U.S. patent application number 13/414427 was filed with the patent office on 2012-09-20 for methods for regulating the growth and/or survival of tumor cells and stem cells by modulating the expression or function of the transcription factors atf5.
Invention is credited to James M. Angelastro, Lloyd A. Greene.
Application Number | 20120238462 13/414427 |
Document ID | / |
Family ID | 46321662 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120238462 |
Kind Code |
A1 |
Greene; Lloyd A. ; et
al. |
September 20, 2012 |
METHODS FOR REGULATING THE GROWTH AND/OR SURVIVAL OF TUMOR CELLS
AND STEM CELLS BY MODULATING THE EXPRESSION OR FUNCTION OF THE
TRANSCRIPTION FACTORS ATF5
Abstract
The present invention provides methods for regulating the growth
and/or survival of tumor cells and stem cells by modulating the
expression or function of ATF5.
Inventors: |
Greene; Lloyd A.;
(Larchmont, NY) ; Angelastro; James M.; (Davis,
CA) |
Family ID: |
46321662 |
Appl. No.: |
13/414427 |
Filed: |
March 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10971483 |
Oct 22, 2004 |
8158420 |
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13414427 |
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10809312 |
Mar 24, 2004 |
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10971483 |
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60460242 |
Apr 4, 2003 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/7.1; 435/7.92 |
Current CPC
Class: |
C12N 5/0618 20130101;
C12N 2501/60 20130101; A61P 35/00 20180101 |
Class at
Publication: |
506/9 ; 435/7.92;
435/7.1; 435/6.11 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C40B 30/04 20060101 C40B030/04; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
No. NS-16036 awarded by NIH/NINCDS. The government has certain
rights in the invention.
Claims
1. A method for determining whether a subject has a tumor,
comprising assaying a diagnostic sample of the subject for ATF5
level, wherein detection of an ATF5 level elevated above normal is
diagnostic of a tumor in the subject.
2. The method of claim 1 wherein the tumor is a neural tumor.
3. The method of claim 2 wherein the neural tumor is a
neuroblasotoma.
4. The method of claim 1 wherein the tumor is a breast, ovary,
enometrium, gastric, colon, liver, pancreas, kidney, bladder,
prostate, testis, skin, esophagus, tongue, mouth, partoid, larynx,
pharynx, lymph node, or lung tumor.
5. The method of claim 1 wherein the ATF5 level corresponds to the
amount of ATF5 protein, ATF5 mRNA, ATF5 cDNA, or ATF5-CRE
interaction present in the diagnostic sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S.
Nonprovisional application Ser. No. 10/971,483, filed Oct. 22,
2004, which is a continuation-in-part of U.S. Nonprovisional
application Ser. No. 10/809,312, filed Mar. 24, 2004; which claims
the benefit of U.S. Provisional Application Ser. No. 60/460,242
filed Apr. 4, 2003; each of which are incorporated by reference in
their entireties herein.
SEQUENCE LISTING
[0003] The specification further incorporates by reference the
Sequence Listing submitted herewith via EFS on Mar. 7, 2012, 2008.
Pursuant to 37 C.F.R. .sctn.1.52(e)(5), the Sequence Listing text
file, identified as "070050.sub.--4478SeqList.txt," is 8,463 bytes
and was created on Mar. 7, 2012. The Sequence Listing,
electronically filed herewith, does not extend beyond the scope of
the specification and thus does not contain new matter.
BACKGROUND OF THE INVENTION
[0004] A key step in the formation of the nervous system is the
determination of proliferating neural progenitor cells to undergo
differentiation into neurons and glia. Despite major advances in
identification and characterization of neural progenitor cells
(Placzek and Furley, Patterning cascades in the neural tube. Neural
development. Curr. Biol., 6:526-29, 1996; Gage, F. H., Mammalian
neural stem cells. Science, 287:1433-38, 2000; Kintner, C.,
Neurogenesis in embryos and in adult neural stem cells. J.
Neurosci., 22:639-43, 2002; Schuurmans and Guillemot, Molecular
mechanisms underlying cell fate specification in the developing
telencephalon. Curr. Opin. Neurobiol., 12:26-34, 2002), the
mechanisms that govern this determination are only partially
understood.
[0005] The selective degeneration of specific types or classes of
neurons of the central nervous system (CNS) underlies many
neurological disorders. This realization has generated interest in
defining populations of progenitor cells that, through manipulation
of the differentiation process, may serve as replenishable sources
of neurons and glia, and, therefore, may present an option for
treating neurodegenerative and demyelinating disorders.
Additionally, it is well recognized that neural tumors and other
cancers develop when cells divide and grow uncontrollably. Thus, a
means of manipulating the proliferation, differentiation and/or
survival of tumor cells may provide a therapy for the treatment of
cancers.
[0006] Neural degeneration may result from neurodegenerative
diseases, CNS traumas, stroke, and the acquired secondary effects
of non-neural dysfunction. Alzheimer's disease is a
neurodegenerative disease characterized by a progressive,
inexorable loss of cognitive function. The pathogenesis of
Alzheimer's disease is associated with an excessive number of
neuritic, or senile, plaques (composed of neurites, astrocytes, and
glial cells around an amyloid core) in the cerebral cortex, and
neurofibrillary tangles (composed of paired helical filaments).
Approximately 4 million Americans suffer from Alzheimer's disease,
at an annual cost of about $90 billion. The disease is about twice
as common in women as in men, and accounts for more than 65% of the
dementias in the elderly. While senile plaques and neurofibrillary
tangles occur with normal aging, they are much more prevalent in
persons with Alzheimer's disease. To date, a cure for Alzheimer's
disease is not available, and cognitive decline is inevitable.
[0007] Demyelination is also a feature of many neurologic
disorders. Demyelinating conditions are manifested in loss of
myelin--the multiple dense layers of lipids and protein which cover
many nerve fibers. Multiple sclerosis (MS) is the most prevalent
demyelinating condition. In Europe and North America, an average of
40-100 people out of every 100,000 have MS. The disease affects
approximately 250,000 people in the United States alone.
Histopathologically, MS is characterized by inflammation, plaques
of demyelination infiltrating cells in the CNS tissue, loss of
oligodendroglia, and focal axonal injury. Typically, the symptoms
of MS include lack of co-ordination, paresthesias, speech and
visual disturbances, and weakness. Current treatments for the
various demyelinating conditions are often expensive, symptomatic,
and only partially effective, and may cause undesirable secondary
effects. Corticosteroids represent the main form of therapy for MS.
While these may shorten the symptomatic period during attacks, they
may not affect eventual long-term disability. Long-term
corticosteroid treatment is rarely justified, and can cause
numerous medical complications, including osteoporosis, ulcers, and
diabetes.
[0008] Approximately one million people are diagnosed with cancer
each year, and many millions of Americans of all ages are currently
living with some form of cancer. At some time during the course of
their lifetime, one out of every two American men and one out of
every three American women will be diagnosed with some form of
cancer. Of the one million Americans diagnosed with cancer
annually, 17,000 are diagnosed with brain tumors. Brain tumors
invade and destroy normal tissue, producing such effects as
impaired sensorimotor and cognitive function, increased
intracranial pressure, cerebral edema, and compression of brain
tissue, cranial nerves, and cerebral vessels. Drowsiness, lethargy,
obtuseness, personality changes, disordered conduct, and impaired
mental faculties are the initial symptoms in 25% of patients with
malignant brain tumors. Treatment of brain tumors is often
multimodal, and depends on pathology and location of the tumors.
For malignant gliomas, multimodal therapy, including chemotherapy,
radiation therapy, and surgery, is used to try to reduce tumor
mass. Regardless of approach, however, prognosis for patients
suffering from these tumors is guarded: the median term of survival
after chemotherapy, radiation therapy, and surgery is only about 1
year, and only 25% of these patients survive for 2 years.
[0009] In particular, malignant astrocytic tumors occur in the
human population at a frequency of 7 per 100,000 per year (Maher,
et al. Malignant glioma: genetics and biology of a grave matter.
Genes Dev., 15: 1311-1333, 2001; Rasheed, et al. Molecular
pathogenesis of malignant gliomas. Curr Opin Oncol., 11: 162-167,
1999), making them the most common form of primary brain tumor.
There is currently no effective curative therapy for patients with
WHO classification Grade IV glioblastomas (also designated
glioblastoma multiforme or GBM) and the average survival time from
diagnosis is approximately 9-11 months (McLendon, et al. Tumors of
central neuroepithelial origin., p. 307-571, 1998; Kleihues, et al,
Histology Typing of Tumours of the Central Nervous System. Berlin:
Springer-Verlag., 1993.).
[0010] Findings that neural progenitor/stem cells may be
experimentally transformed into glioblastomas has supported the
possibility that such tumors may arise from self-renewing
progenitors that have lost the capacity for appropriate regulation
of proliferation and survival (Dai, C. et al. Glioma models.
Biochem. Biophys Acta., 1551: M19-27, 2001). Indeed, GBMs are often
associated with disregulation of pathways that control growth and
survival including those involving p53, Rb, PTEN and growth factor
receptors (reviewed by Collins (Collins, V. P. Brain tumours:
classification and genes. J. Neurol. Neurosurg. Psychiatry, 75
Suppl 2: ii2-11, 2004). Additional novel regulatory genes may also
contribute to blocking GBM cells from undergoing full
differentiation and maintaining them in a state of uncontrolled
growth.
[0011] In view of the foregoing, it is clear that many neural
disorders are related to loss of cells, loss of myelin, or loss of
cell control. An ability to regulate the differentiation of
neuroprogenitor cells into various differentiated neural cells
would provide supplies of neural cells that could be effective in
treating such neural disorders. Additionally, the ability to
regulate the growth and/or survival of tumor cells would be
effective in treating an array of neoplastic disorders. However,
prior to the present invention, manipulating whether or not neural
progenitor cells differentiate into neurons and/or glia continue to
divide and to remain as progenitor cells, as well as the general
regulation of the growth and/or survival of stem cells and tumor
cells has proved difficult.
SUMMARY OF THE INVENTION
[0012] The inventors disclose herein that the b-zip transcription
factor, ATF5, plays a major regulatory role in the differentiation
of neuroprogenitor cells into differentiated neural cells. In
particular, the inventors have discovered that, in the developing
brain, ATF5 expression is high within ventricular zones containing
neural stem cells and neural progenitor cells, but is undetectable
in post-mitotic neurons and glia. In attached clonal neurosphere
cultures, ATF5 is expressed by neural stem cells and neural
progenitor cells, but is undetectable in tau-positive neurons, in
GFAP positive astrocytes and in the nuclei of mature
oligodendroglia. In PC12 cell cultures, nerve growth factor (NGF)
dramatically down-regulates endogenous ATF5 protein and
transcripts, while exogenous ATF5 suppresses NGF-promoted neurite
outgrowth. Such inhibition may require repression of cyclic AMP
(cAMP) responsive element (CRE) DNA-binding sites and/or other ATF5
DNA-binding sites, including those not yet discovered. By contrast,
loss of function conferred by dominant-negative ATF5 accelerates
NGF-promoted neuritogenesis. Exogenous ATF5 suppresses neurogenesis
by cultured nestin-positive telencephalic cells, while
dominant-negative ATF5, and a small interfering RNA targeted to
ATF5, promote this activity. These findings indicate that ATF5
blocks differentiation of neuroprogenitor cells into neurons, and
must be down-regulated to permit this process to occur. Additional
studies carried out in culture, and also in viva, indicate that
ATF5 blocks differentiation of proliferating neural progenitor
cells and oligodendrocyte precursor cells into differentiated
astroglia and oligodendroglia, and that dominant-negative ATF5
accelerates this differentiation. Thus, constitutive expression of
exogenous ATF5 maintains neural progenitor cells in a proliferative
state both in vitro and in vivo and represses their differentiation
in the presence extracellular signals such as NGF, NT3 and CNTF
that otherwise promote differentiation and down-regulation of
endogenous ATF5. By contrast, loss of ATF5 function or expression
achieved with a dominant negative form of ATF5 or with a small
interfering RNA, respectively, accelerates the differentiation of
neural progenitors into non-dividing neurons and glia (Angelastro,
et al. Regulated expression of ATF5 is required for the progression
of neural progenitor cells to neurons. J. Neurosci., 23: 4590-4600,
2003; Angelastro et al., unpublished data).
[0013] The inventors additionally disclose herein that ATF5 is
widely expressed by various tumor types. In particular, the
inventors have shown that ATF5 is expressed not only in highly
proliferative neural tumors, e.g., glioblastomas, but is also
expressed in multiple neoplasias including, but not necessarily
limited to: breast, ovary, endometrium, gastric, colon, liver,
pancrease, kidney, bladder, prostate, testis, skin, esophagus,
tongue, mouth, parotid, larynx, pharynx, lymph node, lung, and
brain tumors. Further, the inventors have demonstrated for the
first time that interfering with the function or expression of ATF5
promotes apoptosis of glioblastoma multiforme tumor cells (GBM) in
vitro and in vivo. The inventors have also shown for the first time
that selective interference with ATF5 function in other carcinoma
types, e.g., breast tumors, also triggers cell death. Importantly,
the effect of ATF5 interference is specific in that interfering
with ATF5 function triggers increased cell death in neoplastic
cells, but not normal cells.
[0014] Accordingly, the present invention provides a method for
regulating the growth and/or survival of tumor cells and stem cells
by modulating the expression or function of ATF5. The invention
additionally provides methods for promoting differentiation of a
neural stem cell or a neural progenitor cell into a differentiated
neural cell, by inhibiting ATF5 function or expression in the cell.
Also provided is a differentiated neural cell produced by this
method.
[0015] The present invention also provides a method for producing
differentiated neural cells by: (a) obtaining or generating a
culture of neural stem cells or neural progenitor cells; (b)
contacting the culture of neural stem cells or neural progenitor
cells with an amount of an ATF5 inhibitor effective to produce
differentiated neural cells; and (c) optionally, contacting the
differentiated neural cells with at least one neurotrophic factor.
Examples of methods for contacting the cells with (treating the
cells with) the ATF5 inhibitor or the neurotrophic factor (in
protein or nucleic acid form) include, without limitation,
absorption, electroporation, immersion, injection, liposome
delivery, transfection, vectors, and other protein-delivery and
nucleic-acid-delivery vehicles and methods. Also provided is a
population of cells, comprising the differentiated neural cells
produced by this method.
[0016] The present invention further provides a method for treating
nervous tissue degeneration in a subject in need of treatment by:
(a) obtaining or generating a culture of neural stem cells or
neural progenitor cells; (b) contacting the culture of neural stem
cells or neural progenitor cells with an amount of an ATF5
inhibitor effective to produce differentiated neural cells; (c)
optionally, contacting the differentiated neural cells with at
least one neurotrophic factor; and (d) transplanting the
differentiated neural cells into the subject in an amount effective
to treat the nervous tissue degeneration.
[0017] Additionally, the present invention provides differentiated
neural cells produced by: (a) obtaining or generating a culture of
neural stem cells or neural progenitor cells; (b) contacting the
neural stem cells or neural progenitor cells with an amount of an
ATF5 inhibitor effective to produce differentiated neural cells;
and (c) optionally, contacting the differentiated neural cells with
at least one neurotrophic factor. Also provided is a transgenic
non-human animal containing these differentiated neural cells, and
uses of these differentiated neural cells in analyzing neuron
development, function, and death, and in monitoring synaptic
differentiation.
[0018] The present invention is also directed to a method for
isolating and/or purifying a population of differentiated neural
cells by: (a) obtaining or generating a culture of neural stem
cells or neural progenitor cells that express enhanced green
fluorescent protein (eGFP); (b) contacting the culture of neural
stem cells or neural progenitor cells with an amount of an ATF5
inhibitor effective to produce differentiated neural cells that
express eGFP; (c) optionally, contacting the differentiated neural
cells with at least one neurotrophic factor; (d) detecting
expression of eGFP in the differentiated neural cells; and (e)
isolating the differentiated neural cells that express eGFP.
[0019] Furthermore, the present invention provides a method for
identifying an agent for use in treating a condition associated
with nervous tissue degeneration by: (a) obtaining or generating a
culture of neural stem cells or neural progenitor cells; (b)
contacting the neural stem cells or neural progenitor cells with an
amount of an ATF5 inhibitor effective to produce neurons, wherein
some or all of the neurons are degenerated; (c) contacting the
degenerated neurons with a candidate agent; and (d) determining if
the agent enhances regeneration or survival of some or all of the
degenerated neurons.
[0020] The present invention also provides a method for suppressing
differentiation of neural stem cells or neural progenitor cells
into differentiated neural cells, by contacting the neural stem
cells or neural progenitor cells with an amount of ATF5 effective
to suppress differentiation in the neural stem cells or neural
progenitor cells.
[0021] Additionally, the present invention is directed to a
therapeutic composition, comprising: (a) a nucleic acid encoding an
ATF5 inhibitor; (b) a vector; and (c) optionally, a
pharmaceutically-acceptable carrier. Also provided is a method for
treating a tumor, e.g., a neural tumor, in a subject in need of
treatment, by administering the therapeutic composition to the
subject.
[0022] The present invention further provides a method for
identifying an agent which inhibits ATF5 by: (a) contacting a
candidate agent with ATF5, in the presence of CRE; and (b)
assessing the ability of the candidate agent to inhibit interaction
between ATF5 and CRE. This method may further comprise the steps
of: (c) contacting the candidate agent with neural stem cells or
neural progenitor cells containing ATF5; and (d) determining if the
agent has an effect on an ATF5-associated biological event in the
cells. Also provided are agents identified by these methods, as
well as methods for promoting differentiation in neural stem cells
or neural progenitor cells, and for treating or preventing a neural
tumor in a subject, using these agents.
[0023] The present invention additionally provides methods for
promoting apoptosis in a neoplastic cell comprising contacting the
neoplastic cell with an ATF5 inhibitor. The neoplastic cell can be
selected from the group consisting of: breast, ovary, endometrium,
gastric, colon, liver, pancrease, kidney, bladder, prostate,
testis, skin, esophagus, tongue, mouth, parotid, larynx, pharynx,
lymph node, lung, and brain. In one embodiment, the neoplastic cell
is selected from the group consisting of glioblastoma, astrocytoma,
glioma, medulloblastoma and neuroblastoma. In other embodiments,
the ATF5 inhibitor is a nucleic acid, which can include, but is not
limited to a dominant negative form of ATF5 (e.g. NTAzip-ATF5), or
ATF5siRNA. The method of the present invention can be performed in
vitro as well as in vivo in a subject.
[0024] The present invention also provides a methods for treating
or preventing a tumor in a subject comprising the steps of: (a)
obtaining or generating a culture of tumor cells; and (b)
contacting the tumor cells with an amount of an ATF5 inhibitor
effective to induce apoptosis in the tumor cells. In one
embodiment, the tumor is selected from the group consisting of:
breast, ovary, endometrium, gastric, colon, liver, pancrease,
kidney, bladder, prostate, testis, skin, esophagus, tongue, mouth,
parotid, larynx, pharynx, lymph node, lung, and brain. In another
embodiment, the tumor is selected from the group consisting of
glioblastoma, astrocytoma, glioma, medulloblastoma and
neuroblastoma. In still another embodiment, the ATF5 inhibitor is a
nucleic acid.
[0025] The invention further provides methods for producing
differentiated tumor cells, comprising the steps of: (a) obtaining
or generating a culture of tumor cells; (b) contacting the culture
of tumor cells with an amount of an ATF5 inhibitor effective to
produce differentiated neural cells; and (c) optionally, contacting
the differentiated neural cells with at least one neurotrophic
factor. The method can be performed in vivo or in vitro.
[0026] The invention also provides a method for determining whether
a subject has a tumor, comprising assaying a diagnostic sample of
the subject for ATF5, wherein detection of an ATF5 level elevated
above normal is diagnostic of a tumor in the subject.
[0027] The invention further provides methods for assessing the
efficacy of therapy to treat a tumor in a subject who has undergone
or is undergoing treatment for a tumor, comprising assaying a
diagnostic sample of the subject for ATF5, wherein a normal level
of ATF5 in the diagnostic sample is indicative of successful
therapy to treat the tumor, and a level of ATF5 elevated above
normal in the diagnostic sample is indicative of a need to continue
therapy to treat the tumor.
[0028] The invention also provides methods for assessing the
prognosis of a subject who has a tumor, comprising assaying a
diagnostic sample of the subject for ATF5, wherein the subject's
prognosis improves with a decreased level of ATF5 in the diagnostic
sample, and the subject's prognosis worsens with an increased level
of ATF5 in the diagnostic sample.
[0029] A therapeutic composition for use in treating or preventing
a tumor is also provided by the present invention, comprising: (a)
a nucleic acid encoding an ATF5 inhibitor; (b) a vector; and (c)
optionally, a pharmaceutically-acceptable carrier.
[0030] Further, the present invention provides a method for
determining whether a subject has a tumor, by assaying a diagnostic
sample of the subject for ATF5, wherein detection of an ATF5 level
elevated above normal is diagnostic of a tumor in the subject. Also
provided are methods for assessing the efficacy of therapy to treat
a tumor in a subject who has undergone or is undergoing treatment
for a tumor, and for assessing the prognosis of a subject who has a
tumor.
[0031] Finally, the present invention provides kits for use in
detecting, treating and preventing tumors.
[0032] Additional aspects of the present invention will be apparent
in view of the description which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows that nerve growth factor (NGF) down-regulates
ATF5 protein in PC12 cells, and demonstrates a reciprocal
relationship with neurite outgrowth. (A) Time course of the effect
of NGF treatment on ATF5 protein expression in PC12 cells. Cells
were exposed to NGF for the indicated times, and 135 .mu.g of whole
cell extracts were subjected to Western immunoblotting, first with
anti-ATF5, then, after stripping, with anti-ERK to normalize for
loading. Numbers at the left of the figure indicate the positions
of molecular weight markers (in kDa). Comparable results were
achieved in 3 independent experiments. (B) Comparison of the
kinetics of NGF-dependent down-regulation of ATF5 expression, and
promotion of neurite outgrowth. The relative levels of ATF5
expression were determined by densitometry, and normalized to
levels of ERK protein in the same sample; the levels are reported
in arbitrary units. Proportions of cells bearing neurites of a
length at least twice the diameter of the cell body were determined
in the same cultures, by scoring at least 200 cells per time
point.
[0034] FIG. 2 shows that overexpression of ATF5 represses neurite
outgrowth in PC12 cells, while NTAzip-ATF5 accelerates
neuritogenesis. (A) Detection and NGF response of PC12 cells
expressing exogenous ATF5. PC12 cells were transiently transfected
with pCMS-eGFP (panels a and b) or pCMS-eGFP expressing FLAG-tagged
ATF5 (panels c and d). Two days after transfection, the cultures
were treated with NGF. Five days after transfection (i.e., after 3
days of NGF exposure), the cells were fixed and co-stained with
rabbit anti-GFP (panels a and c) or mouse anti-FLAG antibody
(panels b and d), with detection by FITC (GFP) and
rhodamine-conjugated secondary antibody (FLAG-ATF5). Scale bar
represents 50 .mu.m (B) Quantification of the effects of exogenous
ATF5 and of NTAzip-ATF5 on NGF-promoted neurite outgrowth. PC12
cells were transiently transfected with pCMS-eGFP, without insert
or expressing FLAG-tagged ATF5 or FLAG-tagged NTAzip-ATF5. Two days
after transfection, the cultures were treated with NGF. Cultures
were fixed at the indicated times, after commencement of NGF
exposure, and immunostained with anti-GFP and anti-FLAG, as above.
Transfected cells (positive for FLAG and/or GFP staining) were
assessed for the presence or absence of neurites. The proportions
of transfected cells bearing neurites are reported .+-.SEM, with
n=3 cultures (and at least 300 transfected cells assessed per
culture). Comparable results were achieved in 4 additional
independent experiments. In all cases (including the data shown),
ANOVA analysis indicated a p value of <0.05 at the 72-h point of
NGF treatment for eGFP vs. ATF5. (C) NTAzip accelerates
NGF-promoted neurite outgrowth. Cultures were transfected, treated,
and assessed as in (B), at 24 h after NGF exposure. Values
represent the mean.+-.SEM for results of 4 independent experiments.
