhances transcription by RXR and GR
reporter genes. PML transactivation on MMTV was most effective at
low concentrations of dexamethasone (10.sup.-8 M) enhancing GR
activation by more than 10-fold. In addition, in CV-1 cells, PML
potentiates Apo14-tk-Luc transcription by both the endogenous and
co-transfected RXR proteins. Finally, in a co-transfection
experiment, the PML delta 231-331 mutant did not activate nuclear
receptor hormone dependent transcription, as exemplified with both
the GR wild-type and fusion Gal-4-RXRalpha F. The strong
transcriptional potentiation of PML compared to CBP on transfected
RXR and GR and its unique ability to transactivate endogenous
nuclear receptors further suggests that PML functions as an
essential nuclear receptor and CBP co-factor and that POD structure
and function is key to maintaining normal cellular activity.
[0104] d) Nuclear Receptors are Recruited to PODs
[0105] CV-1 cells were co-transfected at 70% confluency with
CMX-PML and RSV-GR expression vectors (1 and 3 .mu.g, respectively)
as previously described. Cells were grown in the absence of
dexamethasone or treated with 1 .mu.M dexamethasone for .ltoreq.2
min and fixed 12 hours post-transfection. Localization of PML and
GR was visualized using anti-PML antibodies or anti-GR antibody (GR
135) as described above. In the absence of ligand, GR is located
primarily in the cytoplasm and PML in the nucleus. Treatment with
ligand, however, promotes the nuclear trans-location of GR into a
network-like pattern overlapping with PML. The same results were
obtained in a similar experiment employing the co-transfection of
CMX-PML and CMX-RXR.alpha..
Example 8
Assay for Substances that Increase Nuclear Receptor Activity
[0106] The following example describes assays for identifying
potential therapeutic substances by measuring the ability of the
substance to enhance nuclear receptor-related transcriptional
activity, particularly in the presence of inhibitors of such
activity such as Tax or NF-.kappa.B.
[0107] CV-1 cells, grown as described above, are co-transfected
with expression plasmids containing a nuclear receptor gene (such
as GR), a reporter gene (such as MMTV-Luc) and a repressor of
nuclear receptor activity (such as Tax and/or NF-.kappa.B). The
transiently transfected cells are treated with a test substance,
diluted as described in Examples 1 and 2 above, for approximately
4-16 hours. The cells are subsequently treated with a stimulator of
nuclear receptor activity (such as dexamethasone) and the amount of
nuclear receptor activity measured as a function of the activity of
the reporter gene. Substances that enhance nuclear receptor
activity, as compared to cells that have not been treated with a
test substance, are potential therapeutic agents.
Example 9
Assays for Substances that Modulate POD Structure and/or Function
Via Nuclear Receptor Activity
[0108] The assays methods described above that are utilized in
defining substances that modify nuclear receptor-related
transcriptional activity can additionally be used to identify
substances useful for the modulation of POD structure and/or
function.
[0109] Tells cells, such as CV1 or Hep-2, are grown as described
previously in the presence or absence of a test substance. The
techniques described above, such as detection with specific
antibodies, can be used to determine if the POD structure and/or
function has been disrupted in cells exposed to the test substance
as compared to control cells that have not been so exposed.
[0110] While the foregoing has been with reference to particular
embodiments of the invention, it will be appreciated by those
skilled in the art that changes in these embodiments may be made
without departing from the principles and spirit of the invention,
the scope of which is defined by the appended claims.
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