In each experiment, the data were normalized to the percentage of
neurite-bearing cells transfected with pCMS-eGFP. The average
percentage of such cells was 10.6.+-.3.7. NTAzip vs. eGFP:
p<0.02, Student's t-distribution test.
[0035] FIG. 3 illustrates that ATF5 is differentially expressed in
the ventricular zones of E12-E15 rat brain. (A) Expression of ATF5
message in developing rat brain (panels a and b), In situ
hybridization was carried out using an ATF5 antisense probe in
saggital sections of E15 rat brain. Panel a shows the area around
the fourth ventricle, and panel b shows the telencephalon. There
was no positive signal with a control ATF5 sense probe. Expression
of ATF5 protein is shown in coronal sections of E12 (panels c and
d) and E14 (panels e and f) rat telencephalon. (panel c) Staining
with pre-immune serum. (panels d-f) Co-staining with anti-ATF5
(red) and anti-tubulin .beta. (III) (TUJ1 antibody; green). Arrows
indicate staining of ATF5 in the ventricular zone (VZ). CX=cortex;
scale bar for panel a represents 100 .mu.m (B) High-power confocal
images of reciprocal expression of ATF5 (red) and tubulin .beta.
(III) in coronal sections of E14 rat telencephalon. Immunochemical
staining was carried out as in (A), Images showing the ventricular
zone (panel a) and cortex (panel b) are from the same section, and
were photographed in the same confocal Z-plane section (1.3 .mu.m).
Arrowhead shows a migratory cell undergoing a transition from a
progenitor to a neuron, by exhibiting both ATF5 and tubulin .beta.
(III) staining. Co-localization was confirmed by YZ and XZ confocal
images. Scale bar for panel B represents 20 .mu.m.
[0036] FIG. 4 demonstrates reciprocal expression of ATF5 and
tubulin .beta. (III) in E17 rat brain. (A-C) Expression of ATF5
(red) and tubulin .beta. (III) (green) in the area of the anterior
(A-C) and posterior (D-F) lateral ventricles of the E17 rat brain.
Immunohistochemical staining was carried out as in FIG. 3 and the
Examples. Scale bar represents 100 .mu.m.
[0037] FIG. 5 shows that ATF5 is expressed in neural stem cells and
progenitor cells, but not in mature neurons in attached neurosphere
cultures. Attached clonal neurosphere cultures were established
from the subventricular zone and hippocampal dentate gyrus of
newborn mouse brain, and maintained as described in the Examples.
Cultures were fixed and co-stained as follows: (A) ATF5 (red) and
AC133 (green), a stem cell marker. Thick arrows show examples of
nuclear staining, thin arrows show cytoplasmic staining. (B) ATF5
(red) and nestin (green), a marker for neural progenitor cells.
Arrows indicate nuclear staining. (C, D) ATF5 (red) and NF-M
(green), a marker for the neuronal lineage. Arrows show nuclear
staining in (C) and cell body in (D). (E, F) ATF5 (red) and
anti-tau (green), a neuronal marker. Comparable results were
achieved in 10 independent experiments. Arrows show neurons at the
periphery of the cultures; arrowhead shows stem and neural
progenitor cells at the center of the culture. Stained cells were
examined and photographed by confocal microscopy. The scale bar is
20 .mu.m for (A), and 50 .mu.m for (B-F).
[0038] FIG. 6 illustrates that ATF5 represses, and NTAzip-ATF5
promotes, neurite outgrowth and expression of neuronal markers in
neural progenitor cells. (A) Cultured E14 telencephalic cells were
transiently transfected with pCMS-eGFP containing either no insert
(empty vector), FLAG-ATF5, or NTAzip-ATF5. Three days following
transfection, the cultures were fixed and co-immunostained for GFP
and either nestin or tubulin .beta. (III) (TUJ1 antibody).
Transfected cells (GFP+) were assessed for the presence of
neurite-like processes, and for co-expression of the indicated
markers. Values represent the mean.+-.SEM for 3 cultures in which
at least 300 transfected cells were evaluated per culture.
Comparable results were achieved in 4 independent experiments.
ANOVA analysis of transfected cells: total cells--nestin/eGFP alone
vs. nestin/ATF5, p<0.001; TUJ1/eGFP alone vs. TUJ1/ATF5,
p<0.05; nestin/eGFP alone vs. nestin/NTAzip, and TUJ1/eGFP alone
vs. TUJ1/NTAzip, no significant difference; process-bearing
cells--TUJ1/GFP alone vs. TUJ1/ATF5, p<0.05; nestin/eGFP alone
vs. nestin/NTAzip and TUJ1/eGFP alone vs. TUJ1/NTAzip, no
significant difference. (B) Cultured E14 telencephalic cells were
infected with retroviruses expressing eGFP or FLAG-ATF5 and eGFP.
One week after infection, the cultures were fixed and assessed as
in (A), and assessed for NF-M expression. Comparable results were
achieved in 3 independent experiments. ANOVA analysis: total
cells--nestin/eGFP alone vs. nestin/ATF5, p<0.001; TUJ1/GFP
alone vs. TUJ1/ATF5, p<0.01; NFM/eGFP vs. NFM/ATF5, p<0.001;
process-bearing cells--TUJ1/GFP alone vs. TUJ1/ATF5, p<0.001;
NFM/GFP alone vs. NFM/ATF5, p<0.01; nestin/eGFP alone vs.
nestin/ATF5, no significant difference; TUJ1 vs. NFM, no
significance both with eGFP alone and with eGFP plus ATF5. (C) E14
telencephalon cells were infected with retroviruses expressing
eGFP, eGFP and FLAG-ATF5, or eGFP-FLAG-NTAzip-ATF5. Four days after
infection, the cultures were fixed and evaluated as in (A).
Comparable results were achieved in 2 independent experiments.
ANOVA analysis: nestin/GFP alone vs. nestin/ATF5, p<0.001; total
and process-bearing cells--TUJ1/eGFP alone vs. TUJ1/ATF5,
p<0.01; TUJ1/GFP alone vs. TUJ1/NTAzip, p<0.5. (D) Cultured
E14 telencephalic cells were transiently transfected with
pCMS-eGFP, with or without ATF5 siRNA. Four days following
transfection, the cultures were fixed and co-immunostained either
for GFP and TUJ1 antibody, or with GFP and ATF5 antiserum.
Transfected cells (GFP+) were assessed for the presence of the
neuronal marker, tubulin .beta. (III) (TUJ1), or ATF5. Values
represent the mean.+-.SEM for six cultures in which at least 300
transfected cells were evaluated per culture. Comparable results
were achieved in 3 independent experiments (two experiments with
E14 telencephalon cells cultured with serum plus EGF and FGF2, and
one experiment with only serum). ANOVA analysis: TUJ1/eGFP alone
vs. TUJ1/ATF5 siRNA, p<0.001; ATF5/GFP alone vs. ATF5/ATF5
siRNA, p<0.001. (E) ATF5 suppresses NT3-promoted neuronal
differentiation. E15 telencephalon cells were infected with
retroviruses expressing eGFP, eGFP and FLAG-ATF5, or
eGFP-FLAG-NTAzip-ATF5, all .+-.NT3. Four days after infection and
maintenance .+-.NT3 treatment, the cultures were fixed and
evaluated, as in (A), for eGFP and TUJ1 expression. Comparable
results were achieved in 2 independent experiments. ANOVA analysis:
-NT3/eGFP alone vs. +NT3/GFP alone, p<0.001; -NT3/eGFP alone vs.
-NT3/ATF5, p<0.05; +NT3/eGFP alone vs. +NT3/ATF5, p<0.001;
-NT3/eGFP alone vs. -NT3/NTAzip, p<0.001; +NT3/GFP alone vs.
+NT3/NTAzip, no significant difference.
[0039] FIG. 7 demonstrates that NTAzip-ATF5 and VP16-CREB reverse
ATF5-promoted repression of CRE-mediated gene expression and of
neurite outgrowth. (A) PC12 cells were co-transfected with pG13-CRE
luciferase, pcDNA-LacZ, and 1 .mu.g/culture of pCMS-eGFP expressing
either no insert (empty vector), FLAG-ATF5, FLAG-NTAzip-ATF5, or
VP16-CREB. The cultures were also exposed to NGF for 2 days prior
to and during the time of transfection (for a total of 3 days),
during the time of transfection (1 day), or during the last hour
prior to harvesting. One day after transfection, the cells were
harvested and assessed for luciferase expression and LacZ activity
(.beta.-GAL). Values represent mean normalized CRE-luciferase
activity (in arbitrary units) .+-.SEM (n=3). Comparable results
were achieved in three independent experiments. Student's
t-distribution test: empty vector (eGFP alone) vs. VP16CREB at all
times, p<0.001; GFP alone vs. ATF5, p<0.033 by day 3. (B)
PC12 cells were co-transfected with pG13-CRE luciferase,
pcDNA-LacZ, and the indicated combinations of pCMS-eGFP expressing
either no insert (GFP), FLAG-ATF5 (ATF5), FLAG-NTAzip-ATF5 (AZIP),
or VP16-CREB. The latter vectors were each used at 0.5
.mu.g/culture, and empty vector was added, as needed, to bring the
total DNA level to 1 .mu.g/culture. Cultures were harvested 1 day
later, or assayed for luciferase expression and LacZ activity
(.beta.-GAL). Where indicated, NGF was added to the medium 1 h
before harvesting. Values represent mean normalized CRE-luciferase
activity (in arbitrary units) .+-.SEM (n=6), with data pooled from
2 independent experiments. Student's t-distribution test: -NGF-eGFP
alone vs. ATF5, p<0.003; eGFP alone vs. NTAzip, p<0.0003;
eGFP alone vs. ATF5/NTAzip, no significant difference; eGFP alone
vs. VP16CREB and VP16CREB/ATF5, p<0.0001; +NGF-eGFP alone vs.
ATF5, p<0.0001; eGFP alone vs. NTAzip, p<0.02; eGFP alone vs.
ATF5/NTAzip, p<0.02; eGFP alone vs. VP16CREB and VP16CREB/ATF5,
p<0.0001. (C) PC12 cells were co-transfected with the indicated
constructs, and NGF was added to the medium 2 days later.
Transfected cells (identified for eGFP) were assessed for neurite
outgrowth at the indicated times. Values represent means.+-.SEM of
results for 3 cultures in which at least 300 transfected cells were
scored per culture. Comparable results were obtained in 2
independent experiments. ANOVA analysis after 72 h of
NGF-treatment: eGFP alone vs. ATF5, eGFP p<0.001; eGFP alone vs.
NTAzip-ATF5, NTAzip/ATF5, Vp16CREB, or Vp16CREB/ATF5, no
significant difference.
[0040] FIG. 8 sets forth the nucleotide sequence of ATF5 (SEQ ID
NO:1).
[0041] FIG. 9 sets forth the amino acid sequence of ATF5 (SEQ ID
NO:2).
[0042] FIG. 10 shows expression of ATF5 in human glioblastomas.
Examples of immunostaining for ATF5 (brown DAB product) in paraffin
sections within (A-C, E) and outside of (D) Grade IV glioblastomas.
(A) Arrows show the nuclei of neurons lacking positive ATF5
staining. In contrast, many of the surrounding nuclei of
glioblastoma cells stain positively for ATF5. (B,C) Nuclear ATF5
staining in a giant-cell GBM (B) and in another GBM with
variably-sized nuclei. (D,E) In sections from another patient, ATF5
staining is absent cells in the cortex outside the area of tumor
infiltration (D), but present within the tumor (E). Scale bar is 10
.mu.m for A-C, and 2 .mu.m for D and E.
[0043] FIG. 11 shows expression of ATF5 in glioma cell lines,
cultured astrocytes and HEK 293 cells. (A) Western immunoblot
probed with Anti-ATF5 antiserum reveals expression of ATF5 in
lysates of rat PC12, C6 and RG2 glioma cells, human U251 glioma
cells and human embryonic kidney 293 cells and the absent in low
passage (passage 1-2; LP) neonatal astrocytes. (B) Percentages of
cells in cultures of human and rat glioblastoma lines and of high
passage (passage 5; HP) neonatal rat astrocytes positive for
staining for endogenous ATF5 and for endogenous Ki67. GFP+ cells
were scored 5 days after transfection with pLeGFP-C1. Values
represent the mean.+-.SEM for 3 cultures in which at least 100
transfected cells were evaluated per culture. Inspection of
non-transfected cells revealed a similar level of staining.
[0044] FIG. 12 demonstrates that dominant negative NT-Azip-ATF5
promotes multi-nucleated and apoptosis of U87 cells. U87 cells were
transfected with pLeGFP-C1 (A-D), or transfected with
pLeGFP-C1-NT-Azip-ATF5 (E-H). U87 cells were immunostained with
anti-eGFP (A) and (E); or anti-ATF5-antiserum (B) and (F); Hoechst
dye (C) and (G). All three staining were merged (D) and (H). Arrows
show apoptotic nuclei. Scale bar is 10 .mu.m.
[0045] FIG. 13 shows that dominant negative ATF5 promotes and
apoptosis of U87 cells. U87 cells were transfected with pLeGFP-C1
(A-D), or pLeGFP-C1-NT-Azip-ATF5 (E-H) and immunostained 5 days
later with anti-eGFP (A,E)) or anti-ATF5 (B,F) antisera and with
Hoechst nuclear dye 33258 (C,G). The merged images are shown in
panels D and H. Arrows show apoptotic nuclei. Scale bar is 10
.mu.m.
[0046] FIG. 14 shows (A) dominant negative ATF5 triggers apoptosis
of cultured glioma cells, but not of activated astrocytes. Cultures
were transfected with pLeGFP-C1 or pLeGFP-C1-NT-Azip-ATF5 as
indicated and transfected cells (GFP+) were scored 5 days later for
proportion with condensed apoptotic nuclei. Values represent the
mean.+-.SEM (n=3 cultures in which at least 100 transfected cells
were evaluated per culture). (13) ATF5 siRNA triggers apoptosis of
cultured glioma cells, but not of activated astrocytes. Human cells
were co-transfected with pCMS-eGFP and human ATF5 oligo-duplex
siRNA or pCMS-eGFP. Cultured rat astrocytes were similarly
transfected, but with rat ATF5 oligo-duplex siRNA and pCMS-eGFP or
pCMS-eGFP alone. C6 glioma cells were transfected with
pQcSIREN-zsGreen small hairpin luciferase siRNA (control), or with
pQcSIREN-zsGreen-small hairpin ATF5 siRNA. Five days later,
transfected cells (GFP+ cell, or zsGreen+) were scored for
proportion with condensed apoptotic nuclei. Values represent the
mean.+-.SEM (n=3 cultures in which at least 100 transfected cells
were evaluated per culture).
[0047] FIG. 15 shows that retroviral delivery of dominant negative
NTAzip-ATF5 triggers death of C6 glioma cells in an in vivo tumor
model, but spares cells outside the tumor. Tumors were induced in
adult rat brains by stereotactic injection of approximately
1.times.10.sup.4 C6 glioma cells into the striatum. Ten days later,
retroviruses (1.25.times.10.sup.4 in 5 .mu.l) expressing eGFP
(control) or eGFP-NTAzip-ATF5 were stereotactically injected into
the C6 tumors. Three days after the injection of retrovirus and a
total of 13 days after the injection of C6 cells, the brains were
removed, fixed, sectioned and stained for TUNEL (B,F,J,N) and then
immunostained with rabbit-anti-eGFP antibodies (A,E,I,M) and
stained with Hoechst nuclear dye 33258 (C,G,K,O). A-H. Cells in a
tumor infected with control virus and found outside (A-D) or within
(E-H) the tumor. I-P. Cells in a tumor infected with virus
expressing eGFP-NTAzip-ATF5 found outside (I-L) or within (M-P) the
tumor. Scale bar is 10 .mu.m. L1 and P1 show enlargements of the
areas within the boxes indicated in L and P, respectively. Note the
presence of yellow cells (positive for both TUNEL and eGFP) in the
merged images only in the case of cells within tumors infected with
virus expressing eGFP-NTAzip-ATF5. Scale bar is 10 .mu.m.
[0048] FIG. 16 depicts quantification of the selective
death-promoting effects of NTAzip-ATF5 on cells within C6 brain
tumors. Generation of C6 glioblastoma tumors and their infection
with control eGFP (LeGFP) and NTAzip-ATF5 (LeGFP-Azip) expressing
retroviruses were carried out as in FIG. 6. eGFP-positive cells
were scored for the presence or absence of TUNEL staining. Four
tumors injected with control virus were examined and a total of 252
infected cells were scored within the tumors and 194 outside the
tumors. Five tumors injected with NTAzip-ATF5-expressing virus were
examined and a total of 225 infected cells were scored within the
tumors and 63 outside the tumors. Data represent proportions of
total cells scored in each category that were positive or negative
for TUNEL staining.
DETAILED DESCRIPTION OF THE INVENTION
[0049] As described above, a key step in the formation of the
nervous system is the determination of proliferating neural
progenitor cells to exit the cell cycle and undergo neuronal
differentiation. Despite major advances in identification and
characterization of such progenitor cells, the mechanisms that
govern this determination are only partially understood.
[0050] One system with potential to address this issue is the PC12
line of pheochromocytoma cells (Greene and Tischler, Establishment
of a noradrenergic clonal line of rat adrenal pheochromocytoma
cells which respond to nerve growth factor. Proc. Natl. Acad Sci.
USA, 73:2424-28, 1976; Burstein and Greene, Evidence for RNA
synthesis-dependent and -independent pathways in stimulation of
neurite outgrowth by nerve growth factor. Proc. Natl. Acad. Sci.
USA, 75:6059-63, 1978). In the presence of the neurotrophic factor,
nerve growth factor (NGF), proliferating neuroblast-like PC12 cells
acquire, by means of a transcription-dependent mechanism, a
neuronal phenotype characterized by formation of axons,
up-regulation of a number of neuronal markers, and transition to a
post-mitotic state.
[0051] To identify genes responsible for this neuronal
differentiation, the inventors employed serial analysis of gene
expression (SAGE) to provide a comprehensive profile and comparison
of transcripts present in PC12 cells, before and after 9 days of
treatment with NGF (Angelastro et al., Identification of diverse
nerve growth factor-regulated genes by serial analysis of gene
expression (SAGE) profiling. Proc. Natl. Acad. Sci. USA,
97:10424-29, 2000). Of the approximately 22,000 unique transcripts
detected in the cells, approximately 4% underwent a 6-fold or
greater increase or decrease in expression after NGF exposure.
Among the identified genes with the greatest change in expression
was ATF5, a member of the activating transcription factor
(ATF/CREB) family. In response to NGF, ATF5 transcripts, which were
among the most highly expressed in the cells prior to treatment,
fell by 25-fold in relative expression.
[0052] Relatively few studies have been carried out to characterize
ATF5 (also known as ATFX and ATF-7) and its biological functions
(Nishizawa and Nagata, cDNA clones encoding leucine-zipper proteins
which interact with G-CSF gene promoter element 1-binding protein.
FEBS Lett., 299:36-38, 1992; Pati et al., Human Cdc34 and Rad6B
ubiquitin-conjugating enzymes target repressors of cyclic
AMP-induced transcription for proteolysis. Mol. Cell Biol.,
19:5001-13, 1999; Peters et al., ATF-7, a novel bZIP protein,
interacts with the PRL-1 protein-tyrosine phosphatase. J. Biol.
Chem., 276:13718-726, 2001; Persengiev et al., Inhibition of
apoptosis by ATFx: a novel role for a member of the ATF/CREB family
of mammalian bZIP transcription factors. Genes Dev., 16:1806-14,
2002). ATF5 is a b-zip transcription factor that forms homodimers
that, at least in vitro, bind cyclic AMP (cAMP) responsive element
(CRE) DNA-binding sites. In addition, ATF5 represses cAMP-induced
transcription in intact cells (Pati et al., Human Cdc34 and Rad6B
ubiquitin-conjugating enzymes target repressors of cyclic
AMP-induced transcription for proteolysis. Mol. Cell Biol.,
19:5001-13, 1999; Peters et al., ATF-7, a novel bZIP protein,
interacts with the PRL-1 protein-tyrosine phosphatase. J. Biol.
Chem., 276:13718-726, 2001), and has been shown to inhibit
apoptosis (Persengiev et al., Inhibition of apoptosis by ATFx: a
novel role for a member of the ATF/CREB family of mammalian bZIP
transcription factors. Genes Dev., 16:1806-14, 2002). This raised
the possibility that ATF5 might interfere with the activity of
transcription factors, such as CREB, that appear to promote
neuronal differentiation via CRE-mediated gene activation
(Finkbeiner et al., CREB: a major mediator of neuronal neurotrophin
responses. Neuron, 19:1031-47, 1997; Dawson and Ginty, CREB family
transcription factors inhibit neuronal suicide. Nat. Med.,
8:450-51, 2002; Lonze et al., Apoptosis, axonal growth defects, and
degeneration of peripheral neurons in mice lacking CREB. Neuron,
34:371-85, 2002). These properties, along with its down-regulation
by NGF, suggest that ATF5 is a negative regulator of neuronal
differentiation, via CRE and other (as yet undiscovered)
DNA-binding sites.
[0053] The present invention relates to several findings concerning
the levels of expression of ATF5 in cells of the nervous system. In
particular, the inventors have discovered that ATF5 is highly
expressed in the nuclei of neuroprogenitor cells (in both the
developing and adult nervous systems), and that it functions in
these cells to block their differentiation into neurons, astroglia,
and oligodendroglia. In contrast, ATF5 is only detected outside the
nucleus in oligodendroglia and Schwann cells (myelin-forming cells
in the CNS and the peripheral nervous system (PNS), respectively),
and is not detected in mature neurons or astroglia. Studies also
indicate that ATF5 is highly expressed in human neuroblastoma
cells.
[0054] The inventors have also discovered that ATF5 is widely
expressed by various tumor types. In particular, the inventors have
shown that ATF5 is expressed not only in highly proliferative
neural tumors, e.g., glioblastomas, but is also expressed in
multiple neoplasias including, but not necessarily limited to:
breast, ovary, endometrium, gastric, colon, liver, pancrease,
kidney, bladder, prostate, testis, skin, esophagus, tongue, mouth,
parotid, larynx, pharynx, lymph node, lung, and brain tumors.
Further, the inventors have demonstrated for the first time that
interfering with the function or expression of ATF5 promotes
apoptosis of glioblastoma multiforme tumors (GBM) in vitro and in
vivo. The inventors have also shown for the first time that
selective interference with ATF5 function in other carcinoma types,
e.g., breast tumors, also triggers cell death. Importantly, the
effect of ATF5 interference is specific in that interfering with
ATF5 function triggers increased cell death in neoplastic cells,
but not normal cells.
[0055] The present invention also relates to a role for ATF5 in the
differentiation of progenitor cells, including but not limited to
neuroprogenitor cells. For example, the inventors have observed
that forced constitutive expression of ATF5 protein in
neuroprogenitor cells blocks their differentiation into neurons and
glial cells. The inventors have also observed that specific
suppression of ATF5 protein synthesis, or forced constitutive
expression of a blocking form of the protein, strongly promotes
differentiation of neuroprogenitor cells.
[0056] Furthermore, the present invention relates to regulation of
ATF5 expression. In particular, the inventors' findings indicate
that ATF5 expression is regulated by neurotrophic factors, and,
therefore, is an essential part of the mechanism by which they
promote neuronal differentiation.
[0057] Accordingly, the present invention provides a method for
promoting differentiation of a stem cell or a neural progenitor
cell into a differentiated cell, as well as a differentiated cell
produced by this method. Differentiation is the cellular process by
which cells become structurally and functionally specialized during
development. As used herein, the term "promoting differentiation"
means activating, enhancing, inducing, initiating, or stimulating
differentiation of a stem cell or a progenitor cell. The stem cell
can be a neural stem cell and the progenitor cell can be a neural
progenitor cell.
[0058] Neural stem cells, for example, are cultured cells, derived
from the pluripotent inner cell mass of blastocyst stage embryos,
that are capable of replicating indefinitely. In general, neural
cells have the potential to differentiate into neural cells (i.e.,
they are pluripotent); thus, they may serve as a continuous source
of new neural cells. The neural stem cell of the present invention
may be obtained from any animal, but is preferably obtained from a
mammal (e.g., human, domestic animal, or commercial animal). In one
embodiment of the present invention, the neural stem cell is a
murine neural stem cell. In another, preferred, embodiment, the
neural stem cell is obtained from a human.
[0059] As used herein, a "differentiated neural cell" is a
partially-differentiated or fully-differentiated cell of the
central nervous system (CNS) or peripheral nervous system (PNS),
and includes, without limitation, a fully-differentiated ganglion
cell, glial (or neuroglial) cell (e.g., an astrocyte, astroglial
cell, oligodendrocyte, oligodendroglial cell, or Schwann cell),
granule cell, neuronal cell (or neuron), and stellate cell, as well
as any neural progenitor cells thereof. Progenitor cells are parent
cells which, during development and differentiation, give rise to a
distinct cell lineage by a series of cell divisions. Neural
progenitor cells, for example, are committed to a cell lineage that
will develop, eventually, into fully-differentiated neural cells of
the CNS or PNS; however, such neural progenitor cells may not yet
be dedicated to a particular type, or subclass, of neural cell.
[0060] Initially, neural progenitor cells may acquire a rostral
character (e.g., rostral neural progenitor cells), followed by a
positional identity (e.g., cerebellar progenitor cells, cerebral
progenitor cells, or spinal progenitor cells). Such
partially-differentiated neural progenitor cells may become
committed to a cell line that will differentiate into a specific
type of neural cell (e.g., progenitor cells of astrocytes,
astroglial cells, ganglion cells, granule cells, neurons,
oligodendrocytes, oligodendroglial cells, Schwann cells, or
stellate cells), and, thereafter, give rise to fully-differentiated
neural cells (e.g., astrocytes, astroglial cells, ganglion cells,
granule cells, neurons, oligodendrocytes, oligodendroglial cells,
Schwann cells, or stellate cells). Accordingly, the
partially-differentiated neural cell of the present invention may
be a cell, with a neural identity, that has acquired a directional
or positional character, or that has committed to developing into a
particular class of neural cell, but is not a fully-differentiated
neural cell.
[0061] The neural progenitor cell of the present invention may be
obtained from any animal, but is preferably obtained from a mammal
(e.g., human, domestic animal, or commercial animal). In one
embodiment of the present invention, the neural progenitor cell is
a murine neural progenitor cell. In another, preferred, embodiment,
the neural progenitor cell is obtained from a human.
[0062] A "neuronal cell", or "neuron", as used herein, is a
conducting or nerve cell of the nervous system that typically
consists of a cell body (perikaryon) that contains the nucleus and
surrounding cytoplasm; several short, radiating processes
(dendrites); and one long process (the axon), which terminates in
twig-like branches (telodendrons), and which may have branches
(collaterals) projecting along its course. Examples of neurons
include, without limitation, cerebellar neurons, or neurons from
the cerebellum (e.g., basket cells, Golgi cells, granule cells,
Purkinje cells, and stellate cells); cortical neurons, or neurons
from the cerebral cortex (e.g., pyramidal cells and stellate cells,
including interneurons, midbrain neurons, and neurons of the
substantia nigra); hippocampal cells, or cells from the hippocampus
(including granule cells); cells of the Pons; neurons of the dorsal
root ganglia (DRG); motor neurons; peripheral neurons; sensory
neurons; neurons of the spinal cord; ventral interneurons; and
primary neurons (neurons taken directly from the brain, and, in
general, placed into a tissue culture dish), all of which may be
cholinergic, dopaminergic, GABAergic, or serotonergic.
[0063] Differentiation of neural stem cells and neural progenitor
cells into partially- or fully-differentiated neural cells may be
detected by known cellular or molecular procedures, and assays and
methods disclosed herein. In one embodiment of the present
invention, the differentiated neural cell is a post-mitotic neuron.
The term "post-mitotic", as used herein, refers to a neuron that is
in G0 phase (a quiescent state), and is no longer dividing or
cycling. In another embodiment of the present invention, the
differentiated neural cell is marked, in that it expresses enhanced
green fluorescent protein (eGFP), as described herein. The eGFP
exogenous reporter may be particularly useful in a method for
isolating and/or purifying a population of differentiated neural
cells, as described below.
[0064] The method of the present invention comprises inhibiting
ATF5 in a stem cell, progenitor cell or tumor cell. As used herein,
"ATF5" includes both an "ATF5 protein" and an "ATF5 analogue".
Unless otherwise indicated, "protein" shall include a protein,
protein domain, polypeptide, or peptide, and any fragment thereof.
The ATF5 protein has the amino acid sequence set forth in FIG. 9,
including conservative substitutions thereof. As described below,
Western immunoblotting permitted the inventors to deduce the major
cellular form of ATF5 protein. The ATF5 cDNA sequence predicts two
potential in-frame methionine start sites that would lead to
proteins of approximately 30 and 20 kDa. The inventors' observation
that the major form of ATF5 in cells has an apparent molecular mass
of 20-22 kDa indicates favored utilization of the second site. When
a canonical Kozak initiation consensus sequence was included
upstream of the first methionine, the larger protein was expressed,
thereby indicating that the 22-kDa form is not formed by cleavage
of a 30-kDa precursor. Accordingly, the ATF5 protein of the present
invention further includes both the 22-kDa and 30-kDa isomers
thereof.
[0065] As used herein, "conservative substitutions" are those amino
acid substitutions which are functionally equivalent to a
substituted amino acid residue, either because they have similar
polarity or steric arrangement, or because they belong to the same
class as the substituted residue (e.g., hydrophobic, acidic, or
basic). The term "conservative substitutions" includes
substitutions having an inconsequential effect on the ability of
ATF5 to interact with CRE, particularly in respect of the use of
said interaction for the identification and design of agonists of
ATF5, for molecular replacement analyses, and/or for homology
modeling.
[0066] An "ATF5 analogue", as used herein, is a functional variant
of the ATF5 protein, having ATF5 biological activity, that has 60%
or greater (preferably, 70% or greater) amino-acid-sequence
homology with the ATF5 protein. As further used herein, the term
"ATF5 biological activity" refers to the activity of a protein or
peptide that demonstrates an ability to associate physically with,
or bind with, CRE (i.e., binding of approximately two fold, or,
more preferably, approximately five fold, above the background
binding of a negative control), under the conditions of the assays
described herein, although affinity may be different from that of
ATF5.
[0067] it will be obvious to the skilled practitioner that the
numbering of amino acid residues in ATF5, or in the ATF5 analogues
or peptidomimetics covered by the present invention, may be
different than that set forth herein, or may contain certain
conservative amino acid substitutions that produce the same
ATF5-CRE associating activity as that described herein.
Corresponding amino acids and conservative substitutions in other
isoforms or analogues are easily identified by visually inspecting
the relevant amino acid sequences, or by using commercially
available homology software programs.
[0068] In accordance with methods described herein, ATF5 may be
inhibited in a stem cell, progenitor cell or neoplastic cell by
disabling, disrupting, or inactivating the function or activity of
ATF5 in the cell, or by diminishing the amount or level of ATF5 in
the cell. For example, ATF5 in a cell may be inhibited by targeting
ATF5 directly. Additionally, activity of ATF5 in a cell may be
inhibited indirectly, by targeting an enzyme or other endogenous
molecule that regulates or modulates the functions or levels of
ATF5 in the cell. ATF5 expression may also be inhibited by
engineering the ATF5 gene so that ATF5 is expressed on an inducible
promoter. In such a case, ATF5 expression would be sustained in the
presence of a suitable inducing agent, but would shut down once the
supply of inducer was depleted, thereby resulting in a decrease in
the amount or level of ATF5 in the cell.
[0069] Preferably, activity of the ATF5 in the cell is inhibited or
decreased by at least 10% in the method of the present invention.
More preferably, activity of the ATF5 is decreased by at least 20%.
Activity of the ATF5 is inhibited in the stem, progenitor or tumor
cell by an amount effective to promote differentiation of the stem
cell, progenitor cell, tumor cell. This amount may be readily
determined by the skilled artisan, based upon known procedures,
including analysis of titration curves established in vivo, and
methods disclosed herein.
[0070] By way of example, activity of the ATF5 in a neuron may be
inhibited by directly or indirectly inactivating, interfering with,
or down-regulating the CRE-binding function of ATF5 in the neural
stem cell or neural progenitor cell (e.g., by the modulation or
regulation of proteins that interact with ATF5). The ATF5 in a
neural stem cell or neural progenitor cell may be inactivated, for
example, by contacting the neural stem cell or neural progenitor
cell with a small molecule or protein mimetic that inhibits ATF5 or
that is reactive with (i.e., has affinity for, binds to, or is
directed against) ATF5. Examples of methods for contacting the cell
with (treating the cell with) a molecule or protein mimetic
include, without limitation, absorption, electroporation,
immersion, injection, liposome delivery, transfection, vectors, and
other protein-delivery and nucleic-acid-delivery vehicles and
methods, as described below.
[0071] Activity of ATF5 in a neural stem cell or neural progenitor
cell also may be inhibited by directly or indirectly causing,
inducing, or stimulating the down-regulation of ATF5 expression
within the cell. Accordingly, in one embodiment of the present
invention, activity of ATF5 is inhibited in a neural stem cell or
neural progenitor cell by contacting the cell with a modulator of
ATF5 expression, in an amount effective to promote differentiation
of the cell. The modulator may be a protein, polypeptide, peptide,
nucleic acid (including DNA or RNA), antibody, Fab fragment,
F(ab').sub.2 fragment, molecule, compound, antibiotic, drug, or an
agent reactive with (i.e., has affinity for, binds to, or is
directed against) ATF5, that inhibits or down-regulates ATF5
expression. A Fab fragment is a univalent antigen-binding fragment
of an antibody, which is produced by papain digestion. An
F(ab').sub.2 fragment is a divalent antigen-binding fragment of an
antibody, which is produced by pepsin digestion.
[0072] Modulators of ATF5 may be identified using a simple
screening assay. For example, to screen for candidate modulators of
ATF5, neural progenitor cells may be plated onto microtiter plates,
then contacted with a library of drugs. Any resulting decrease in,
or down-regulation of, ATF5 expression then may be detected using a
luminescence reporter, nucleic acid hybridization, and/or
immunological techniques known in the art, including an ELISA.
Additional modulators of ATF5 expression may be identified using
screening procedures well known in the art or disclosed herein.
Modulators of ATF5 will include those drugs which inhibit or
down-regulate expression of ATF5. In this manner, candidate
modulators also may be screened for their ability to promote
differentiation of neural stem cells or neural progenitor cells,
and, therefore, their ability to treat neural tumors. as discussed
below.
[0073] In one embodiment of the present invention, ATF5 in a neural
stem cell or neural progenitor cell is inhibited by contacting the
cell with an ATF5 inhibitor. As used herein, "an ATF5 inhibitor"
shall include a protein, polypeptide, peptide, nucleic acid
(including DNA, RNA, and an antisense oligonucleotide), antibody
(monoclonal and polyclonal, as described above), Fab fragment (as
described above), F(ab').sub.2 fragment (as described above),
molecule, compound, antibiotic, drug, and any combinations thereof,
and may be an agent reactive with ATF5, as defined above. By way of
example, the ATF5 inhibitor of the present invention may be a
neurotrophic factor. As used herein, a "neurotrophic factor" is a
factor involved in the nutrition or maintenance of neural tissue.
Neurotrophic factors, may further the development and
differentiation of committed neural progenitor cells, or they may
induce or enhance the growth and survival of differentiated neural
cells. A classic example of a neurotrophic factor is NGF (nerve
growth factor). Other examples of neurotrophic factors for use in
the present invention include, without limitation, GDNF, NT3, CNTF,
and BDNF, as well as cognate receptors thereof (including TrkB and
TrkC). These factors may be obtained from R&D Systems, Inc.
(Minneapolis, Minn.).
[0074] Additionally, the ATF5 inhibitor of the present invention
may be an ATF5 transgene, comprising the ATF5 gene and an inducible
promoter, in the absence of a suitable inducer. In a cell
containing such a transgene, ATF5 expression would be sustained in
the presence of a suitable inducing agent; however, ATF5 expression
would be shut down once the supply of inducer was depleted. Thus,
an ATF5 transgene, comprising the ATF5 gene and an inducible
promoter, would, in the absence of a suitable inducer, effectively
bring about a decrease in the amount or level of ATF5 in the cell,
thereby functioning as an ATF5 inhibitor.
[0075] The ATF5 inhibitor of the present invention also may be an
interfering RNA, or RNAi, including ATF5 small interfering RNA
(siRNA), as disclosed herein. As used herein, "RNAi" refers to a
double-stranded RNA (dsRNA) duplex of any length, with or without
single-strand overhangs, wherein at least one strand, putatively
the antisense strand, is homologous to the target mRNA to be
degraded. As further used herein, a "double-stranded RNA" molecule
includes any RNA molecule, fragment, or segment containing two
strands forming an RNA duplex, notwithstanding the presence of
single-stranded overhangs of unpaired nucleotides. Additionally, as
used herein, a double-stranded RNA molecule includes
single-stranded RNA molecules forming functional stem-loop
structures, such that they thereby form the structural equivalent
of an RNA duplex with single-strand overhangs. The double-stranded
RNA molecule of the present invention may be very large, comprising
thousands of nucleotides; preferably, however, it is small, in the
range of 21-25 nucleotides. In a preferred embodiment, the RNAi of
the present invention comprises a double-stranded RNA duplex of at
least 19 nucleotides.
[0076] In one embodiment of the present invention, RNAi is produced
in vivo by an expression vector containing a gene-silencing
cassette coding for RNAi (see, e.g., U.S. Pat. No. 6,278,039, C.
elegans deletion mutants; U.S. Patent Application No. 2002/0006664,
Arrayed transfection method and uses related thereto; WO 99/32619,
Genetic inhibition by double-stranded RNA; WO 01/29058, RNA
interference pathway genes as tools for targeted genetic
interference; WO 01/68836, Methods and compositions for RNA
interference; and WO 01/96584, Materials and methods for the
control of nematodes). In another embodiment of the present
invention, RNAi is produced in vitro, synthetically or
recombinantly, and transferred into the microorganism using
standard molecular-biology techniques. Methods of making and
transferring RNAi are well known in the art. See, e.g., Ashrafi et
al., Genome-wide RNAi analysis of Caenorhabditis elegans fat
regulatory genes. Nature, 421:268-72, 2003; Cottrell et al.,
Silence of the strands: RNA interference in eukaryotic pathogens.
Trends Microbiol., 11:37-43, 2003; Nikolaev et al., Parc. A
Cytoplasmic Anchor for p53. Cell, 112:29-40, 2003; Wilda et al.,
Killing of leukemic cells with a BCR/ABL fusion gene RNA
interference (RNAi), Oncogene, 21:5716-24, 2002; Escobar et al.,
RNAi-mediated oncogene silencing confers resistance to crown gall
tumorigenesis. Proc. Natl. Acad Sci. USA, 98:13437-42, 2001; and
Billy et al., Specific interference with gene expression induced by
long, double-stranded RNA in mouse embryonal teratocarcinoma cell
lines. Proc. Natl. Acad Sci. USA, 98:14428-33, 2001.
[0077] Furthermore, the ATF5 inhibitor of the present invention may
be an oligonucleotide antisense to ATF5. Oligonucleotides antisense
to ATF5 may be designed based on the nucleotide sequence of ATF5,
which is readily available (FIG. 8). For example, a partial
sequence of the ATF5 nucleotide sequence (generally, 18-20 base
pairs), or a variation sequence thereof, may be selected for the
design of an antisense oligonucleotide. This portion of the ATF5
nucleotide sequence may be within the 5' domain. A nucleotide
sequence complementary to the selected partial sequence of the ATF5
gene, or the selected variation sequence, then may be chemically
synthesized using one of a variety of techniques known to those
skilled in the art, including, without limitation, automated
synthesis of oligonucleotides having sequences which correspond to
a partial sequence of the ATF5 nucleotide sequence, or a variation
sequence thereof, using commercially-available oligonucleotide
synthesizers, such as the Applied Biosystems Model 392 DNA/RNA
synthesizer.
[0078] Once the desired antisense oligonucleotide has been
prepared, its ability to inhibit ATF5 then may be assayed. For
example, the oligonucleotide antisense to ATF5 may be contacted
with neural progenitor cells, and the levels of ATF5 expression or
activity in the cells may be determined using standard techniques,
such as Western-blot analysis and immunostaining. Alternatively,
the antisense oligonucleotide may be delivered to neural progenitor
cells using a liposome vehicle, then the levels of ATF5 expression
or activity in the cells may be determined using standard
techniques, such as Western-blot analysis. Where the level of ATF5
expression in the cells is reduced in the presence of the designed
antisense oligonucleotide, it may be concluded that the
oligonucleotide could be a useful ATF5 inhibitor.
[0079] It is within the confines of the present invention that
oligonucleotide antisense to ATF5 or ATF5 interfering RNA (e.g.,
siRNA) may be linked to another agent, such as a drug or a
ribozyme, in order to increase the effectiveness of treatments
using ATF5 inhibition, increase the efficacy of targeting, and/or
increase the efficacy of degradation of ATF5 RNA. Examples of
antineoplastic drugs to which the antisense oligonucleotide may be
linked include, without limitation, carboplatin, cyclophosphamide,
doxorubicin, etoposide, and vincristine. Moreover, oligonucleotide
antisense to ATF5 may be prepared using modified bases (e.g., a
phosphorothioate) to make the oligonucleotide more stable and
better able to withstand degradation.
[0080] The ATF5 inhibitor of the present invention also may be a
dominant-negative form of the protein (e.g., NTAzip-ATF5), as
disclosed herein. In one embodiment, the dominant-negative form of
ATF5 is expressed on an inducible promoter.
[0081] Additional ATF5 inhibitors may be identified using screening
procedures well known in the art, and methods described below.
[0082] The present invention contemplates the use of proteins and
protein analogues generated by synthesis of polypeptides in vitro,
e.g., by chemical means or in vitro translation of mRNA. For
example, ATF5 and inhibitors thereof may be synthesized by methods
commonly known to one skilled in the art (Modern Techniques of
Peptide and Amino Acid Analysis (New York: John Wiley & Sons,
1981); Bodansky, M., Principles of Peptide Synthesis (New York:
Springer-Verlag New York, Inc., 1984). Examples of methods that may
be employed in the synthesis of the amino acid sequences, and
analogues of these sequences, include, but are not limited to,
solid-phase peptide synthesis, solution-method peptide synthesis,
and synthesis using any of the commercially-available peptide
synthesizers. The amino acid sequences of the present invention may
contain coupling agents and protecting groups, which are used in
the synthesis of protein sequences, and which are well known to one
of skill in the art.
[0083] A method of the present invention comprises inhibiting ATF5
in a stem cell or progenitor cell by contacting the cell with an
ATF5 inhibitor. The inhibitor is provided in an amount effective to
produce a differentiated cell. This amount may be readily
determined by the skilled artisan, based upon known procedures and
methods disclosed herein. The inventors have demonstrated herein
that neurons cultured in the presence of neurotrophic factors
survive and elaborate processes. Accordingly, in another
embodiment, the method of the present invention further comprises
the step of contacting a neural stem cell or neural progenitor cell
with at least one neurotrophic factor, contemporaneously with, or
following, inhibition of ATF5. The neurotrophic factors of the
present invention are provided in amounts effective to produce a
fully-differentiated neural cell of the CNS or PNS (e.g., a
neuron). These amounts may be readily determined by the skilled
artisan, based upon known procedures and methods disclosed
herein.
[0084] In the method of the present invention, neural stem cells or
neural progenitor cells may be contacted with effective amounts of
an ATF5 inhibitor and neurotrophic factors in vitro, or in vivo in
a subject. The inhibitor and factors may be contacted with a neural
stem cell or neural progenitor cell by introducing the inhibitor
and factors into the cell. Where contacting is effected in vitro,
the inhibitor and factors may be added directly to the culture
medium, as described herein. Alternatively, the inhibitor and
factors may be contacted with a neural stem cell or neural
progenitor cell in vivo in a subject, by introducing the inhibitor
and factors into the subject (e.g., by introducing the inhibitor
and factors into cells of the subject), or by administering the
inhibitor and factors to the subject. The subject may be any neural
or developed animal, but is preferably a mammal (e.g., a human,
domestic animal, or commercial animal). More preferably, the
subject is a human.
[0085] Where the inhibitor and factors are contacted with the cell
in vivo, the subject is preferably an embryo. However, it is within
the confines of the present invention for the cells to be
transplanted into a fully-grown human or animal subject, and for
the inhibitor and factors then to be administered to the human in
order to effect differentiation of the neural stem cells or neural
progenitor cells into differentiated neural cells in vivo in the
subject. The cells may be contained in nervous tissue of a subject,
and may be detected in nervous tissue of the subject by standard
detection methods readily determined from the known art, examples
of which include, without limitation, immunological techniques
(e.g., immunohistochemical staining), fluorescence imaging
techniques, and microscopic techniques.
[0086] The inhibitor and factors of the present invention may be
contacted with neural stem cells or neural progenitor cells, either
in vitro or in vivo in a subject, by known techniques used for the
introduction and administration of proteins, nucleic acids, and
other drugs. Examples of methods for contacting the cells with
(i.e., treating the cells with) an ATF5 inhibitor or a neurotrophic
factor (in protein or nucleic acid form) include, without
limitation, absorption, electroporation, immersion, injection,
introduction, liposome delivery, transfection, transfusion,
vectors, and other protein-delivery and nucleic-acid-delivery
vehicles and methods. When target cells are localized to a
particular portion of a subject, it may be desirable to introduce
the inhibitor and factors directly to the cells, by injection or by
some other means (e.g., by introducing the inhibitor and factors
into the blood or another body fluid).
[0087] Where the inhibitor or neurotrophic factor is a protein or
other molecule, it may be introduced into a neural stem cell or
neural progenitor cell directly, in accordance with conventional
techniques and methods disclosed herein. Additionally, a protein
inhibitor or factor may be introduced into a neural stem cell or
neural progenitor cell indirectly, by introducing into the cell a
nucleic acid encoding the inhibitor or factor, in a manner
permitting expression of the protein inhibitor or factor. The
inhibitor or factor may be introduced into neural stem cells or
neural progenitor cells, in vitro or in vivo, using conventional
procedures known in the art, including, without limitation,
electroporation, DEAE Dextran transfection, calcium phosphate
transfection, monocationic liposome fusion, polycationic liposome
fusion, protoplast fusion, creation of an in vivo electrical field,
DNA-coated microprojectile bombardment, injection with recombinant
replication-defective viruses, homologous recombination, in viva
gene therapy, ex vivo gene therapy, viral vectors, and naked DNA
transfer, or any combination thereof. Recombinant viral vectors
suitable for gene therapy include, but are not limited to, vectors
derived from the genomes of such viruses as retrovirus, HSV,
adenovirus, adeno-associated virus, Semiliki Forest virus,
cytomegalovirus, lentivirus, and vaccinia virus. The amount of
nucleic acid to be used is an amount sufficient to express an
amount of protein effective to produce a differentiated neural
cell. These amounts may be readily determined by the skilled
artisan. It is also within the confines of the present invention
that a nucleic acid encoding a protein inhibitor or factor may be
introduced into suitable neural stem cells or neural progenitor
cells in vitro, using conventional procedures, to achieve
expression of the protein inhibitor or factor in the cells. Cells
expressing protein inhibitor or factor then may be introduced into
a subject to produce a differentiated neural cell in vivo.
[0088] In accordance with the method of the present invention, ATF5
inhibitors and neurotrophic factors may be administered to a human
or animal subject by known procedures, including, without
limitation, oral administration, parenteral administration, and
transdermal administration. Preferably, the inhibitors or factors
are administered parenterally, by intracranial, intraspinal,
intrathecal, or subcutaneous injection. The inhibitors and factors
of the present invention also may be administered to a subject in
accordance with any of the above-described methods for effecting in
vivo contact between neural stem cells/neural progenitor cells and
ATF5 inhibitors/neurotrophic factors.
[0089] For oral administration, an inhibitor or factor formulation
may be presented as capsules, tablets, powders, granules, or as a
suspension. The formulation may have conventional additives, such
as lactose, mannitol, corn starch, or potato starch. The
formulation also may be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch, or gelatins.
Additionally, the formulation may be presented with disintegrators,
such as corn starch, potato starch, or sodium
carboxymethylcellulose. The formulation also may be presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation may be presented with lubricants, such as
talc or magnesium stearate.
[0090] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal), an
inhibitor or factor may be combined with a sterile aqueous solution
that is preferably isotonic with the blood of the subject. Such a
formulation may be prepared by dissolving a solid active ingredient
in water containing physiologically-compatible substances, such as
sodium chloride, glycine, and the like, and having a buffered pH
compatible with physiological conditions, so as to produce an
aqueous solution, then rendering said solution sterile. The
formulation may be presented in unit or multi-dose containers, such
as sealed ampoules or vials. The formulation may be delivered by
any mode of injection, including, without limitation, epifascial,
intracapsular, intracranial, intracutaneous, intrathecal,
intramuscular, intraorbital, intraperitoneal, intraspinal,
infrasternal, intravascular, intravenous, parenchymatous,
subcutaneous, or sublingual.
[0091] For transdermal administration, an inhibitor or factor may
be combined with skin penetration enhancers, such as propylene
glycol, polyethylene glycol, isopropanol, ethanol, oleic acid,
N-methylpyrrolidone, and the like, which increase the permeability
of the skin to the inhibitor or factor, and permit the inhibitor or
factor to penetrate through the skin and into the bloodstream. The
inhibitor/enhancer or factor/enhancer compositions also may be
further combined with a polymeric substance, such as
ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate,
polyvinyl pyrrolidone, and the like, to provide the composition in
gel form, which may be dissolved in solvent, such as methylene
chloride, evaporated to the desired viscosity, and then applied to
backing material to provide a patch.
[0092] The present invention provides a method for promoting
differentiation of neural stem cells or neural progenitor cells
into differentiated neural cells, and for purifying and isolating
the neural cells so generated using enhanced green fluorescent
protein (eGFP) as a genetic marker. The method described herein for
promoting differentiation of neural stem cells or neural progenitor
cells in vitro provides a source of neurons, or other neural cells
of the CNS or PNS, that are available for transplant into a
subject. Thus, this method is particularly useful for producing
neural cells for use in treating conditions associated with nervous
tissue degeneration.
[0093] The term "nervous tissue", as used herein, refers to tissue
of the nervous system, which includes the differentiated neural
cells of the present invention and progenitors thereof. As further
used herein, "nervous tissue degeneration" means a condition of
deterioration of nervous tissue, wherein the nervous tissue changes
to a lower or less functionally-active form. It is believed that,
by promoting differentiation of neural stem cells or neural
progenitor cells, the method described herein will be useful in
repopulating various injured and/or degenerated nervous tissues in
a subject, through production of differentiated neural cells and
subsequent transplant thereof into a subject in need of such
transplantation.
[0094] Accordingly, the present invention provides a method for
treating nervous tissue degeneration in a subject in need of
treatment for nervous tissue degeneration, comprising promoting
differentiation of neural stem cells or neural progenitor cells
into differentiated neural cells, in accordance with the methods
described herein, and transplanting the differentiated neural cells
into the subject, thereby treating the nervous tissue degeneration.
By way of example, the method of the present invention may comprise
the following steps: (a) obtaining or generating a culture of
neural stem cells or neural progenitor cells; (b) contacting the
culture of neural stem cells or neural progenitor cells with an
amount of an ATF5 inhibitor effective to produce differentiated
neural cells; (c) optionally, contacting the differentiated neural
cells with at least one neurotrophic factor; and (d) transplanting
the differentiated neural cells into the subject, in an amount
effective to treat the nervous tissue degeneration. In one
embodiment of the invention, the subject is an embryo. In another
embodiment of the invention, the subject is a human. Preferably,
the subject has nervous tissue degeneration.
[0095] Nervous tissue degeneration may arise in the CNS or PNS, and
may be caused by, or associated with, a variety of disorders,
conditions, and factors, including, without limitation, primary
neurologic conditions (e.g., neurodegenerative diseases),
demyelinating conditions, CNS and PNS traumas and injuries, and
acquired secondary effects of non-neural dysfunction (e.g., neural
loss secondary to degenerative, pathologic, or traumatic events).
Examples of CNS traumas include, without limitation, blunt trauma,
hypoxia, and invasive trauma. Examples of acquired secondary
effects of non-neural dysfunction include, without limitation,
cerebral palsy, congenital hydrocephalus, muscular dystrophy,
stroke, and vascular dementia, as well as neural degeneration
resulting from any of the following: an injury associated with
cerebral hemorrhage, developmental disorders (e.g., a defect of the
brain, such as congenital hydrocephalus, or a defect of the spinal
cord, such as spina bifida), diabetic encephalopathy, hypertensive
encephalopathy, intracranial aneurysms, ischemia, kidney
dysfunction, subarachnoid hemorrhage, trauma to the brain and
spinal cord, treatment by such therapeutic agents as chemotherapy
agents and antiviral agents, vascular lesions of the brain and
spinal cord, and other diseases or conditions prone to result in
nervous tissue degeneration.
[0096] In one embodiment of the present invention, the nervous
tissue degeneration is a peripheral neuropathy in the PNS. As
defined herein, the term "peripheral neuropathy" refers to a
syndrome of sensory loss, muscle weakness, muscle atrophy,
decreased deep-tendon reflexes, and/or vasomotor symptoms. In a
subject who has a peripheral neuropathy, myelin sheaths (or Schwann
cells) may be primarily affected, or axons may be primarily
affected. The peripheral neuropathy may affect a single nerve
(mononeuropathy), two or more nerves in separate areas (multiple
mononeuropathy), or many nerves simultaneously
(polyneuropathy).
[0097] Examples of peripheral neuropathies that may be treated by
the methods disclosed herein include, without limitation,
peripheral neuropathies associated with acute or chronic
inflammatory polyneuropathy, amyotrophic lateral sclerosis (ALS),
collagen vascular disorder (e.g., polyarteritis nodosa, rheumatoid
arthritis, Sjogren's syndrome, or systemic lupus erythematosus),
diphtheria, Guillain-Barre syndrome, hereditary peripheral
neuropathy (e.g., Charcot-Marie-Tooth disease (including type I,
type II, and all subtypes), hereditary motor and sensory neuropathy
(types I, II, and III, and peroneal muscular atrophy), hereditary
neuropathy with liability to pressure palsy (HNPP), infectious
disease (e.g., acquired immune deficiency syndrome (AIDS)), Lyme
disease (e.g., infection with Borrelia burgdorferi), invasion of a
microorganism (e.g., leprosy--the leading cause of peripheral
neuropathy worldwide, after neural trauma), leukodystrophy,
metabolic disease or disorder (e.g., amyloidosis, diabetes
mellitus, hypothyroidism, porphyria, sarcoidosis, or uremia),
neurofibromatosis, nutritional deficiencies, paraneoplastic
disease, peroneal nerve palsy, polio, porphyria, postpolio
syndrome, Proteus syndrome, pressure paralysis (e.g., carpal tunnel
syndrome), progressive bulbar palsy, radial nerve palsy, spinal
muscular atrophy (SMA), a toxic agent (e.g., barbital, carbon
monoxide, chlorobutanol, dapsone, emetine, heavy metals,
hexobarbital, lead, nitrofurantoin, orthodinitrophenal, phenytoin,
pyridoxine, sulfonamides, triorthocresyl phosphate, the vinca
alkaloids, many solvents, other industrial poisons, and certain
AIDS drugs (including didanosine and zalcitabine), trauma
(including neural trauma--the leading cause of peripheral
neuropathy, worldwide), and ulnar nerve palsy (Beers and Berkow,
eds., The Merck Manual of Diagnosis and Therapy, 17.sup.th ed.
(Whitehouse Station, N.J.: Merck Research Laboratories, 1999) chap.
183). In a preferred embodiment of the present invention, the
peripheral neuropathy is ALS or SMA.
[0098] In another embodiment of the present invention, the nervous
tissue degeneration is a neurodegenerative disease. Examples of
neurodegenerative diseases that may be treated by the methods
disclosed herein include, without limitation, Alzheimer's disease,
amyotrophic lateral sclerosis (Lou Gehrig's Disease), Binswanger's
disease, Huntington's chorea, multiple sclerosis, myasthenia
gravis, Parkinson's disease, and Pick's disease.
[0099] It is also within the confines of the present invention for
the method described herein to be used to treat nervous tissue
degeneration that is associated with a demyelinating condition.
Demyelinating conditions are manifested in loss of myelin--the
multiple dense layers of lipids and protein which cover many nerve
fibers. These layers are provided by oligodendroglia in the CNS,
and Schwann cells in the PNS. In patients with demyelinating
conditions, demyelination may be irreversible; it is usually
accompanied or followed by axonal degeneration, and often by
cellular degeneration. Demyelination can occur as a result of
neuronal damage or damage to the myelin itself--whether due to
aberrant immune responses, local injury, ischemia, metabolic
disorders, toxic agents, or viral infections.
[0100] Central demyelination (demyelination of the CNS) occurs in
several conditions, often of uncertain etiology, that have come to
be known as the primary demyelinating diseases. Of these, multiple
sclerosis is the most prevalent. Other primary demyelinating
diseases include adrenoleukodystrophy (ALD), adrenomyeloneuropathy,
AIDS-vacuolar myelopathy, HTLV-associated myelopathy, Leber's
hereditary optic atrophy, progressive multifocal
leukoencephalopathy (PML), subacute sclerosing panencephalitis, and
tropical spastic paraparesis. In addition, there are acute
conditions in which demyelination can occur in the CNS, e.g., acute
disseminated encephalomyelitis (ADEM) and acute viral encephalitis.
Furthermore, acute transverse myelitis, a syndrome in which an
acute spinal cord transection of unknown cause affects both gray
and white matter in one or more adjacent thoracic segments, can
also result in demyelination. Finally, there are animal models
which mimic features of human demyelinating diseases. Examples
include experimental autoimmune neuritis (EAN), demyelination
induced by Theiler's virus, and experimental autoimmune
encephalomyelitis (EAE)--an autoimmune disease which is
experimentally induced in a variety of species and which resembles
MS in its clinical and neuropathological aspects.
[0101] The differentiated neural cells of the present invention may
be transplanted into a subject in need of treatment by standard
procedures known in the art, as well as the methods described
herein. By way of example, neural stem cells or neural progenitor
cells may be induced with an ATF5 inhibitor, to produce
differentiated neural cells. At an appropriate time post-induction
(e.g., 3-4 days after induction), the cells may be prepared for
transplantation (e.g., partially triturated), and then transplanted
into a subject (e.g., into the spinal cord of a chick, HH stage
15-17). To accommodate transplanted tissue, the subject may be
suction-lesioned prior to implantation. In one embodiment of the
present invention, the differentiated neural cells are transplanted
into the spinal cord of a subject, thereby repopulating the
subject's spinal cord, and the nervous tissue degeneration is a
peripheral neuropathy associated with ALS or SMA.
[0102] In another embodiment of the present invention, the neural
stem cells or neural progenitor cells contain an ATF5 transgene
that has been engineered to express ATF5 on an inducible promoter.
In this embodiment of the present invention, ATF5 may be expressed
in the presence of a suitable inducing agent, thereby permitting
propagation of the neural stem cells or neural progenitor cells in
vitro. Once the cells are transplanted into the subject, however,
the inducing agent would be withdrawn, resulting in decreased ATF5
expression, and thereby promoting differentiation of the
transplanted cells. Expression of ATF5 would be sustained in the
presence of the inducer, and would be shut down once the supply of
inducer was depleted (e.g., upon transplant into a subject).
[0103] In an alternative embodiment, a dominant-negative form of
ATF5 (an ATF5 inhibitor) may be introduced into the neural stem
cells or neural progenitor cells on an inducible promoter. The
transgene could be maintained in an uninduced state in vitro,
permitting propagation of the cells, and then induced with a
suitable inducing agent, in vivo in a subject, thereby promoting
differentiation of the neural stem cells or neural progenitor
cells.
[0104] In the method of the present invention, the differentiated
neural cells are transplanted into a subject in need of treatment
in an amount effective to treat the nervous tissue degeneration. As
used herein, the phrase "effective to treat the nervous tissue
degeneration" effective to ameliorate or minimize the clinical
impairment or symptoms of the nervous tissue degeneration. For
example, where the nervous tissue degeneration is a peripheral
neuropathy, the clinical impairment or symptoms of the peripheral
neuropathy may be ameliorated or minimized by alleviating vasomotor
symptoms, increasing deep-tendon reflexes, reducing muscle atrophy,
restoring sensory function, and strengthening muscles. The amount
of differentiated neural cells effective to treat nervous tissue
degeneration in a subject in need of treatment will vary depending
upon the particular factors of each case, including the type of
nervous tissue degeneration, the stage of the nervous tissue
degeneration, the subject's weight, the severity of the subject's
condition, the type of differentiated neural cells, and the method
of transplantation. This amount may be readily determined by the
skilled artisan, based upon known procedures, including clinical
trials, and methods disclosed herein.
[0105] In view of the above-described method for promoting
differentiation of neural stem cells and neural progenitor cells
into differentiated neural cells, the present invention further
provides a method for producing differentiated neural cells,
comprising the steps of: (a) obtaining or generating a culture of
neural stem cells or neural progenitor cells; (b) contacting the
culture of neural stem cells or neural progenitor cells with an
amount of an ATF5 inhibitor effective to produce a subclass of
differentiated neural cells; and (c) optionally, contacting the
differentiated neural cells with at least one neurotrophic factor.
The present invention also provides a population of cells,
comprising the differentiated neural cells produced by this method.
In one embodiment, some or all of the cells express eGFP.
[0106] In the method of the present invention, any of steps (b)-(c)
may be performed in vitro, or in vivo in a subject. Following any
in vitro steps, cells may be transplanted into a subject such that
the remaining steps are performed in vivo. Accordingly, the method
of the present invention further comprises the step of
transplanting the neural progenitor cells or the differentiated
neural cells into a subject. For example, the neural stem cells or
neural progenitor cells may contain an ATF5 transgene that has been
engineered to express ATF5 on an inducible promoter. In this
embodiment of the present invention, ATF5 would be expressed in the
presence of a suitable inducing agent, thereby permitting
propagation of the neural stem cells or neural progenitor cells in
vitro. Thereafter, the cells may be transplanted into a subject,
such that steps (b) and (c) are carried out in vivo. Because the
inducing agent would be withdrawn upon transplantation of the cells
into the subject, ATF5 expression would be decreased, thereby
promoting differentiation of the transplanted cells. Similarly, a
culture of neural stem cells or neural progenitor cells may be
contacted with an ATF5 inhibitor in vitro, to produce
differentiated neural cells. The neural cells so produced then may
be transplanted into a subject, such that step (c) is carried out
in vivo. In an alternative method, a culture of neural stem cells
or neural progenitor cells may be contacted with an ATF5 inhibitor
in vitro, to produce differentiated neural cells; and, optionally,
the differentiated neural cells may be contacted with at least one
neurotrophic factor in vitro. The differentiated neural cells then
may be transplanted into a subject. In one embodiment of the
present invention, the neurons are transplanted into the spinal
cord of the subject.
[0107] Because the selective degeneration of specific classes of
CNS neurons underlies many neurological disorders, research into
the growth, survival, and activity of neurons remains a priority.
Unfortunately, however, live neurons are not readily available for
such studies. For this reason, the present invention will be of
particular importance to researchers in the fields of neuroscience
and neurology, as it provides a potentially-unlimited source of
neural cells to be studied. Accordingly, the present invention also
provides for uses of the above-described neural progenitor cells
and differentiated neural cells in particular areas of
research.
[0108] The neural progenitor cells and differentiated neural cells
of the present invention will be useful in the analysis of neuron
development, function, and death--research which is critical to a
complete understanding of neurological diseases. Furthermore, the
neural progenitor cells and differentiated neural cells of the
present invention will be useful in monitoring synaptic
differentiation at sites of contact with target muscles. Finally,
the neural progenitor cells and differentiated neural cells of the
present invention will facilitate a direct comparison of normal,
healthy neurons with degenerated neurons. For such a comparison,
both the healthy and the diseased neural cells may be produced
using well-known techniques and methods described herein.
[0109] The present invention further provides a method for
isolating a pure population of differentiated neural cells and/or
purifying a population of differentiated neural cells, comprising
the steps of: (a) obtaining or generating a culture of neural stem
cells or neural progenitor cells that express enhanced green
fluorescent protein (eGFP); (b) contacting the culture of neural
stem cells or neural progenitor cells with an amount of an ATF5
inhibitor effective to produce differentiated neural cells, wherein
some or all of the differentiated neural cells also express eGFP;
(c) optionally, contacting the differentiated neural cells with at
least one neurotrophic factor; (d) detecting expression of eGFP in
the differentiated neural cells; and (e) isolating the
differentiated neural cells that express eGFP. Neural stem cells or
neural progenitor cells that express eGFP may be made in accordance
with methods disclosed herein.
[0110] According to the method of the present invention, expression
of eGFP may be detected in differentiated neural cells by either in
vitro or in vivo assay. As used herein, "expression" refers to the
transcription of the eGFP gene into at least one mRNA transcript,
or the translation of at least one mRNA into an eGFP protein. The
differentiated neural cells may be assayed for eGFP expression by
assaying for eGFP protein, eGFP eDNA, or eGFP mRNA. The appropriate
form of eGFP will be apparent based on the particular techniques
discussed herein.
[0111] Differentiated neural cells may be assayed for eGFP
expression, and eGFP expression may be detected in differentiated
neural cells, using assays and detection methods well known in the
art. Because eGFP provides a non-invasive marker for labeling cells
in culture and in vivo, expression of eGFP is preferably detected
in differentiated neural cells using various imaging techniques
such as phase, and fluorescence imaging techniques, as disclosed
herein. Differentiated neural cells expressing high levels of eGFP
then may be isolated from a cell suspension by sorting (e.g., by
FACS sorting, using a Beckman-Coulter Altra flow cytometer), based
upon their eGFP fluorescence and forward light scatter, as
described below.
[0112] Other methods also may be used to detect eGFP expression in
the differentiated neural cells of the present invention. Examples
of such detection methods include, without limitation,
hybridization analysis, imaging techniques, immunological
techniques, immunoprecipitation, radiation detection, Western-blot
analysis, and any additional assays or detection methods disclosed
herein. For example, differentiated neural cells may be assayed for
eGFP expression using an agent reactive with eGFP protein or eGFP
nucleic acid. As used herein, "reactive" means the agent has
affinity for, binds to, or is directed against eGFP. As further
used herein, an "agent" shall include a protein, polypeptide,
peptide, nucleic acid (including DNA or RNA), antibody, Fab
fragment, F(ab').sub.2 fragment, molecule, compound, antibiotic,
drug, and any combinations thereof. In one embodiment of the
present invention, the agent reactive with eGFP is an antibody
(e.g., .alpha.GFP (Molecular Probes, Inc., Eugene, Oreg.; BD
Biosciences Clontech, Palo Alto, Calif.)).
[0113] Following detection of eGFP expression in differentiated
neural cells, the extent of eGFP expression in the cells may be
measured or quantified, if desired, using one of various
quantification assays. Such assays are well known to one of skill
in the art, and may include immunohistochemistry,
immunocytochemistry, flow cytometry, mass spectroscopy,
Western-blot analysis, or an ELISA for measuring amounts of eGFP
protein.
[0114] The present invention further provides a method for
identifying an agent for use in treating a condition associated
with nervous tissue degeneration, as defined above. Examples of
conditions associated with nervous tissue degeneration include
peripheral neuropathies, demyelinating conditions, and the primary
neurologic conditions (e.g., neurodegenerative diseases), CNS and
PNS traumas and injuries, and acquired secondary effects of
non-neural dysfunction (e.g., neural loss secondary to
degenerative, pathologic, or traumatic events) described
herein.
[0115] The method of the present invention comprises the steps of:
(a) obtaining or generating a culture of neural stem cells or
neural progenitor cells; (b) contacting the culture of cells with
an amount of an ATF5 inhibitor effective to produce neurons,
wherein some or all of the neurons are degenerated; (c) contacting
the degenerated neurons with a candidate agent; and (d) determining
if the agent enhances regeneration or survival of some or all of
the degenerated neurons. As used herein, the term "enhance
regeneration" means augment, improve, or increase partial or full
growth (or regrowth) of a neuron (including neurites and the myelin
sheath) that has degenerated. As further used herein, the term
"growth" refers to an increase in diameter, length, mass, and/or
thickness of a neuron (including neurites and the myelin sheath).
Regeneration of the neuron may take place in neurons of both the
central nervous system and the peripheral nervous system.
Additionally, as used herein, the term "enhance survival" of a
neuron means increasing the duration of the neuron's viable
lifespan, either in vitro or in vivo. In one embodiment of the
present invention, the agent enhances regeneration or survival of
degenerated motor neurons.
[0116] In the method of the present invention, degenerated neurons
may be contacted with a candidate agent by any of the methods of
effecting contact between inhibitors or factors or agents and
cells, and any modes of introduction and administration, described
herein. Regeneration, and enhanced regeneration, of neurons may be
measured or detected by known procedures, including Western
blotting for myelin-specific and axon-specific proteins, electron
microscopy in conjunction with morphometry, and any of the methods,
molecular procedures, and assays known to one of skill in the art.
In addition, growth of myelin may be assayed using the g-ratio--one
measure of the integrity of the axon:myelin association. The
g-ratio is defined as the axonal diameter divided by the total
diameter of the axon and myelin. This ratio provides a reliable
measure of relative myelination for an axon of any given size
(Bieri et al., Abnormal nerve conduction studies in mice expressing
a mutant faun of the POU transcription factor, SCIP. J. Neurosci.
Res., 50:821-28, 1997). Numerous studies have documented that a
g-ratio of 0.6 is normal for most fibers (Waxman and Bennett,
Relative conduction velocities of small myelinated and
nonmyelinated fibres in the central nervous system. Nature New
Biol., 238:217, 1972). In one embodiment of the present invention,
the degenerated neurons express enhanced green fluorescent protein
(eGFP). It is expected that such neurons will allow for inhibited
high-throughput drug screening.
[0117] The inventors have demonstrated herein that neuronal
differentiation may be induced by CRE-mediated gene activation, and
that such activation is repressed in neural progenitor cells by
factors such as ATF5. Accordingly, the present invention further
provides a method for suppressing differentiation of neural stem
cells or neural progenitor cells into differentiated neural cells,
where such cells might otherwise be determined to differentiate.
The method of the present invention comprises contacting the neural
stem cells or neural progenitor cells with an amount of ATF5, or a
peptidomimetic thereof, effective to suppress differentiation in
the neural stem cells or neural progenitor cells. This method will
permit a pool of these undifferentiated cells to be generated under
conditions in which they might otherwise differentiate and cease
proliferation. The ATF5 or mimetic may be in the form of a protein,
or a nucleic acid encoding the protein, and may be contacted with
the cells in accordance with methods previously described.
[0118] The present invention also provides therapeutic
compositions, comprising a nucleic acid encoding an ATF5 inhibitor,
a vector, and, optionally, a pharmaceutically-acceptable carrier.
The pharmaceutically-acceptable carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
composition, and not deleterious to the recipient thereof. The
pharmaceutically-acceptable carrier employed herein is selected
from various organic or inorganic materials that are used as
materials for pharmaceutical formulations, and which may be
incorporated as analgesic agents, buffers, binders, disintegrants,
diluents, emulsifiers, excipients, extenders, glidants,
solubilizers, stabilizers, suspending agents, tonicity agents,
vehicles, and viscosity-increasing agents. If necessary,
pharmaceutical additives, such as antioxidants, aromatics,
colorants, flavor-improving agents, preservatives, and sweeteners,
may also be added. Examples of acceptable pharmaceutical carriers
include carboxymethyl cellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methyl cellulose, powders,
saline, sodium alginate, sucrose, starch, talc, and water, among
others.
[0119] The formulations of the present invention may be prepared by
methods well-known in the pharmaceutical arts. For example, the
ATF5 inhibitor protein or nucleic acid may be brought into
association with a carrier or diluent, as a suspension or solution.
Optionally, one or more accessory ingredients (e.g., buffers,
flavoring agents, surface active agents, and the like) also may be
added. The choice of carrier will depend upon the route of
administration. The pharmaceutical composition would be useful for
administering the ATF5 inhibitor of the present invention to a
subject to treat a neural tumor, as discussed below. The ATF5
inhibitor is provided in an amount that is effective to treat the
neural tumor in a subject to whom the pharmaceutical composition is
administered. That amount may be readily determined by the skilled
artisan, as described above.
[0120] As disclosed herein, the inventors have determined that ATF5
expression is elevated in neural and other tumor types, including
but not limited to breast, ovary, endometrium, gastric, colon,
liver, pancrease, kidney, bladder, prostate, testis, skin,
esophagus, tongue, mouth, parotid, larynx, pharynx, lymph node,
lung, and brain tumors. The inventors have also demonstrated for
the first time that interfering with the function or expression of
ATF5 promotes apoptosis of glioblastoma multiforme tumors in vitro
and in vivo. Additionally, the inventors have shown for the first
time that selective interference with ATF5 function in other
carcinoma types, e.g., breast tumors, also triggers cell death.
Therefore, the pharmaceutical composition of the present invention
may be useful for treating a neural tumor in a subject. As used
herein, the term "tumor" refers to a pathologic proliferation of
cells, and includes a neoplasia. The term "neoplasia", and related
terms as further used herein, refers to the uncontrolled and
progressive multiplication of tumor cells under conditions that
would not elicit, or would cause cessation of, multiplication of
normal cells. Neoplasia results in the formation of a "neoplasm",
which is defined herein to mean any new and abnormal growth,
particularly a new growth of tissue, in which the growth of cells
is uncontrolled and progressive. As used herein, neoplasms include,
without limitation, morphological irregularities in cells in tissue
of a subject, as well as pathologic proliferation of cells in
tissue of a subject, as compared with normal proliferation in the
same type of tissue. Additionally, neoplasms include benign tumors
and malignant tumors. Malignant neoplasms are distinguished from
benign in that the former show a greater degree of anaplasia, or
loss of differentiation and orientation of cells, and have the
properties of invasion and metastasis. Thus, neoplasia includes
"cancer", which herein refers to a proliferation of tumor cells
having the unique trait of loss of normal controls, resulting in
unregulated growth, lack of differentiation, local tissue invasion,
and metastasis.
[0121] Additionally, as used herein, the term "neural tumor" refers
to a tumorigenic form of neural cells (i.e., transformed neural
cells), and includes astrocytoma cells (i.e., cells of all
astrocytomas, including, without limitation, Grades I-IV
astrocytomas, anaplastic astrocytoma, astroblastoma, astrocytoma
fibrillare, astrocytoma protoplasmaticum, gemistocytic astrocytoma,
and glioblastoma multiforme), gliomas, medulloblastomas,
neuroblastomas, and other brain tumors. Brain tumors invade and
destroy normal tissue, producing such effects as impaired
sensorimotor and cognitive function, increased intracranial
pressure, cerebral edema, and compression of brain tissue, cranial
nerves, and cerebral vessels. Metastases may involve the skull or
any intracranial structure. The size, location, rate of growth, and
histologic grade of malignancy determine the seriousness of brain
tumors. Nonmalignant tumors grow slowly, with few mitoses, no
necrosis, and no vascular proliferation. Malignant tumors grow more
rapidly, and invade other tissues. However, they rarely spread
beyond the CNS, because they cause death by local growth.
Drowsiness, lethargy, obtuseness, personality changes, disordered
conduct, and impaired mental faculties are the initial symptoms in
25% of patients with malignant brain tumors.
[0122] Brain tumors may be classified by site (e.g., brain stem,
cerebellum, cerebrum, cranial nerves, ependyma, meninges,
neuroglia, pineal region, pituitary gland, and skull) or by
histologic type (e.g., meningioma, primary CNS lymphoma, or
astrocytoma). Common primary childhood tumors are cerebellar
astrocytomas and medulloblastomas, ependymomas, gliomas of the
brain stem, neuroblastomas, and congenital tumors. In adults,
primary tumors include meningiomas, schwannomas, and gliomas of the
cerebral hemispheres (particularly the malignant glioblastoma
multiforme and anaplastic astrocytoma, and the more benign
astrocytoma and oligodendroglioma). Overall incidence of
intracranial neoplasms is essentially equal in males and females,
but cerebellar medulloblastoma and glioblastoma multiforme are more
common in males.
[0123] Gliomas are tumors composed of tissue representing neuroglia
in any one of its stages of development. They account for 45% of
intracranial tumors. Gliomas can encompass all of the primary
intrinsic neoplasms of the brain and spinal cord, including
astrocytomas, ependymomas, and neurocytomas. Astrocytomas are
tumors composed of transformed astrocytes, or astrocytic tumor
cells. Such tumors have been classified in order of increasing
malignancy: Grade I consists of fibrillary or protoplasmic
astrocytes; Grade II is an astroblastoma, consisting of cells with
abundant cytoplasm and two or three nuclei; and Grades III and IV
are forms of glioblastoma multiforme, a rapidly growing tumor that
is usually confined to the cerebral hemispheres and composed of a
mixture of astrocytes, spongioblasts, astroblasts, and other
astrocytic tumor cells. Astrocytoma, a primary CNS tumor, is
frequently found in the brain stem, cerebellum, and cerebrum.
Anaplastic astrocytoma and glioblastoma multiforme are commonly
located in the cerebrum.
[0124] The present invention additionally provides methods for
promoting apoptosis in a neoplastic cell comprising contacting the
neoplastic cell with an ATF5 inhibitor. The neoplastic cell can be
selected from the group consisting of: breast, ovary, endometrium,
gastric, colon, liver, pancrease, kidney, bladder, prostate,
testis, skin, esophagus, tongue, mouth, parotid, larynx, pharynx,
lymph node, lung, and brain. In one embodiment, the neoplastic cell
is selected from the group consisting of glioblastoma, astrocytoma,
glioma, medulloblastoma and neuroblastoma. In other embodiments,
the ATF5 inhibitor is a nucleic acid, which can include, but is not
limited to a dominant negative form of ATF5 (e.g. NTAzip-ATF5), or
ATF5siRNA. The method of the present invention can be performed in
vitro as well as in vivo in a subject. As used herein, "apoptosis"
refers to cell death which is wholly or partially genetically
controlled.
[0125] In view of the foregoing, the present invention further
provides a method for treating or preventing a tumor in a subject
in need of treatment, comprising administering to the subject a
pharmaceutical composition comprising an ATF5 inhibitor and,
optionally, a pharmaceutically-acceptable carrier. The ATF5
inhibitor is provided in an amount that is effective to treat the
tumor in a subject to whom the composition is administered. As used
herein, the phrase "effective" means effective to ameliorate or
minimize the clinical impairment or symptoms of the tumor. For
example, the clinical impairment or symptoms of the tumor may be
ameliorated or minimized by diminishing any pain or discomfort
suffered by the subject; by extending the survival of the subject
beyond that which would otherwise be expected in the absence of
such treatment; by inhibiting or preventing the development or
spread of the tumor; or by limiting, suspending, terminating, or
otherwise controlling the maturation and proliferation of cells in
the tumor. The amount of ATF5 inhibitor effective to treat a tumor
in a subject in need of treatment will vary depending upon the
particular factors of each case, including the type of tumor, the
stage of the tumor, the subject's weight, the severity of the
subject's condition, and the method of administration. This amount
can be readily determined by the skilled artisan. In one embodiment
of the present invention, the pharmaceutical composition comprises
a nucleic acid encoding an ATF5 inhibitor, a viral vector, and,
optionally, a pharmaceutically-acceptable carrier.
[0126] As disclosed herein, according to one proposed pathway, ATF5
binding to CRE DNA-binding sites suppresses differentiation of
neural stem cells and neural progenitor cells into differentiated
neural cells. Thus, effective ATF5 inhibitors can be designed to
replace CRE in its interaction with ATF5. A candidate agent having
the ability to bind ATF5 may, as a consequence of this binding,
prevent ATF5 binding to CRE through steric hindrance. According to
other proposed pathways, ATF5 may act by affecting additional
classes of transcription binding sites on DNA. Accordingly,
effective ATF5 inhibitors can also be designed to replace these
additional binding sites. A candidate agent having the ability to
bind ATF5 may, as a consequence of this binding, prevent ATF5
binding to these additional DNA binding sites through steric
hindrance.
[0127] Accordingly, the present invention also provides a method
for identifying an agent that inhibits ATF5, by assessing the
ability of a candidate agent to inhibit interaction between ATF5
and CRE. The method of the present invention comprises the steps
of: (a) contacting a candidate agent with ATF5, in the presence of
CRE; and (b) assessing the ability of the candidate agent to
inhibit interaction between ATF5 and CRE. An agent that inhibits
interaction between ATF5 and CRE may be either natural or
synthetic, and may be an agent reactive with ATF5 (i.e., has
affinity for, binds to, or is directed against ATF5). An agent that
is reactive with ATF5, as disclosed herein, may have the ability to
inhibit interaction between ATF5 and CRE by binding to ATF5. A
candidate agent having the ability to bind to ATF5 may, as a
consequence of this binding, inhibit ATF5 activity through steric
interactions (without binding to CRE itself). A CRE-luciferase
reporter assay may be used to gauge such interactions, as described
herein (Example 11).
[0128] In accordance with the method of the present invention, a
CRE-like agent that binds ATF5 may be identified using an in vitro
assay (e.g., a direct binding assay, competitive binding assay,
etc.). In a direct binding assay, for example, the binding of a
candidate agent to ATF5 or a peptide fragment thereof may be
measured directly. A candidate agent may be supplied by a peptide
library, for example. Alternatively, in a competitive binding
assay, standard methodologies may be used in order to assess the
ability of a candidate agent to bind ATF5, and thereby inhibit
CRE-ATF5 interaction. In such a competitive binding assay, the
candidate agent competes with CRE for binding to ATF5 (but does not
bind directly to CRE). Once bound to ATF5, the candidate agent
could sterically hinder binding of CRE to ATF5, thereby preventing
interaction between CRE and ATF5. A competitive binding assay
represents a convenient way to assess inhibition of CRE-ATF5
interaction, since it allows the use of crude extracts containing
ATF5 and CRE.
[0129] A competitive binding assay may be carried out by adding
ATF5, or an extract containing ATF5 biological activity (as defined
above), to a mixture containing the candidate agent and labeled
CRE, both of which are present in the mixture in known
concentrations. After incubation, the ATF5-agent complex may be
separated from the unbound labeled CRE and unlabeled candidate
agent, and counted. The concentration of the candidate agent
required to inhibit 50% of the binding of the labeled CRE to ATF5
(IC.sub.50) then may be calculated.
[0130] The binding assay formats described herein employ labeled
assay components. Labeling of CRE or ATF5 may be accomplished using
one of a variety of different chemiluminescent and radioactive
labels known in the art. The label of the present invention may be,
for example, a nonradioactive or fluorescent marker, such as
biotin, fluorescein (FITC), acridine, cholesterol, or
carboxy-X-rhodamine, which can be detected using fluorescence and
other imaging techniques readily known in the art. Alternatively,
the label may be a radioactive marker, including, for example, a
radioisotope. The radioisotope may be any isotope that emits
detectable radiation, including, without limitation, .sup.35S,
.sup.32P, .sup.125I, .sup.3H, or .sup.14C. The label may also be
luciferase, for use in a CRE-luciferase reporter assay, as
described below (Example 11).
[0131] Qualitative results of the above-described assays may be
obtained by competitive autoradiographic-plate binding assays;
alternatively, Scatchard plots may be used to generate quantitative
results. The labels of the present invention may be coupled
directly or indirectly to the desired component of the assay,
according to methods well known in the art. The choice of label
depends on a number of relevant factors, including the sensitivity
required, the ease of conjugation with the compound to be labeled,
stability requirements, and available instrumentation.
[0132] Both direct and competitive binding assays may be used in a
variety of different configurations. In one competitive binding
assay, for example, the candidate agent may compete against labeled
CRE (the labeled analyte) for a specific binding site on ATF5 (the
capture agent) that is bound to a solid substrate, such as a column
chromatography matrix or tube. Alternatively, the candidate agent
may compete for a specific binding site on labeled ATF5 (the
labeled analyte) against wild-type CRE or a fragment thereof (the
capture agent) that is bound to a solid substrate. The capture
agent is bound to the solid substrate in order to effect separation
of bound labeled analyte from the unbound labeled analyte. In
either type of competitive binding assay, the concentration of
labeled analyte that binds the capture agent bound to the solid
substrate is inversely proportional to the ability of a candidate
agent to compete in the binding assay. The amount of inhibition of
labeled analyte by the candidate agent depends on the binding assay
conditions and on the concentrations of candidate agent, labeled
analyte, and capture agent that are used.
[0133] Another competitive binding assay, for use in detecting
agents that bind to ATF5, may be conducted in a liquid phase. In
this type of assay, any of a variety of techniques known in the art
may be used to separate the bound labeled analyte (which may be
either CRE or ATF5) from the unbound labeled analyte. Following
such separation, the amount of bound labeled analyte may be
determined. The amount of unbound labeled analyte present in the
separated sample is inversely proportional to the amount of bound
labeled analyte.
[0134] In the further alternative, a homogeneous binding assay may
be performed, in which a separation step is not needed. In this
type of binding assay, the label on the labeled analyte (which may
be either CRE or ATF5) is altered by the binding of the analyte to
the capture agent. This alteration in the labeled analyte results
in a decrease or increase in the signal emitted by the label, so
that measurement of the label at the end of the binding assay
allows for detection or quantification of the analyte.
[0135] Under specified assay conditions, a candidate agent is
considered to be capable of inhibiting the binding of CRE to ATF5
in a competitive binding assay if the amount of binding of the
labeled analyte to the capture agent is decreased by 50% or more
(preferably 90% or more). Where a direct binding assay
configuration is used, a candidate agent is considered to bind ATF5
when the signal measured is twice the background level or higher.
Furthermore, as proof of the specificity of the candidate agent
identified using an ATF5 competitive binding assay, binding
competition also may be performed using purified ATF5 in the
presence of washed ribosomes. A functional assay, such as a
luciferase assay, also may be used to screen for ATF5 inhibitors,
as described herein.
[0136] As disclosed herein, ATF5 has been implicated in a number of
biological events in neural stem cells, neural progenitor cells,
and neuroblastoma cells. For example, it has been shown that ATF5
plays a role in the differentiation of neural stem and progenitor
cells, and may be associated with uncontrolled cell proliferation
in neuroblastomas and other neural tumors. Accordingly, it is clear
that therapeutics designed to inhibit ATF5 (i.e., those which bind
to, or are otherwise reactive with, ATF5) may be useful in
regulation of a number of ATF5-associated biological events,
including differentiation of neural stem cells and neural
progenitor cells, and control of proliferation of neural tumor
cells.
[0137] Thus, once the candidate agent of the present invention has
been screened, and has been determined to have a suitable
inhibitory effect on ATF5 (i.e., it is reactive with ATF5, it binds
ATF5, or it otherwise inactivates ATF5), it may be evaluated for
its effect on differentiation of neural stem cells or neural
progenitor cells, or on tumor-cell proliferation. In particular,
the candidate agent may be assessed for its ability to act as a
promoter of differentiation, or as an inhibitor of tumor-cell
division proliferation, or to otherwise function as an appropriate
tumor-suppressing agent. It is expected that the ATF5 inhibitor of
the present invention will be useful for promoting differentiation
of neural stem cells and neural progenitor cells, and for treating
neural tumors, including those disclosed herein. Furthermore, the
inventors propose that the ATF5 inhibitor of the present invention
might be useful for restoring proliferation control in tumor
cells.
[0138] Accordingly, the present invention further comprises the
steps of: (c) contacting the candidate agent with neural stem cells
or neural progenitor cells containing ATF5; and (d) determining if
the agent has an effect on an ATF5-associated biological event in
the neural stem cells or neural progenitor cells. As used herein,
an "ATF5-associated biological event" includes a biochemical or
physiological process in which ATF5 levels or activity have been
implicated. As disclosed herein, examples of ATF5-associated
biological events include, without limitation, binding to, and
interaction with, CRE; regulation of differentiation in neural stem
cells or neural progenitor cells; and proliferation of neural tumor
cells. As further used herein, a cell "containing ATF5" is a cell
in which ATF5, or a derivative or homologue thereof, is naturally
expressed or naturally occurs.
[0139] According to this method of the present invention, a
candidate agent may be contacted with one or more neural stem cells
or neural progenitor cells in vitro. For example, a culture of
cells may be incubated with a preparation containing the candidate
agent. The candidate agent's effect on an ATF5-associated
biological event then may be assessed by any biological assays or
methods known in the art, including histological analyses. In one
embodiment of the present invention, the neural stem cells or
neural progenitor cells express luciferase (see Examples 3 and
11).
[0140] The present invention is further directed to agents
identified by the above-described identification methods. Such
agents may be useful for promoting differentiation of neural stem
cells or neural progenitor cells, and for treating an
ATF5-associated condition. As used herein, an "ATF5-associated
condition" is a condition, disease, or disorder in which ATF5
levels or activity have been implicated, and includes the
following: an ATF5-associated biological event, and neural tumors.
The ATF5-associated condition may be treated in the subject by
administering to the subject an amount of the agent effective to
treat the ATF5-associated condition in the subject. This amount may
be readily determined by one skilled in the art.
[0141] Accordingly, in one embodiment, the present invention
provides a method for promoting differentiation in neural stem
cells or neural progenitor cells, by contacting the cells with the
above-described agent, in an amount effective to promote
differentiation in the cells. In another embodiment, the present
invention provides a method for treating or preventing a neural
tumor in a subject, by administering to the subject the
above-described agent, in an amount effective to treat or prevent
the neural tumor in the subject. In a preferred embodiment of the
present invention, the neural tumor is a neuroblastoma.
[0142] The present invention also provides a pharmaceutical
composition comprising the agent identified by the above-described
identification method and a pharmaceutically-acceptable carrier.
Examples of suitable pharmaceutically-acceptable carriers, and
methods of preparing pharmaceutical formulations and compositions,
are described above. The pharmaceutical composition of the present
invention would be useful for contacting neural stem cells or
neural progenitor cells with an agent that inhibits interaction
between CRE and ATF5, in order to promote differentiation of the
cells, and would also be useful for treating an ATF5-associated
condition. In such a case, the pharmaceutical composition is
administered to a subject in an amount effective to treat the
ATF5-associated condition.
[0143] The inventors have demonstrated herein that ATF5 expression
is elevated in neuroblastoma cells. Thus, ATF5 represents a marker
for neuroblastoma. Accordingly, the present invention further
provides a method for determining whether a subject has a neural
tumor, thereby permitting the diagnosis of such a neural tumor in
the subject. The subject may be any of those described above.
Preferably, the subject is a human. Examples of neural tumors have
been previously discussed. In one embodiment of the present
invention, the neural tumor is a neuroblastoma. The method of the
present invention comprises assaying a diagnostic sample of the
subject for ATF5, wherein detection of an ATF5 level elevated above
normal is diagnostic of a neural tumor in the subject. As used
herein, "ATF5" includes both an ATF5 protein and an ATF5 analogue,
as discussed above.
[0144] In accordance with the method of the present invention, the
diagnostic sample of a subject may be assayed in vitro or in vivo.
Where the assay is performed in vitro, a diagnostic sample from the
subject may be removed using standard procedures. The diagnostic
sample may be any nervous tissue, including brain tissue, which may
be removed by standard biopsy. In addition, the diagnostic sample
may be any tissue known to have a neural tumor, any tissue
suspected of having a neural tumor, or any tissue believed not to
have a neural tumor. In a preferred embodiment of the present
invention, the diagnostic sample contains post-mitotic cells. More
preferably, the diagnostic sample contains neural-tumor cells.
[0145] Protein may be isolated and purified from the diagnostic
sample of the present invention using standard methods known in the
art, including, without limitation, extraction from a tissue (e.g.,
with a detergent that solubilizes the protein) where necessary,
followed by affinity purification on a column, chromatography
(e.g., FTLC and HPLC), immuno-precipitation (with an antibody to
ATF5), and precipitation (e.g., with isopropanol and a reagent such
as Trizol). Isolation and purification of the protein may be
followed by electrophoresis (e.g., on an SDS-polyacrylamide gel).
Nucleic acid may be isolated from a diagnostic sample using
standard techniques known to one of skill in the art.
[0146] In accordance with the method of the present invention, a
neural tumor in a subject is diagnosed by assaying a diagnostic
sample of the subject for ATF5. The level of ATF5 in the sample,
for example, may be detected by measuring ATF5 amounts in the
sample. A diagnostic sample may be assayed for the level of ATF5 by
assaying for ATF5 protein, ATF5 cDNA, or ATF5 mRNA. The appropriate
form of ATF5 will be apparent based on the particular techniques
discussed herein. Preferably, the diagnostic sample of the present
invention is assayed for the level of ATF5 protein. It is
contemplated that the diagnostic sample may be assayed for
expression of any or all forms of ATF5 protein (including precursor
forms, endoproteolytically-processed forms, the 22-kDa and 30-kDa
forms, and other forms resulting from post-translational
modification) in order to determine whether a subject or patient
has a neural tumor.
[0147] Alternatively, the level of ATF5 in the sample may be
detected by detecting above-normal interaction of ATF5 and CRE.
Accordingly, in one embodiment of the present invention, the level
of ATF5 elevated above normal is detected by detecting above-normal
interaction of ATF5 and CRE. Methods for detecting interaction
between CRE and ATF5 have been discussed above.
[0148] As used herein, the term "elevated above normal" means that
ATF5 is detected at a level that is significantly greater than the
level expected for the same type of diagnostic sample taken from a
nondiseased subject or patient (i.e., one who does not have a
neural tumor) of the same gender and of similar age. As further
used herein, "significantly greater" means that the difference
between the level of ATF5 that is elevated above normal, and the
expected (normal) level of ATF5, is of statistical significance.
Preferably, the level of ATF5 elevated above normal is a level that
is at least 10% greater than the level of ATF5 otherwise expected
in the diagnostic sample. Where ATF5 is expected to be absent from
a particular diagnostic sample taken from a particular subject or
patient, the normal level of ATF5 for that subject or patient is
nil. Where a particular diagnostic sample taken from a particular
subject or patient is expected to have a low, constitutive level of
ATF5, that low level is the normal level of ATF5 for that subject
or patient. As disclosed herein, ATF5 is generally present at lower
levels in post-mitotic neurons, than in neural stem cells, neural
progenitor cells, or neural tumor cells.
[0149] Expected or normal levels of ATF5 for a particular
diagnostic sample taken from a subject or patient may be easily
determined by assaying nondiseased subjects of a similar age and of
the same gender. For example, diagnostic samples may be obtained
from at least 30 normal, healthy men between the ages of 25 and 80,
to determine the normal quantity of ATF5 in males. A similar
procedure may be followed to determine the normal quantity of ATF5
in females. Once the necessary or desired samples have been
obtained, the normal quantity of ATF5 in men and women may be
determined using a standard assay for quantification, such as flow
cytometry, Western-blot analysis, or an ELISA for measuring protein
quantities, as described below. For example, an ELISA may be run on
each sample in duplicate, and the mean and standard deviation of
the quantity of ATF5 may be determined. If necessary, additional
subjects may be recruited before the normal quantity of ATF5 is
determined. A similar type of procedure may be used to determine
the expected or normal level of interaction between ATF5 and CRE
for a particular diagnostic sample taken from a subject or
patient.
[0150] In accordance with the method of the present invention, a
diagnostic sample of a subject may be assayed for ATF5 (or for
interaction between ATF5 and CRE), and ATF5 (or interaction between
ATF5 and CRE) may be detected in a diagnostic sample, using assays
and detection methods readily determined from the known art (e.g.,
immunological techniques, hybridization analysis, fluorescence
imaging techniques, and/or radiation detection, etc.), as well as
any assays and detection methods disclosed herein (e.g.,
immunoprecipitation, Western-blot analysis, etc.). For example, a
diagnostic sample of a subject may be assayed for ATF5 using an
agent reactive with ATF5. The agent may include any of those
described above. Preferably, the agent of the present invention is
labeled with a detectable marker or label.
[0151] In one embodiment of the present invention, the agent
reactive with ATF5 is an antibody. As used herein, the antibody of
the present invention may be polyclonal or monoclonal. In addition,
the antibody of the present invention may be produced by techniques
well known to those skilled in the art. Polyclonal antibody, for
example, may be produced by immunizing a mouse, rabbit, or rat with
purified ATF5 or with a short peptide sequence thereof. Monoclonal
antibody then may be produced by removing the spleen from the
immunized mouse, and fusing the spleen cells with myeloma cells to
form a hybridoma which, when grown in culture, will produce a
monoclonal antibody.
[0152] The antibodies used herein may be labeled with a detectable
marker or label. Labeling of an antibody may be accomplished using
one of a variety of labeling techniques, including peroxidase,
chemiluminescent labels known in the art, and radioactive labels
known in the art. The detectable marker or label of the present
invention may be, for example, a nonradioactive or fluorescent
marker, such as biotin, fluorescein (FITC), acridine, cholesterol,
or carboxy-X-rhodamine, which can be detected using fluorescence
and other imaging techniques readily known in the art.
Alternatively, the detectable marker or label may be a radioactive
marker, including, for example, a radioisotope. The radioisotope
may be any isotope that emits detectable radiation, such as 35S,
32P, 125I, 3H, or 14C. Radioactivity emitted by the radioisotope
can be detected by techniques well known in the art. For example,
gamma emission from the radioisotope may be detected using gamma
imaging techniques, particularly scintigraphic imaging. Preferably,
the agent of the present invention is a high-affinity antibody
labeled with a detectable marker or label.
[0153] Where the agent of the present invention is an antibody
reactive with ATF5, a diagnostic sample taken from the subject may
be purified by passage through an affinity column which contains an
anti-ATF5 antibody as a ligand attached to a solid support, such as
an insoluble organic polymer in the form of a bead, gel, or plate.
The antibody attached to the solid support may be used in the form
of a column. Examples of suitable solid supports include, without
limitation, agarose, cellulose, dextran, polyacrylamide,
polystyrene, sepharose, or other insoluble organic polymers. The
antibody may be further attached to the solid support through a
spacer molecule, if desired. Appropriate binding conditions (e.g.,
temperature, pH, and salt concentration) for ensuring binding of
the agent and the antibody may be readily determined by the skilled
artisan. In a preferred embodiment, the antibody is attached to a
sepharose column, such as Sepharose 4B.
[0154] Where the agent is an antibody, a diagnostic sample of the
subject may be assayed for ATF5 using binding studies that utilize
one or more antibodies immunoreactive with ATF5, along with
standard immunological detection techniques. For example, the ATF5
protein eluted from the affinity column may be subjected to an
ELISA assay, Western-blot analysis, flow cytometry, or any other
immunostaining method employing an antigen-antibody interaction.
Preferably, the diagnostic sample is assayed for ATF5 using Western
blotting.
[0155] Alternatively, a diagnostic sample of a subject may be
assayed for ATF5 using hybridization analysis of nucleic acid
extracted from the diagnostic sample taken from the subject.
According to this method of the present invention, the
hybridization analysis may be conducted using Northern blot
analysis of mRNA. This method also may be conducted by performing a
Southern blot analysis of DNA using one or more nucleic acid
probes, which hybridize to nucleic acid encoding ATF5. The nucleic
acid probes may be prepared by a variety of techniques known to
those skilled in the art, including, without limitation, the
following: restriction enzyme digestion of ATF5 nucleic acid; and
automated synthesis of oligonucleotides having sequences which
correspond to selected portions of the nucleotide sequence of the
ATF5 nucleic acid, using commercially-available oligonucleotide
synthesizers, such as the Applied Biosystems Model 392 DNA/RNA
synthesizer.
[0156] The nucleic acid probes used in the present invention may be
DNA or RNA, and may vary in length from about 8 nucleotides to the
entire length of the ATF5 nucleic acid. The ATF5 nucleic acid used
in the probes may be derived from mammalian ATF5. The nucleotide
sequence for human ATF5, rat ATF5, and mouse ATF5, for example, are
known. Using this sequence as a probe, the skilled artisan could
readily clone a corresponding ATF5 cDNA from other species. In
addition, the nucleic acid probes of the present invention may be
labeled with one or more detectable markers or labels. Labeling of
the nucleic acid probes may be accomplished using one of a number
of methods known in the art--e.g., nick translation, end labeling,
fill-in end labeling, polynucleotide kinase exchange reaction,
random priming, or SP6 polymerase (for riboprobe
preparation)--along with one of a variety of labels--e.g.,
radioactive labels, such as 35S, 32P, or 3H, or nonradioactive
labels, such as biotin, fluorescein (FITC), acridine, cholesterol,
or carboxy-X-rhodamine (ROX). Combinations of two or more nucleic
acid probes (or primers), corresponding to different or overlapping
regions of the ATF5 nucleic acid, also may be used to assay a
diagnostic sample for ATF5, using, for example, PCR or RT-PCR.
[0157] The detection of ATF5 (or interaction between ATF5 and CRE)
in the method of the present invention may be followed by an assay
to measure or quantify the extent of ATF5 in a diagnostic sample of
a subject. Such assays are well known to one of skill in the art,
and may include immunohistochemistry/immunocytochemistry, flow
cytometry, mass spectroscopy, Western-blot analysis, or an ELISA
for measuring amounts of ATF5 protein. For example, to use an
immunohistochemistry assay, histological (paraffin-embedded)
sections of tissue may be placed on slides, and then incubated with
an antibody against ATF5. The slides then may be incubated with a
second antibody (against the primary antibody), which is tagged to
a dye or other colorimetric system (e.g., a fluorochrome, a
radioactive agent, or an agent having high electron-scanning
capacity), to permit visualization of ATF5 present in the
sections.
[0158] It is contemplated that the diagnostic sample in the present
invention frequently will be assayed for ATF5 (or interaction
between ATF5 and CRE) not by the subject or patient, nor by his/her
consulting physician, but by a laboratory technician or other
clinician. Accordingly, the method of the present invention further
comprises providing to a subject's or patient's consulting
physician a report of the results obtained upon assaying a
diagnostic sample of the subject or patient for ATF5.
[0159] The present invention further provides a method for
assessing the efficacy of therapy to treat a neural tumor in a
subject or patient who has undergone or is undergoing treatment for
a neural tumor. The method of the present invention comprises
assaying a diagnostic sample of the subject or patient for ATF5,
wherein a normal level of ATF5 in the diagnostic sample is
indicative of successful therapy to treat a neural tumor, and a
level of ATF5 elevated above normal in the diagnostic sample is
indicative of a need to continue therapy to treat a neural tumor.
In one embodiment of the present invention, a level of ATF5
elevated above normal is detected by detecting above-normal
interaction between ATF5 and CRE. The neural tumor may be any of
those described above. The diagnostic sample may be assayed for
ATF5 (or interaction between ATF5 and CRE) in vitro or in vivo. In
addition, the diagnostic sample may be assayed for ATF5 (or
interaction between ATF5 and CRE) using all of the various assays
and methods of detection and quantification described above. This
method of the present invention provides a means for monitoring the
effectiveness of therapy to treat a neural tumor by permitting the
periodic assessment of levels of ATF5 (or interaction between ATF5
and CRE) in a diagnostic sample taken from a subject or
patient.
[0160] According to the method of the present invention, a
diagnostic sample of a subject or patient may be assayed, and
levels of ATF5 (or interaction between ATF5 and CRE) may be
assessed, at any time following the initiation of therapy to treat
a neural tumor. For example, levels of ATF5 (or interaction between
ATF5 and CRE) may be assessed while the subject or patient is still
undergoing treatment for a neural tumor. Where levels of ATF5
detected in an physician may choose to continue with the subject's
or patient's treatment for the neural tumor. Where levels of ATF5
in an assayed diagnostic sample of the subject or patient decrease
through successive assessments, it may be an indication that the
treatment for a neural tumor is working, and that treatment doses
could be decreased or even ceased. Where levels of ATF5 in an
assayed diagnostic sample of the subject or patient do not rapidly
decrease through successive assessments, it may be an indication
that the treatment for a neural tumor is not working, and that
treatment doses could be increased. Where ATF5 is no longer
detected in an assayed diagnostic sample of a subject or patient at
a level elevated above normal, a physician may conclude that the
treatment for a neural tumor has been successful, and that such
treatment may cease.
[0161] It is within the confines of the present invention to assess
levels of ATF5 (or interaction between ATF5 and CRE) following
completion of a subject's or patient's treatment for a tumor, in
order to determine whether the tumor has recurred in the subject or
patient. Accordingly, an assessment of levels of ATF5 (or
interaction between ATF5 and CRE) in an assayed diagnostic sample
may provide a convenient way to conduct follow-ups of patients who
have been diagnosed with a tumors. Furthermore, it is within the
confines of the present invention to use assessed levels of ATF5
(or interaction between ATF5 and CRE) in an assayed diagnostic
sample as a clinical or pathologic staging tool, as a means of
determining the extent of a tumor in the subject or patient, and as
a means of ascertaining appropriate treatment options.
[0162] A correlation exists, in general, between levels of ATF5 in
post-mitotic neural cells and neuroblastoma. Therefore, it is also
contemplated in the present invention that assaying a diagnostic
sample of a subject for ATF5 may be a useful means of providing
information concerning the prognosis of a subject or patient who
has a neural tumor. Accordingly, the present invention further
provides a method for assessing the prognosis of a subject who has
a neural tumor, comprising assaying a diagnostic sample of the
subject for ATF5, wherein the subject's prognosis improves with a
decreased level of ATF5 in the diagnostic sample, and the subject's
prognosis worsens with an increased level of ATF5 in the diagnostic
sample. In one embodiment of the present invention, the level of
ATF5 elevated above normal is detected by detecting above-normal
interaction between ATF5 and CRE. Suitable diagnostic samples,
assays, and detection and quantification methods for use in the
method of the present invention have already been described. This
method of the present invention provides a means for determining
the prognosis of a subject or patient diagnosed with a neural tumor
based upon the level of ATF5, or interaction between ATF5 and CRE,
in an assayed diagnostic sample of the subject or patient.
[0163] According to the method of the present invention, a
diagnostic sample of a subject or patient may be assayed, and
levels of ATF5 (or interaction between ATF5 and CRE) may be
assessed, at any time during or following the diagnosis of a neural
tumor in the subject or patient. For example, levels of ATF5 (or
interaction between ATF5 and CRE) in an assayed diagnostic sample
may be assessed before the subject or patient undergoes treatment
for a neural tumor, in order to determine the subject's or
patient's initial prognosis. Additionally, levels of ATF5 (or
interaction between ATF5 and CRE) in an assayed diagnostic sample
may be assessed while the subject or patient is undergoing
treatment for a neural tumor, in order to determine whether the
subject's or patient's prognosis has become more or less favorable
through the course of treatment.
[0164] For example, where the level of ATF5 detected in an assayed
diagnostic sample of the subject or patient is, or continues to
remain, significantly high, a physician may conclude that the
subject's or patient's prognosis is unfavorable. Where the level of
ATF5 in an assayed diagnostic sample of the subject or patient
decreases through successive assessments, it may be an indication
that the subject's or patient's prognosis is improving. Where the
level of ATF5 in an assayed diagnostic sample of the subject or
patient does not decrease significantly through successive
assessments, it may be an indication that the subject's or
patient's prognosis is not improving. Finally, where the level of
ATF5 is low, or is normal, in a diagnostic sample of the subject or
patient, a physician may conclude that the subject's or patient's
prognosis is favorable.
[0165] The discovery that ATF5 can be detected in a wide variety of
tumor cells provides a means of identifying patients with a tumor,
and presents the potential for commercial application in the form
of a test for the diagnosis of a tumor. The development of such a
test could provide general screening procedures. Such procedures
can assist in the early detection and diagnosis of a tumor, and can
provide a method for the follow-up of patients in whom a level of
ATF5 elevated above normal has been detected.
[0166] Accordingly, the present invention further provides a kit
for use as an assay of a tumor, comprising an ATF5-specific agent
and reagents suitable for detecting ATF5. The ATF5-specific agent
may be any agent reactive with ATF5 protein or nucleic acid,
including a nucleic acid probe which hybridizes to nucleic acid
encoding ATF5, an antibody, and any of the agents described above.
The agent may be used in any of the above-described assays or
methods for detecting or quantifying levels of ATF5. Preferably,
the agent of the present invention is labeled with a detectable
marker or label.
[0167] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention, and should not be construed to limit in any way the
scope of the invention as defined in the claims which follow
thereafter.
EXAMPLES
Example 1
Reagents
[0168] Cell-culture media, RPMI 1640 and DMEM, and molecular
biology reagents, Taq platinum DNA polymerase, SuperScript II
reverse transcriptase, and LipofectAMINE 2000, were obtained from
Invitrogen, Inc. (Carlsbad, Calif.). Donor horse and fetal bovine
serum were obtained from JRH Biosciences, Inc. (Lenexa, Kans.). The
Marathon cDNA amplification library kit was obtained from Clontech
(Palo Alto, Calif.), and PCR primers were obtained from Integrated
DNA Technologies or Life Technologies, Inc. Anti-FLAG M2 antibody
was from Sigma Corp. (St. Louis, Mo.).
Cell Culture
[0169] PC12 cells were grown on collagen-coated dishes, as
previously described (Greene et al., Establishment of a
noradrenergic clonal line of rat adrenal pheochromocytoma cells
which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA,
73:2424-28, 1998), with or without human recombinant nerve growth
factor (NGF) (Genentech, Inc.). Dissociated cultures of
telencephalic cells were prepared from E14 Sprague-Dawley rats.
Telencephalic cells were trypsinized (0.05% in 0.53 mM EDTA;
Invitrogen, Inc.) for 30 min (Li et al., Neuronal differentiation
of precursors in the neocortical ventricular zone is triggered by
BMP. J. Neurosci., 18:8853-62, 1998), and dissociated cells were
centrifuged and re-suspended in DMEM containing 5% FBS, 10 ng/ml
EGF, and 20 ng/ml bFGF, then plated on 24-well dishes coated with
polylysine at 3-5.times.10.sup.5 cells per well (Laywell et al.,
Multipotent neurospheres can be derived from forebrain subependymal
zone and spinal cord of adult mice after protracted postmortem
intervals. Exp. Neurol., 156:430-33, 1999). The presence of bFGF
promotes proliferation of the progenitor cells, but does not
interfere with their differentiation into neurons (Ghosh and
Greenberg, Distinct roles for bFGF and NT-3 in the regulation of
cortical neurogenesis. Neuron, 15:89-03, 1995).
[0170] Adherent clonal neurosphere cultures were prepared from
newborn mouse subependymal zone cells, as previously described
(Kukekov et al., A nestin-negative precursor cell from the adult
mouse brain gives rise to neurons and glia. Glia, 21:399-07, 1997;
Kukekov et al., Multipotent stem/progenitor cells with similar
properties arise from two neurogenic regions of adult human brain.
Exp. Neurol., 156:333-44, 1999). The cell suspension used to
generate neurospheres was filtered through sterile gauze, and
visually verified to contain only single cells.
Cloning of Full-Length rATF5 and Plasmid Constructs
[0171] SAGE tag, CATGAGAACCTAGTC (SEQ ID NO:3), was found in rat
EST, UI-R-G0-ur-g-10-0-UI (GenBank.TM./EBI accession number
AI576016), which, in turn, showed high homology with the 3' end of
murine ATF5. To clone the open-reading frame of rat ATF5, PCR
antisense primer 5'-CTTGGTTTCTCAGTTGCAC-3' (SEQ ID NO:4) (derived
from the sequence of the above EST) was used for 5' RACE PCR, using
the Clontech Marathon kit according to the manufacturer's protocol.
The first-strand cDNA PCR template was prepared from 5 .mu.g of
PC12 cell total RNA, by reverse transcription with Superscript II.
The products of the 5' RACE PCR included the second of 2 potential
Kozak start sites.
[0172] Cloning of the rATF5 open-reading frame that included the
first potential start site was achieved with sense PCR primer,
5'-TGCACCTGTGCCTCAGCCATGTC-3' (SEQ ID NO:5). This sequence was
obtained from an EST sequence (GenBank.TM./EBI accession number
AW917099) that overlapped with the 5' end of the 5' RACE PCR
product described above. Both potential rATF5 forms were
FLAG-tagged, by PCR, with sense primers,
5'-CTCGAGAACCATGGACTACAAGGACGATGATGACAAAGGATCACTCCTGGCGAC CCT-3'
(SEQ ID NO:6), and 5'-CTCGAGAAGCATGGACTACAAGGACGATGATG
ACAAAGGAGCATCCCTACTCAAGAA-3' (SEQ ID NO:7). 5'-GAATTCTCGAG
CTTGGTTTCTCAGTTGCAC-3' (SEQ ID NO:8) was the antisense primer for
both ATF5s. NTAzip-ATF5 was constructed by overlapping PCR, using
FLAG-tagged ATF5 (potential start site 2 form) as the template.
[0173] PCR product 1 was produced with 5'-CTCGAGAAGCATGGACTACAA
GGACGATGATGACAAAGGAGCATCCCTACTCAAGAA-3' (SEQ ID NO:7) and
5'-TTCTTCTGCTTCTTTTTCTAGTAGTTCTTCGTTTTCTCTTGCTAGTTCTTCTGCTCTTTG
TTCGAGG GTGCTGGCAGGACTAGGATA-3' (SEQ ID NO:9) as primers, and PCR
product 2 was made with
5'-GCAAGAGAAAACGAAGAACTACTAGAAAAAGAAGCAGAAGAACTAGAACAAGA
AATGCAGAGCTAGAGGGCGAGTGCCAAGGG-3' (SEQ ID NO:10) and
5'-GAATTCTCGAGCTTG GTTTCTCAGTTGCAC-3' (SEQ ID NO:11) as primers.
Products 1 and 2 were mixed, and the product (FL-NTAzip-ATF5) was
PCR amplified with
5'-CTCGAGAAGCATGGACTACAAGGACGATGATGACAAAGGAGCATCCCTACTCAAGA A-3'
(SEQ ID NO:7) and 5'-GAATTCTCGAGCTTGGTTT CTCAGTTGCAC-3' (SEQ ID
NO:8). To generate NTAzip-ATF5, the activation domain was removed
from FL-NTAzip-ATF5 by PCR, using primers
5'-GAATTCAACCATGGACTACAAGGACGA
TGATGACAAAATGGCATCTATGACTGGAGGACAACAAATGGGAAGAGACCCA
GACCTCGAACAAAGAGCAGAA-3' (SEQ ID NO:11) (sense) and 5'-GAATTCT
CGAGCTTGGTTTCTCA GTTGCAC-3' (SEQ ID NO:8) (antisense).
[0174] NTAzip-ATF5 was N-terminal FLAG-tagged with a predicted
open-reading frame of
MDYKDDDDKMASMTGGQQMGRDPDLEQRAEELRENEELLEKEAEELE
QENAELEGECQGLEARNRELRERAESVEREIQYVKDLLIEVYKARSQRTRSA (SEQ ID
NO:12), where the DNA binding motif was replaced with an
amphipathic acidic .alpha.-helical sequence, as marked in bold
(Moll et al., Attractive interhelical electrostatic interactions in
the proline- and acidic-rich region (PAR) leucine zipper subfamily
preclude heterodimerization with other basic leucine zipper
subfamilies. J. Biol. Chem., 275:34826-832, 2000). All PCR products
were subcloned into the Topo II pCR 2.1 vector, and were sequenced
to verify identity. After confirmation, all full-length constructs
were subcloned into the EcoR1 sites of the pCMS-eGFP vector.
[0175] Retrovirus plasmids were constructed by blunt ligation of
eGFP into the XhoI site of QCX (Julius et al., Q vectors,
bicistronic retroviral vectors for gene transfer. Biotechniques,
28:702-08, 2000). Subsequently, full-length FLAG-ATF5 was blunt
ligated into the BsiWI site of QCX-eGFP, to form the bicistronic Q
vector construct (QC-FLAG-ATF5-eGFP) for retrovirus production.
[0176] The CRE-luciferase reporter plasmid was constructed by
annealing synthetic oligo
5'-TCGAGTCATGGTAAAAATGACGTCATGGTAATTATCATGGTAAAAAT
GACGTCATGGTAATTATCATGGTAAAAATGACGTCATGGTAATTA-3' (SEQ ID NO:13) to
5'-AGC TTAATTACCATGACGTCATTTTTACCATGATAATTACCATGACGTCATT TTTACCA
TGATAATTACCATGACGTCATTTTTACCATGAC-3' (SEQ ID NO:14), to form a
double-stranded DNA (Peters et al., ATF-7, a novel bZIP protein,
interacts with the PRL-1 protein-tyrosine phosphatase. J. Biol.
Chem., 276:13718-26, 2001). The annealed DNA was ligated into the
XhoI and HindIII sites of the GL3 plasmid. VPI6-CREB (Columbia
University) was subcloned into the EcoRI and XbaI sites of the
pCMS-eGFP vector.
ATF5 Antiserum
[0177] The CTRGDRKQKKRDQNK (SEQ ID NO:15) peptide, corresponding to
ATF5 DNA-binding-domain I (plus an N-terminal cysteine, for
conjugation to keyhole limpet hemocyanin), was used as the antigen
for production of rabbit antiserum.
Western-Blot Analysis
[0178] Cultured cells and adult mouse cortex were harvested in
Laemmli sample buffer. The protein concentrations were measured by
the Bradford assay (Bio-Rad, Hercules, Calif.), and cell proteins
were resolved by SDS-PAGE on a 12% gel. The separated proteins were
electrophoretically transferred from the gel to Hybond P membrane
(Amersham) (Towbin et al., Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proc. Natl. Acad. Sci. USA, 76:4350-54, 1979).
The membranes were blocked for 1 h in PBS containing 5% milk and 1%
BSA, and immunolabeled overnight with ATF5 antipeptide antiserum,
at 1:1000, in PBS containing 5% milk and 1% BSA. For detection, the
blots were washed and probed with goat anti-rabbit HRP-conjugated
antibody (Pierce), and then visualized on film using an enhanced
chemiluminescence detection kit (ECL) (Amersham). For the PC12 cell
NGF time course, to normalize for protein loading, the blots were
stripped of immunocomplexes, as described by Amersham, and reprobed
with ERK1 C-16 antibody (Santa Cruz) and goat anti-rabbit
HRP-conjugated antibody, followed by ECL film visualization.
Densitometry was carried out with NIH Image 1.62 software.
Immunochemistry
[0179] For PC12 cells, fluorescence immunohistochemistry was
carried out, as previously described (Angelastro et al.,
Characterization of a novel isoform of caspase-9 that inhibits
apoptosis. J. Biol. Chem., 276:12190-200, 2001). For dissociated
telencephalic cultures, the cells were fixed with 4%
paraformaldehyde and 2% sucrose in PBS, for 15 min. After 3 washes
in PBS, the cells were blocked in 10% non-immune goat serum and
0.3% Triton X-100 for 1 h. The cultures were immunolabeled
separately with the following combinations: (1) rabbit anti-GFP
(1:1000 dilution; Clontech) and mouse anti-nestin (1:500; rat-401
from the DSBH antibody collection, University of Iowa); (2) rabbit
anti-GFP (1:1000 dilution) and mouse TUJ1 (1:2000 dilution;
Covance); (3) mouse GFP (1:500; Sigma) and rabbit
anti-neurofilament 160 kDa (1:200; Columbia University); or (4)
mouse GFP (1:500) and rabbit anti-GFAP (1:500; Dako) antibody, in
10% non-immune goat serum and 0.3% Triton X100, for 1 h, followed
by secondary labeling with goat FITC-conjugated anti-rabbit or
rhodamine-conjugated anti-mouse antibodies (Alexa) at 1:5000.
[0180] For immunolabeling, embryos were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer, overnight, then
cryoprotected in 30% sucrose; they were then frozen in O.C.T.
compound (Tissue-TEK). Cryosectioned (14 .mu.m) embryos were
blocked for 1 h in 10% non-immune goat serum and 0.3% Triton X-100.
The sections were then incubated with ATF5 antiserum (1:500) and
TUJ1 antibody (1:2000), in 2.5% nonimmune goat serum and 0.3%
Triton X-100, overnight. The sections were subsequently incubated
for 1 h with goat FITC-conjugated anti-rabbit and
rhodamine-conjugated anti-mouse antibodies, in 10% non-immune goat
serum and 0.3% Triton X-100.
[0181] For adherent neurospheres, cells were fixed with 4%
para-formaldehyde in PBS/2% sucrose for 10 min, at room
temperature, and then permeabilized for 5 min with 0.5% Triton
X-100 in ice-cold PBS/2% sucrose. After blocking with 25% goat or
bovine serum in PBS, for 20 min, the cultures were incubated with
primary antibodies (diluted in 25% serum in PBS for 30 min, at room
temperature, followed by 3 washes with PBS), and then incubated
with the appropriate secondary goat anti-rabbit or anti-mouse
antibodies conjugated with FITC (Alexa Fluor 488, Molecular Probes,
A 11001) or Texas Red-X (Molecular Probes, T 6391) for 30 min, at
room temperature, at 1:200. The cultures were then incubated with
the second set of primary and secondary antibodies, as above.
Immunochemical reagents were anti-AC133/2 antibody (Miltenyl
Biotech), anti-neurofilament 160 (clone NN18, Sigma), anti-beta
tubulin isotype III (clone SDL.3D10, Sigma), and goat anti-tau
antiserum (clone C-17, Santa Cruz), all diluted according to the
manufacturers' recommendations.
[0182] Confocal microscopy was carried out on either a Zeiss LSM
410 confocal laser scanning microscope (neural brain sections) or
on a Bio-Rad Confocal Microscope System 1024ES (neurosphere
cultures). Images were obtained under conditions that were
identical for both fluorochromes. Confocal images of XY and YZ
planes confirmed co-localization in brain sections.
In Situ Hybridization
[0183] Non-radioactive in situ hybridization of sections was
carried out as previously described (Mendelsohn et al., Stromal
cells mediate retinoid-dependent functions essential for renal
development. Development, 126:1139-48, 1999). The antisense ATF5
probe was synthesized using T3 RNA polymerase, and the
pCMS-eGFP-ATF5 construct was digested with NheI as the template.
The corresponding sense probe was synthesized using T7 RNA
polymerase, and the pCMS-eGFP-ATF5 construct was digested with NotI
as the template.
Transient Transfections
[0184] For PC12 cells, transfection was carried out using 0.5 .mu.g
of plasmid well and 6 .mu.l/well of LipofectAMINE 2000, for 9 h.
Thereafter, the cells were re-fed with fresh culture medium, and
then handled as described. For telencephalic cells, transfection
was performed with 2.0 .mu.g of plasmid/well and 2 .mu.l/well of
LipofectAMINE 2000 for 7 h followed by an exchange of medium. For
transfection of ATF5 siRNA (AAN19; AAG UCA GCU GCU CUC AGG UAC (SEQ
ID NO:16)), 6.67 .mu.g/well of pCMS-EGFP vector were mixed with 80
pmol/well of siRNA in 100 .mu.l of DMEM medium. An equal amount of
DMEM medium, premixed with 1 .mu.l of LipofectAMINE 2000/well, was
added to, and mixed with, the vector and siRNA. After 30 min, the
final mixture was added to 1/6 the volume containing the cells, and
the cells were re-fed with fresh culture medium after 7 h of
transfection. For the control, telencephalic cells were transfected
with pCMS-EGFP vector alone.
Retrovirus Production and Infection of Telencephalic Cells
[0185] Nonreplicating retrovirus was made by transfecting
subconfluent GP2 293 cells (grown in DMEM plus 10% FBS) with 5
.mu.g of QCX-eGFP or pLeGFP, and 5 .mu.g of pVSV-G, for production
of empty eGFP retrovirus (as described by Clontech). Similarly, GP2
293 cells were transfected with 5 .mu.g of QC-FLAG-ATF5-eGFP or
pLeGFP-FLAG-NTAzip-ATF5, and 5 .mu.g of pVSV-G, to make the
bicistronic FLAG-ATF5-eGFP or fusion eGFP-FLAG-NTAzip-ATF5
retroviruses, respectively. After 48 h, medium was collected, and
the virus was concentrated by centrifugation at 50,000.times.g, at
4.degree. C. The final titer was approximately 1.times.10.sup.6
virus particles per ml. The telencephalic cells were infected with
5-10 .mu.l of retrovirus, one day after plating, and the cells were
fixed 7 days after infection.
Scoring of Neuronal Differentiation
[0186] Transfected cells were detected by positive immunostaining
for eGFP. Co-staining with anti-FLAG established that the
GFP-positive PC12 cells also expressed ATF5 constructs. NGF-treated
PC12 cells (transfected unless otherwise noted) were scored for
processes of length greater than two cell diameters (about 20
.mu.m) (Greene et al., Culture and Experimental Use of the PC12 Rat
Pheochromocytoma Cell Line. In: Culturing Nerve Cells, 2.sup.nd
ed., Goslin, G. K., ed. (Cambridge, Mass.: The MIT Press, 1998) pp.
161-87. Transfected telencephalic neurons were scored for the
presence of processes with lengths greater than two cell diameters
(about 20 .mu.m) and for co-staining with TUJ1, nestin, or NF-M
antisera antibodies.
CRE-Luciferase Reporter Assay
[0187] PC12 cells were co-transfected with 1 .mu.g of pCMS-eGFP
(empty, or containing FLAG-tagged-ATF5 or FLAG-tagged NTAzip-ATF5)
and with 0.2 .mu.g of pGl3-CRE-luciferase reporter and 1 .mu.g of
LacZ plasmid per well; cells were then transfected with 2
.mu.l/well of LipofectAMINE 2000 24 h prior to harvesting. The
cells were treated with NGF for a total of 1 h to 3 days.
Luciferase levels were assayed using the Promega Luciferase System
with Reporter Lysis Buffer, as described by the manufacturer. The
level of LacZ activity was measured, as previously described
(Sambrook et al., Molecular Cloning. In: A Laboratory Manual,
2.sup.nd ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press, 1989) pp. 16-66.
Statistical Analysis
[0188] Multiple comparisons among the data from different plasmid
transfections and retrovirus infections were achieved using Tucky's
one way ANOVA test. Comparisons for pairs of data were conducted
with Student's t-distribution test.
[0189] Results
Reciprocal Effects of NGF on ATF5 Protein Expression and Neurite
Outgrowth
[0190] The inventors' previous findings revealed that long-term NGF
treatment promotes a 25-fold down-regulation of ATF5 transcripts in
PC12 cells (Angelastro et al., Identification of diverse nerve
growth factor-regulated genes by serial analysis of gene expression
(SAGE) profiling. Proc. Natl. Acad. Sci. USA, 97:10424-429, 2000).
To determine whether this is reflected at the level of protein
expression, the inventors cloned the coding sequence of rat ATF5
(GenBank/EBI accession number AY123225), and an antiserum was
raised against a peptide corresponding to a portion of the deduced
sequence of the DNA-binding domain. Western immunoblotting with
this antiserum detected a single major band in extracts of PC12
cells (FIG. 1A), and in extracts of HEK-293 cells, primary human
neuroblastoma, and mouse brain (data not shown), with an apparent
molecular mass of 20-22 kDa. The nucleotide sequence of rat ATF5
indicates two potential in-frame Kozak start sites, and the
apparent molecular mass of 20-22 kDa indicates preferential use of
the second.
[0191] A time course of ATF5 protein expression in PC12 cells, in
response to NGF treatment, revealed a drop in levels by 1 day, and
a progressive fall thereafter, with relatively little detectable
expression by day 10 (FIGS. 1A and 113). Quantification of neurite
outgrowth in the same sets of cultures revealed a reciprocal
relationship with ATF5 expression (FIG. 1B).
Exogenous ATF5 Represses NGF-Promoted Neurite Outgrowth, while a
Dominant-Negative ATF5 Accelerates Initial Neuritogenesis
[0192] The inverse behaviors of ATF5 expression and neurite
outgrowth suggested a possible causal relationship. To test this,
FLAG-tagged ATF5 was subcloned into the pCMS-eGFP vector, and
transfected into PC12 cells. Two days later, NGF was added, and the
transfected cells (expressing eGFP and tagged ATF5) were assessed
over time for the appearance of neurites. In contrast to cells
transfected with empty vector, those expressing exogenous ATF5
showed markedly repressed genesis of neurites over a 5-day time
course (FIGS. 2A and 2B).
[0193] To assess the possibility that exogenous ATF5 might act, at
least in part, by non-physiologically sequestering and "squelching"
the actions of binding partners, the inventors also prepared a
construct encoding an N-terminally-truncated form of FLAG-tagged
ATF5, possessing an enhanced b-Zip domain (NTAzip-ATF5). This was
achieved by deleting the N-terminal acidic activation domain, and
replacing the DNA-binding domain with an amphipathic acidic
.alpha.-helical sequence containing leucine repeats at each seventh
residue.
[0194] Without activation and DNA-binding domains, NTAzip-ATF5 does
not interact with DNA or directly affect gene transcription.
However, because this protein includes the intact ATF5 leucine
zipper, it retains specific interactions with endogenous ATF5 and
with heterologous binding partners. In addition, the Azip
amphipathic acidic .alpha.-helical domain should tightly associate
with the basic DNA-interaction domains of ATF5-binding partners,
thereby blocking their functions (Vinson et al., Dimerization
specificity of the leucine zipper-containing bZIP motif on DNA
binding: prediction and rational design. Genes Dev., 7:1047-58,
1993; Kxylov et al., Extending dimerization interfaces: the bZIP
basic region can form a coiled coil. EMBO J., 14:5329-37, 1995;
Moitra et al., Life without white fat: a transgenic mouse. Genes
Dev., 12:3168-81, 1998; Moll et al., Attractive interhelical
electrostatic interactions in the proline- and acidic-rich region
(PAR) leucine zipper subfamily preclude heterodimerization with
other basic leucine zipper subfamilies. J. Biol. Chem.,
275:34826-832, 2000). Thus, if exogenous ATF5 acts by non-specific
squelching, rather than by binding to DNA, NTAzip-ATF5 should have
a similar effect. However, in contrast to ATF5, NTAzip-ATF5 did not
block NGF-promoted neurite outgrowth (FIG. 2B), thus ruling out a
non-specific action of the former.
[0195] In addition to serving as a control for non-specific
squelching, NTAzip-ATF5 acts as a dominant-negative for ATF5,
thereby permitting evaluation of the consequences of ATF5
loss-of-function. In the absence of NGF, transfected NTAzip-ATF5
did not stimulate neurite outgrowth (data not shown). However,
cells transfected with NTAzip-ATF5, and then exposed to NGF, showed
a significantly faster (two-fold) initial appearance of neurites,
as compared with controls (FIG. 2C). This reinforces the notion
that a physiologic function of ATF5 is suppression of neurite
outgrowth, and that its down-regulation is required for this
process to occur. After the first 1-2 days of NGF treatment, the
effect of NTAzip-ATF5 is much less apparent, presumably due to
down-regulation of endogenous ATF5.
ATF5 is Highly Expressed in Ventricular Zones of Developing
Brain
[0196] The suppression of neurite outgrowth by ATF5 in PC12 cells,
and the potential suitability of this system for modeling the
transition of neural progenitor cells to differentiated
post-mitotic neurons, led the inventors to examine expression of
ATF5 in the developing nervous system. In situ hybridization
revealed specific expression of ATF5 transcripts in E12-15 rat
neural nasal epithelium (see, also, Hansen et al., Mouse Atf5:
molecular cloning of two novel mRNAs, genomic organization, and
odorant sensory neuron localization. Genomics, 80:344-50, 2002),
dorsal root and trigeminal ganglia, and brain (FIG. 3 and data not
shown). The only signal of comparable strength detected outside the
nervous system at these stages was in liver (data not shown).
Within E12-15 rat brain, expression was highest in the ventricular
zone (VZ) of the neural epithelium adjacent to the lateral
ventricles and the fourth ventricle--sites of intense proliferation
of neural cell precursors--and was decreased in overlying
structures containing migrating and post-mitotic neurons (FIG. 3A,
panels a and b).
[0197] In view of the pattern of ATF5 transcripts in developing
brain, the inventors next examined ATF5 protein expression in
developing brain, using immunohistochemistry. ATF5 protein was
strongly expressed in the VZ of E12 and E14 telencephalon, and fell
to undetectable levels toward the surface of the developing cortex
(FIG. 3A, panels c-f; FIG. 3B). Double staining with the TUJ1
antibody that recognizes tubulin .beta.III, a marker for
post-mitotic neurons (Lee et al., Posttranslational modification of
class III beta-tubulin. Proc. Natl. Acad. Sci. USA, 87:7195-99, 1
990), showed a converse pattern of staining (FIGS. 3A and 3B),
indicating that ATF5 is highly expressed in proliferating neural
progenitor cells and undetectable in differentiated neurons. A
comparable pattern was also observed in E14 rat embryo
telencephalon, at higher magnification, using confocal microscopy
(FIG. 3B). At E17, ATF5 expression remained largely confined to the
VZ, in contrast to the large expansion of TUJ1-positive staining in
the cortical area (FIGS. 4A-4F).
ATF5 is a Marker for Neural Stem/Progenitor Cells, but not for
Mature Neurons in Clonal Neural Progenitor Cell Cultures
[0198] The above findings indicate that ATF5 is highly expressed in
proliferating PC12 cells and in VZ progenitor cells, but not in
post-mitotic neurons. To further examine the correlation between
ATF5 expression and neuronal differentiation, the inventors
prepared cultures of neural progenitor cells from the neurogenic
subventricular zone or hippocampal dentate gyrus of newborn mouse
brain. Clones derived from single-cell suspensions were expanded
and cultured as neurospheres, under non-adherent conditions, in the
presence of EGF, bFGF, and insulin, and then plated onto
poly-L-ornithine and laminin, with 10% fetal bovine serum, to
trigger substrate attachment and neurogenesis (Kukekov et al.,
Multipotent stem/progenitor cells with similar properties arise
from two neurogenic regions of adult human brain. Exp. Neurol.,
156:333-44, 1999; Laywell et al., Identification of a multipotent
astrocytic stem cell in the immature and adult mouse brain. Proc.
Natl. Acad. Sci. USA, 97:13883-888, 2000). Cells at the centers of
the cultured neurospheres proliferate as stem/progenitor cells,
while those that migrate to the culture periphery differentiate
into neurons and glia (FIG. 5E).
[0199] ATF5 expression was very high at the 3-dimensional core of
the cultures. Co-staining with antibodies to the AC133 antigen, a
marker for hematopoietic and neural stem cells (Yin et al., AC133,
a novel marker for human hematopoietic stem and progenitor cells.
Blood, 90:5002-12, 1997; Uchida et al., Direct isolation of human
central nervous system stem cells. Proc. Natl. Acad Sci. USA,
97:14720-25, 2000; Bhatia, AC133 expression in human stem cells.
Leukemia, 15:1685-88, 2001; Yu et al., AC133-2, a novel isoform of
human AC133 stem cell antigen. J. Biol. Chem., 277:20711-716,
2002), revealed extensive co-expression with ATF5 in this region
(FIG. 5A). AC133 antigen localization appeared to be largely at the
cell surface and plasma membrane, while ATF5 appeared to be mainly
localized to nuclei. ATF5 was also extensively expressed in cells
positive for nestin (FIG. 5B), an intermediate filament expressed
by neuroectodermal progenitors (Lendahl et al., CNS stem cells
express a new class of intermediate filament protein. Cell,
60:585-95, 1990).
[0200] Co-localization experiments were also carried out with ATF5
and neuronal markers. The 160-kDa neurofilament protein, NF-M, was
detected in cells outgrowing towards the culture periphery. A
sub-population of such cells, which generally appeared to have
short, neurite-like processes, co-stained for nuclear ATF5 (FIG.
5C). For such cells, staining of ATF5 and NF-M appeared to be of
relatively low intensity, indicating that these were immature
neuronal cells in transition with rising levels of NF-M expression
and falling levels of ATF5. Another population of cells, with more
advanced neuronal morphology, strongly stained for NF-M, but was
negative for expression of ATF5 (FIG. 5D). Finally, co-staining
with antiserum, for the neuronal marker, tau (Takemura et al., In
situ localization of tau mRNA in developing rat brain.
Neuroscience, 44:393-07, 1991), revealed a set of tau-positive
cells, at the periphery of the cultures, with clear neuronal
morphology (FIGS. 5E and 5F). Unlike the progenitor cells in the
centers of the cultures, that were positive for ATF5 expression and
negative for tau, the tau-positive cells in the periphery did not
co-stain for ATF5. Taken together, these observations indicate that
ATF5 is expressed in neural stem (AC133+) and progenitor (nestin+)
cells, including those committed to the neuronal lineage, and are
down-regulated in differentiated, post-mitotic neurons (tau+).
ATF5 Represses, but Dominant-Negative ATF5 and ATF5 Small
Interfering RNA Accelerate, Neuronal Differentiation of Neural
Progenitor Cells
[0201] The above-described expression pattern of ATF5 raised the
possibility that the presence of this protein, as in PC12 cells,
may block proliferating neural progenitor cells from undergoing
neuronal differentiation. To assess this, rat E14 telencephalic
cell cultures containing a mixture of proliferating progenitor
cells and post-mitotic neurons, and a small number of glial cells
(Ghosh and Greenberg, Distinct roles for bFGF and NT-3 in the
regulation of cortical neurogenesis. Neuron, 15:89-03, 1995), were
transfected with pCMS-eGFP containing either no insert, FLAG-ATF5,
or FLAG-NTAzip-ATF5. Transfected cells (identifiable by eGFP
expression) were scored 3 days later for neuronal morphology and
expression of nestin and tubulin .beta.III (FIG. 6A). In contrast
with the cells transfected with empty vector, few cells transfected
with ATF5 exhibited neuronal morphology.
[0202] In addition, ATF5 greatly repressed expression of the
neuronal marker, tubulin .beta.III. On the other hand, ATF5
significantly increased the proportion of cells expressing nestin,
a marker for neural progenitor cells. NTAzip-ATF5 did not mimic
ATF5, ruling out a potential non-physiological squelching action of
ATF5, as in the case of PC12 cells. In comparison with control
transfectants, somewhat fewer cells transfected with NTAzip-ATF5
expressed nestin, although a greater number tended to express
neuronal markers.
[0203] To ensure initial expression only in proliferating cells of
the inventors' telencephalic cell cultures, and to permit transgene
delivery at an early point after establishment of the cultures
(which was technically unfeasible with the inventors' transfection
conditions), the inventors constructed, and infected the cells at 1
clay in vitro with, retroviral vectors expressing either eGFP,
eGFP-FLAG-NTAzip-ATF5, or FLAG-ATF5 and eGFP. In this paradigm,
ATF5 once again suppressed neurite outgrowth and expression of
neuronal markers (NF-M and TUJ1), and led to an increase in
proportion of nestin-positive cells at either 7 (FIG. 6B) or 4
(FIG. 6C) days after infection. Moreover, loss of function of
endogenous ATF5, promoted by NTAzip-ATF5, significantly enhanced
the genesis of neurite-bearing, TUJ1-positive cells in cultures
assessed at 3 (data not shown) and 4 (FIG. 6C) days following viral
exposure. The double-negative construct also promoted a fall in
nestin positive cells, which presumably reflected the increase in
neuronal differentiation. The increase in TUJ1-positive cells was
greater than can be accounted for by the fall of nestin-positive
cells, indicating either that the antibody the inventors employed
led to an underestimation of the numbers of nestin-positive
progenitor cells in the cultures, or that at least some neurons
were generated from a population of nestin-expressing
progenitors.
[0204] To corroborate the inventors' findings that NTAzip-ATF5
accelerates neurogenesis by specifically interfering with the
function of endogenous ATF5, rather than through non-specific
actions, the inventors employed small interfering RNA (siRNA) to
selectively down-regulate endogenous ATF5. After 3 days in vitro,
E14 telencephalic cells were transfected with GFP or with GFP plus
ATF5 siRNA. On the fourth day after transfection with the siRNA,
the proportion of transfected cells with detectable endogenous ATF5
fell by 96%, as compared with controls (FIG. 6D). Significantly,
the reduction of endogenous ATF5 resulted in a 3.4-fold increase in
neurogenesis, as judged by the appearance of TUJ1 staining (FIG.
6D) and neurite outgrowth (data not shown). In contrast, an
irrelevant siRNA synthesized to target down-regulation of the
protein, POSH, had no effect on development of neuronal markers or
processes. Taken together, these findings support a model in which
ATF5 suppresses the transition between neural progenitor cells and
post-mitotic neurons, and in which loss of, or interference with,
ATF5 function accelerates neuronal differentiation.
[0205] The limited degree of neuronal differentiation in the
telencephalic cultures appears to occur in response to endogenous
factors. To determine whether ATF5 can also regulate CNS neuronal
differentiation promoted by a defined trophic agent, the inventors
tested the effects of exogenous ATF5 and NTAzip-ATF5 in the
presence and absence of NT3, a neurotrophin previously reported to
drive telencephalic progenitor cell differentiation into neurons
(Ghosh and Greenberg, Distinct roles for bFGF and NT-3 in the
regulation of cortical neurogenesis. Neuron, 15:89-03, 1995). As
shown in FIG. 6E, NT3 nearly tripled the level of neurogenesis in
the cultures, and ATF5 suppressed this by 5- to 6-fold.
Contrastingly, NTAzip-ATF5 had no significant effect on
neurogenesis in the presence of NT3--unlike its marked promotion of
neuronal differentiation in the absence of NT3. The latter
observation would suggest that neuronal differentiation in the
cultures is maximally stimulated by NT3, and cannot be further
promoted by interfering with endogenous ATF5 activity. Furthermore,
it appears that NT3 leads to down-regulation of endogenous ATF5, as
none of the neurons formed in its presence exhibited detectable
ATF5 immunostaining (data not shown). In conclusion, these findings
indicate that, as in the case of NGF, NT3 promotes neurogenesis by
a mechanism that can be suppressed by exogenous ATF5, and which
includes loss of endogenous ATF5 expression.
Inhibition of Neurite Outgrowth by ATF5 Involves Repression of CRE
Transactivation
[0206] The work of Peters et al (ATF-7, a novel bZIP protein,
interacts with the PRL-1 protein-tyrosine phosphatase. J. Biol.
Chem., 276:13718-26, 2001) has established that ATF5 homodimers
specifically bind to CRE elements, and that there is evidence that
CRE plays an important role in neuronal differentiation and
maintenance (Finkbeiner et al., CREB: a major mediator of neuronal
neurotrophin responses. Neuron, 19:1031-47, 1997). Hence, the
inventors next determined whether ATF5 regulates CRE activity in
neuronal cells, and whether this action plays a role in
ATF5-mediated suppression of neuronal differentiation.
[0207] The inventors also wished to determine whether the presence
of NGF would affect the capacity of ATF5 to regulate CRE activity.
Accordingly, PC12 cells were co-transfected with a CRE-luciferase
reporter construct, a lacZ expression construct (for normalization
of transfection efficiency), and pCMS-eGFP containing either no
insert, FLAG-ATF5, or FLAG-NTAzip-ATF5. One day later, the cells
were harvested and assessed for reporter activity. A portion of the
cultures were treated with NGF for 2 days prior to, and during, the
24 h after transfection (3-day NGF treatment); others were either
unexposed to NGF, or exposed to the factor at the time of
transfection (1-day NGF treatment) or during the last hour before
harvesting (1-h NGF treatment).
[0208] Without NGF treatment, or after 1 h of NGF treatment, there
was relatively little constitutive CRE transactivation. The effect
of exogenous ATF5 was somewhat variable at this time, with
suppression of activity in some experiments and not others (FIGS.
7A and 7B), possibly reflecting cell-culture conditions. At 1 day
with NGF, there was a small (50%), but statistically significant,
increase in CRE activity, in comparison with naive cells; this was
reduced to baseline by exogenous ATF5. At day 3, there was a
10-fold increase in CRE reporter activity, as compared with
untreated cells, and this was again substantially reduced by
exogenous ATF5. NTAzip-ATF5 did not reduce CRE activity, thereby
making it unlikely that ATF5 interferes with CRE transactivation by
non-physiologic interaction with CRE-regulatory proteins. Moreover,
neither ATF5 nor NTAzip-ATF5 expression suppressed expression of a
SRE reporter (data not shown). In addition to establishing that
ATF5 suppresses CRE transactivation in intact neuronal cells, these
findings indicate that NGF elevates basal CRE activity, and that
this occurs at a time when endogenous ATF5 levels have fallen by
about 2/3 (FIG. 1).
[0209] If ATF5 suppresses neuronal differentiation by binding to
CRE and inhibiting its transactivation, then one might predict that
this action should be reversed, either by a dominant-negative ATF5
protein without DNA binding or activation sites, or by a strong
competitive CRE activator. The former characteristics are fulfilled
by NTAzip-ATF5, which should form tight heterodimers with ATF5, but
does not bind DNA. In support of the inventors' hypothesis,
co-expression of NTAzip-ATF5 blocked inhibition of CRE reporter
activity by ATF5 (FIG. 7B), and reversed ATF5-dependent suppression
of NGF-promoted neurite outgrowth (FIG. 7C).
[0210] With respect to a competitive CRE activator, the inventors
employed VP16-CREB, a constitutively-active form of the CRE-binding
protein, CREB (Lu et al., The herpesvirus transactivator VP16
mimics a human basic domain leucine zipper protein, luman, in its
interaction with HCF. J. Viral., 72:6291-97, 1998; Barco et al.,
Expression of constitutively active CREB protein facilitates the
late phase of long-term potentiation by enhancing synaptic capture.
Cell, 108:689-03, 2002). Co-transfection of pCMS-eGFP-VP16-CREB
into PC12 cells produced strong transactivation of the CRE reporter
(FIG. 7A), and this was essentially unaffected by the additional
co-transfection of FLAG-ATF5 (FIG. 7B).
[0211] The inventors next assessed whether driving CRE with
VP16-CREB would reverse the actions of ATF5 on neurite outgrowth.
Transfection of PC12 cells with pCMS-eGFP-VP16-CREB alone did not
elicit neurite outgrowth in the absence of NGF, and, as in the ease
of FLAG-NTAzip-ATF5, enhanced the initial rate of neuritogenesis in
the presence of NGF (FIG. 7C). Significantly, co-transfection of
VP16-CREB, along with FLAG-ATF5, reversed the suppression of
NGF-stimulated neurite outgrowth that was achieved with ATF5 alone
(FIG. 7C). Taken together, these findings further support a model
in which CRE transactivation is required for neuronal
differentiation, but is reversibly blocked by ATF5.
Regulation of Endogenous ATF5 Protein in PC12 Cells and Neural
Progenitor Cells
[0212] In consonance with their past observations of ATF5
transcripts, the inventors found that ATF5 protein is expressed in
PC12 cells, and drops to nearly undetectable levels during
NGF-promoted neuronal differentiation. Similarly, both ATF5
transcripts and protein are highly expressed in neural progenitor
cells, and absent from post-mitotic neurons. The observed decrease
in ATF5 protein expression most likely reflects the down-regulation
of ATF5 transcripts. ATF5 has been reported to be a substrate for
ubiquitin-conjugating enzymes, including Cdc34 (Pati et al., Human
Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of
cyclic AMP-induced transcription for proteolysis. Mol. Cell Biol.,
19:5001-13, 1999); thus, it is likely to have a relatively rapid
turnover that would produce efficient loss of expression following
transcriptional down-regulation.
[0213] Western immunoblotting permitted the inventors to deduce the
major cellular form of ATF5 protein. The ATF5 cDNA sequence
predicts two potential in-frame methionine start sites that would
lead to proteins of approximately 30 and 20 kDa. The inventors'
observation that the major form of ATF5 in cells has an apparent
molecular mass of 20-22 kDa indicates favored utilization of the
second site. When a canonical Kozak initiation consensus sequence
was included upstream of the first methionine, the larger protein
was expressed (data not shown), thereby indicating that the 22-kDa
form is not formed by cleavage of a 30-kDa precursor.
ATF5 Represses Neuronal Differentiation of Neural Progenitor
Cells
[0214] The down-regulation of ATF5 expression by NGF in PC12 cells,
the progressive loss of ATF5 expression that occurs as cells leave
the ventricular zone and enter the developing cortex, and the
presence of ATF5 in neural stem and progenitor cells, but not in
well-differentiated neurons in neurosphere cultures, suggested to
the inventors that this factor may play a causal role in regulating
neuronal differentiation. In support of this supposition, exogenous
ATF5 suppressed both neurite outgrowth in PC12 cell cultures and
differentiation of cultured neural progenitor cells. Conversely,
loss of ATF5 function (evoked by NTAzip, an ATF5 dominant-negative)
nearly doubled the initial rate of NGF-promoted neuritogenesis by
PC12 cells, and significantly enhanced neurogenesis in
telencephalic cell cultures. In particular, an ATF5 siRNA that
effectively reduced endogenous ATF5 levels also promoted a 3.6-fold
enhancement of neurogenesis by cultured telencephalic cells.
[0215] The effect of exogenous ATF5 does not appear to be limited
solely to neurite outgrowth, as virally-induced ATF5 expression in
proliferating progenitor cells also blocked the appearance of
several neuronal markers and led to an increase in numbers of cells
that expressed nestin--a marker for neural progenitor cells. The
increase in numbers of nestin-positive cells induced by exogenous
ATF5 appeared to be greater than could be accounted for merely by
simply blocking progenitor-cell differentiation. One possible
explanation is that nestin-positive cells expressing exogenous ATF5
continued to proliferate, instead of leaving the cell cycle and
differentiating.
[0216] Taken together, the inventors' observations with developing
rat brain and neurosphere cultures indicate a scenario in which
ATF5 is highly expressed in neural stem cells and neuroprogenitor
cells, and suppresses their differentiation. The action of
appropriate neurotrophic factors leads to down-regulation of ATF5,
thereby permitting differentiation of neural progenitor cells into
neurons. Therefore, the inventors' present findings suggest that
ATF5 acts in a permissive, rather than instructional, manner, in
that it does not appear to play a role in directly specifying cell
fate per se; rather, it appears to act as a negative suppressor
that must be down-regulated to permit the transition of neural
progenitor cells to neurons. In this role, ATF5 would function to
prevent stem cells and progenitor cells from undergoing terminal
differentiation until stimulated by appropriate neurotrophic
agents.
[0217] Further support for the notion that ATF5 acts as a negative
permissive regulator, rather than as an instructional factor, comes
from the inventors' observations with NTAzip-ATF5. This modified
form of ATF5 should act as a dominant-negative that prevents
interaction of ATF5 with DNA as well as with other potential
protein-binding partners. This is borne out by the capacity of
NTAzip-ATF5 to reverse the effect of ATF5 on CRE reporter activity.
Nevertheless, when expressed in PC12 cells, NTAzip did not promote
neurite outgrowth in the absence of NGF. Thus, although ATF5
down-regulation appears to be necessary for neuronal
differentiation, loss of ATF5 activity does not appear to be
sufficient to promote this process. Factors such as NGF appear to
down-regulate negative permissive agents such as ATF5 and to
provide instructional information that actively promotes neuronal
differentiation. In the CNS neuroprogenitor cultures employed
herein, down-regulation and instructional activity were likely to
be supplied by endogenously-synthesized and released factors, such
as NT3 and BDNF (Ghosh and Greenberg, Distinct roles for bFGF and
NT-3 in the regulation of cortical neurogenesis. Neuron, 15:89-03,
1995).
[0218] The expression pattern of NGF during embryogenesis makes it
unlikely that this factor is a key regulator of ATF5 expression in
developing brain. However, many other potential neurotrophic
factors are present there that could fulfill a similar role. For
instance, BDNF and NT3, and their cognate receptors, TrkB and TrkC,
are present in rat ventricular progenitor cells at E13 and E15
(Fukumitsu et al., Simultaneous expression of brain-derived
neurotrophic factor and neurotrophin-3 in Cajal-Retzius, subplate
and ventricular progenitor cells during early development stages of
the rat cerebral cortex. Neuroscience, 84:115-27, 1998). BDNF
(Ahmed et al., BDNF enhances the differentiation but not the
survival of CNS stem cell-derived neuronal precursors. J.
Neurosci., 15:5765-78, 1995) and NT3 (Ghosh and Greenberg, Distinct
roles for bFGF and NT-3 in the regulation of cortical neurogenesis.
Neuron, 15:89-03, 1995) promote differentiation of cultured
neuronal progenitor cells.
[0219] The inventors' experiments were focused on neuronal
differentiation, and did not establish whether ATF5 also affects
glial cell differentiation. However, the following evidence
suggests that ATF5 may also be a negative regulator of astrocyte
differentiation: the localization of ATF5 in brain areas that also
give rise to glial progenitor cells; the co-localization of ATF5
with nestin, which is present in progenitor cells for both neurons
and glia; and the inventors' preliminary observations that ATF5
co-localizes with GFAP in neuroprogenitor cell cultures and that
exogenous ATF5 suppresses GFAP expression. Although ATF5 expression
negatively correlates with neuronal differentiation, this may not
be the case universally for differentiation of other cell types.
Peters et al. (ATF-7, a novel bZIP protein, interacts with the
PRL-1 protein-tyrosine phosphatase. J. Biol. Chem., 276:13718-26,
2001) reported that ATF5 transcripts were markedly elevated when
human Caco-2 cells reached confluency and spontaneously
differentiated into a brush-border-bearing polarized cell
layer.
Suppression of Neuronal Differentiation by ATF5 Involves CRE
[0220] Based on reports that ATF5 homodimers bind CRE, but not
C/EBP or API, sites (Peters et al., ATF-7, a novel bZIP protein,
interacts with the PRL-1 protein-tyrosine phosphatase. J. Biol.
Chem., 276:13718-726, 2001), and that ATF5 represses cAMP-mediated
activation of a CRE reporter in JEG3 cells (Pati et al., Human
Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of
cyclic AMP-induced transcription for proteolysis. Mol. Cell Biol.,
19:5001-13, 1999), the inventors examined the effect of ATF5 on the
activity of a CRE reporter in PC12 cells. The inventors' findings
confirm that ATF5 suppresses cellular CRE transactivation. As
discussed above, it is significant that NTAzip-ATF5 did not mimic
the suppressive actions of ATF5 on neurite outgrowth and CRE
activity; rather, it antagonized these effects, thereby indicating
that ATF5 acts by binding to DNA, instead of by non-specific
"squelching" of binding partners.
[0221] The inventors observed that basal CRE activity substantially
increased by 3 days of NGF treatment. One potential cause for this
is the concurrent fall in endogenous ATF5 expression, and
subsequent loss of ATF5-mediated CRE repression; however, the
inventors cannot rule out the possibility that NGF regulates
additional proteins that affect CRE activity.
[0222] Although NTAzip-ATF5 blocked the inhibitory effects of
exogenous ATF5 on its own, it had no, or relatively little, effect
on CRE reporter activity. If, as the inventors propose,
CRE-dependent gene activation is suppressed by endogenous ATF5,
then it might have been anticipated that basal CRE activation would
be elevated in response to NTAzip-ATF5. Since this was not the
case, this raises the possibility that one or more factors, in
addition to ATF5, act to suppress CRE in neural progenitor cells,
and that these are also down-regulated during neuronal
differentiation.
[0223] To assess whether interference with CRE-mediated gene
regulation might account for the mechanism by which ATF5 interferes
with neuronal differentiation, the inventors co-expressed it with
VP16-CREB, a constitutively-active fusion protein that includes the
CREB DNA-binding domain and transactivation domain of the HSV VP16
protein. VP16-CREB potently activated the CRE reporter, and this
effect was not blocked by co-expression of ATF5. Significantly,
co-expressed VP16-CREB overrode ATF5-mediated inhibition of neurite
outgrowth. This finding supports a model in which neuronal
differentiation requires CRE-mediated gene activation, and in which
such activation is repressed in neural progenitor cells by factors
such as ATF5. In this light, it is of interest that PACAP, a potent
activator of adenylate cyclase, promotes mitotic exit and neuronal
differentiation of cultured cortical neuron precursor cells
(Dicicco-Bloom et al., The PACAP ligand/receptor system regulates
cerebral cortical neurogenesis. Ann. N.Y. Acad. Sci., 865:274-89,
1998), and that NGF-promoted differentiation of PC12 cells is
synergized by cell-permeant cAMP derivatives (Gunning et al.,
Differential and synergistic actions of nerve growth factor and
cyclic AMP in PC12 cells. J. Cell Biol., 89:240-45, 1981).
[0224] In summary, the inventors' findings indicate that both
positive and negative regulators govern the transition of neural
progenitor cells to neurons. On one hand, ATF5 is highly expressed
in neural stem cells and neuroprogenitor cells, and suppresses
their neuronal differentiation, apparently by competing for binding
to CREs. On the other hand, neuronal differentiation is accompanied
by, and appears to require, down-regulation of ATF5 expression.
This can be accomplished by neurotrophic factors such as NGF and
NT3. Though such down-regulation may be necessary, it is not
sufficient to permit neuronal differentiation. The latter also
appears to require instructive signals that may be imparted by
neurotrophic factors and/or activators of adenylate cyclase.
Example 2
[0225] Materials and Methods
Reagents
[0226] Cell culture medium DMEM, molecular biology reagents and
LipofectAMINE 2000 were from Invitrogen, Inc. Fetal bovine serum
was from JRH Biosciences. Mouse monoclonal anti-GFP IgG.sub.1 was
from Sigma. Goat anti-mouse Alexa 488, Alexa 568, goat anti-mouse
IgG.sub.2a Alexa 488, goat anti-mouse IgG.sub.1 Alexa 568, goat
anti-rabbit Alexa 488 and 568, and mouse IgG.sub.2a anti-GFP
antibody were from Molecular Probes. Rabbit anti-GFP antibody was
from BD Biosciences (Clontech). Rabbit anti-Ki67 was from
Novacastra. Normal 10% goat-serum was from Zymed. Polyclonal rabbit
anti-ATF5 was as previously described (7).
Immunostaining of Human Glioblastomas
[0227] Paraffin sections (10 .mu.m) of surgically excised
glioblastoma multiforme tumors (WHO, Grade IV) were provided by the
Department of Pathology, Columbia University. Paraffin was removed
by heating the sections at 60.degree. C. for one to two hours
followed by 3 incubations in 100% xylene for 5 min each. Subsequent
incubations were in 100%, 95%, 75% and 50% and 0% ethanol for 5 min
each. The sections were then subjected to antigen retrieval by
incubation in 10 mM citrate buffer (pH=6.0) at 100.degree. C. in a
Black & Decker HS 800 steamer for 40 min. Endogenous peroxidase
was blocked by incubation with 0.3% hydrogen peroxide for 10 min
followed by 3 washes in water. The tissue was then permeabilized by
incubation with 0.04% Tween 20 in TBS (3.times. for 5 min each) and
immunostained with ATF5 antiserum (1:600) in PBS containing 1% BSA
for one hour at room temperature. Visualization was achieved with
DAB reagent following the manufacturer's protocol (DAKO, Envision
System kit). The sections were counterstained with light
hematoxylin to reveal nuclei and cellular morphology. Antiserum
against the Ki67 antigen (1:1000) was used as a positive
immunostaining control.
Cell Culture
[0228] Glioma cell lines rat C6 (13) and RG2 (14), and human U87
(15), U373 (15), U251 (15), T98 (16), U138 (15), and DBTRG-05 (17)
were grown in DMEM medium supplemented with 10% fetal bovine serum.
Cells were passaged into 24-well culture dishes for transfections.
Primary astrocytes were obtained by the method of Levison and
McCarthy (18) and were passaged up to 5 times with trypsin and
grown in DMEM medium plus 10% fetal bovine serum.
Transient Transfections
[0229] pLeGFP mock, pLeGFPfusionFlag-Tagged-NTAzip-ATF5,
pSIREN-RetroQ-ZsGreen encoding 21 bp complementary hairpin
loop-siRNA Luciferase mock control and pSIREN-RetroQ-ZsGreen-U 21
bp complementary hairpin loop-siRNA rat ATF5
(GATCCGTCAGCTGCTCTCAGGTACTTCAAGAGAGTACCTGAGAGCAGCTGACCTTTT TTCTAGAG
(SEQ ID NO:17) were transfected into cell monolayers in 24-well
dishes using 1 .mu.g of plasmid/well and 2 .mu.l/well of
LipofectAMINE 2000 for 9 hours, after which time the cells were
re-fed with fresh culture medium.
[0230] For transient transfections of oligo ribonucleotide
duplexes, 80 pmole/well of ATF5 siRNA (AAN.sub.19; rat AAG UCA GCU
GCU CUC AGG UAC (SEQ ID NO:18) or human AAG UCG GCG GCU CUG AGG
UAC) (SEQ ID NO:19) oligo ribonucleotide duplexes (Qiagen) and 1
.mu.g/well of pCMS-EGFP vector were incubated with cells in 100
.mu.l of DMEM medium and 2 .mu.l/well of LipofectAMINE 2000 for 9
hours followed by an exchange of medium. For the control, cells
were transfected with pCMS-EGFP vector alone.
In Vivo Induction of ATF5 Loss-of-Function
[0231] Non-replicating retroviruses encoding eGFP or
Flag-Tagged-NTAzip-ATF5 were prepared as previously described
(Angelastro, et al. Regulated expression of ATF5 is required for
the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003). Adult rats were deeply
anesthetized using ketamine and xylazine, and access to the brain
was achieved by drilling a 0.5 mm diameter hole into the skulls of
the animals 1 mm anterior and 3 mm lateral to the bregma on the
right side and stereotactically injecting cells 1.times.10.sup.4 in
5 .mu.l and at a depth of 3.5 mm. After 10 days of tumor growth,
the retroviruses (1.25.times.10.sup.4 CFU in 5 .mu.l) was
stereotactically injected into the growing tumors, using the same
coordinates. Three days later, the rats were deeply anesthetized
using ketamine and xylazine and were perfused transcardially with
PBS, which was followed by 4% paraformaldehyde in PBS. The brains
were removed and were post-fixed overnight in 4% paraformaldehyde,
and were subsequently cryoprotected in 30% sucrose for two days.
The brains were frozen in O.C.T. compound (Tissue-TEK) and then
cryosectioned (10 .mu.m coronal sections). Sections were stained
for TUNEL following the manufacturer's protocol (Roche in Situ Cell
Death Detection Kit, TMR Red, Cat. No. 2 156 792). The sections
were blocked overnight at 4.degree. C. with 10% goat serum in PBS
containing 0.3% Triton X-100 (PBS-T) and then incubated overnight
at 4.degree. C. with rabbit anti-GFP antibody (1:500, Clontech) in
PBS-T. After washing with PBS-T, the sections were then
immunostained with goat anti-rabbit Alexa 488 secondary antibody
(1:1000) for 2 hr at room temperature and then washed with PBS-T.
Nuclei were stained with Hoechst dye 33342 (1 .mu.g/ml) for 5 min
and the sections were coverslipped with Gel/mount slide mounting
medium.
Quantitative Assessment of Cell Death
[0232] Transfected cell cultures were fixed and immunostained for
the expression of eGFP as previously described Angelastro et al.
(Angelastro, et al. Regulated expression of ATF5 is required for
the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003) and then incubated with Hoechst dye
33342 at 1 .mu.g/ml in PBS and 0.3% Triton X-100 for 5 min at room
temperature to detect apoptotic nuclei (Angelastro, et al.
Characterization of a novel isoform of caspase-9 that inhibits
apoptosis. J. Biol. Chem., 276: 12190-12200, 2001). eGFP+ cells
possessing condensed nuclei and fragmented chromatin were scored as
apoptotic. For brain sections, eGFP+ cells were located and scored
for the presence or absence of TUNEL labeling. Only cells with
TUNEL positive nuclei (as indicated by co-staining with Hoechst dye
33342) were scored. Tumors were well demarcated and examination of
the sections by phase microscopy and with respect to nuclear
staining indicated whether cells were within or outside of the
margins of the tumors. Separate staining of additional sections for
either eGFP or TUNEL alone revealed no cross-over of signals. This
was also verified in the double-stained sections by the presence of
cells that were either TUNEL+ and eGFP- or vice versa.
Human Glioblastomas Express Nuclear ATF5
[0233] As discussed above, proliferative neural progenitor cells
express high levels of nuclear ATF5 whereas mature neurons and glia
express little or no detectable levels of this protein (Angelastro,
et al. Regulated expression of ATF5 is required for the progression
of neural progenitor cells to neurons, J. Neurosci., 23: 4590-4600,
2003). Therefore, the inventors assessed whether ATF5 might be
expressed in highly proliferative glial tumors. A series of 29
surgically resected human glioblastoma multiforme tumors (GBM, WHO
Grade IV) were immunostained with ATF5 antiserum (McLendon, et al.
Tumors of central neuroepithelial origin., p. 307-571, 1998;
Kleihues, et al. Histology Typing of Tumours of the Central Nervous
System, Berlin: Springer-Verlag, 1993). Positive specific nuclear
staining was seen in the majority of glioma cells within all 29
tumors (FIG. 10). Tumor cells were identified on the basis of
cytologic atypia. ATF5 staining was also seen in some cells with
relatively round nuclei, which may represent reactive astrocytes,
and in some endothelial cells in regions of microvascular
proliferation (not shown). In contrast, there was little or no
staining of neurons in the surrounding tissue.
[0234] We also examined ATF5 expression by 6 well-characterized
human and 2 rat glioma cell lines. All eight lines expressed
nuclear ATF5 with 60-100% of the cells showing positive staining
(FIG. 11). Western blotting confirmed the presence of ATF5 protein
in these lines as a single 22 kDa band (FIG. 11 and data not
shown). In contrast to the gliomas lines, cultured normal
non-neoplasmic rat astrocytes isolated from neonatal rats, in first
or second passage, as in vivo, showed little or no ATF5 expression
as assessed by immunostaining and western immunoblotting (FIG.
11A). However, 60% of the cultured astrocytes expressed the protein
when activated by 4-5 passages in vitro (FIG. 11B).
Interfering with the Function or Expression of ATF5 Promotes
Apoptosis of Glioma Cells, but not Activated Astrocytes, In
Vitro
[0235] We have observed that interfering with the expression or
function of ATF5 in neural progenitor cells causes them to exit the
cell cycle and to undergo accelerated differentiation (Angelastro,
et al. Regulated expression of ATF5 is required for the progression
of neural progenitor cells to neurons. J. Neurosci., 23: 4590-4600,
2003). We therefore next determined whether glioma cells respond
similarly. To interfere with function, we transfected the glioma
lines with a dominant negative ATF5 construct (eGFP-NTAzip-ATF5)
(Angelastro, et al. Regulated expression of ATF5 is required for
the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003). Surprisingly, all 7 lines tested
responded to the d/n construct by showing high levels of death
compared with cells transfected with a control construct expressing
eGFP (FIG. 12, 13A). By 5 days, 25-40% of the cells transfected
with the d/n construct exhibited condensed chromatin indicative of
apoptotic death (as compared with 2-8% of such cells transfected
with the control construct). There was also significant increased
amount of floating cellular debris in the cultures transfected with
d/n construct, suggesting that the level of cell death was even
higher than measured. To confirm that death was apoptotic and to
determine whether it was caspase-dependent, C6 cells were
transfected with d/n ATF5 in the presence and absence of the
general caspase inhibitor BAF (Deshmukh, et al. Genetic and
metabolic status of NGF-deprived sympathetic neurons saved by an
inhibitor of ICE family proteases. J. Cell Biol., 135: 1341-1354,
1996). This resulted in a 4-fold reduction in cell death. (data not
shown).
[0236] To corroborate our findings with NTAzip-ATF5 and to rule out
possible non-specific actions of din ATF5, we also employed a small
interfering RNA oligoduplex (siRNA) that selectively down-regulates
ATF5 expression (Angelastro, et al. Regulated expression of ATF5 is
required for the progression of neural progenitor cells to neurons.
J. Neurosci., 23: 4590-4600, 2003). Compared with a control
construct, the ATF5 siRNA promoted death of all 4 human glioma
lines tested (FIG. 13B). We also used a construct that expresses a
short hairpin ATF5 siRNA driven by a U6 promoter and that reduced
by 80% the proportion of transfected cells (as compared with cells
transfected with a control construct) that were positive for ATF5
immunostaining. In comparison with the control short hairpin
siRNA-luciferase construct, the short hairpin ATF5 siRNA construct
significantly elevated death in cultures of C6 rat gliomas cells
(FIG. 13B). As in the case of cultures transfected with d/n ATF5,
the cultures transfected with ATF5 siRNA constructs contained large
amounts of floating debris, presumably derived from dead cells.
[0237] Death of cultured glioma cells caused by loss of ATF5
function or expression appeared to independent of p53. Although U87
and C6 cells express wild-type p53, lines U138, U251, U273 and T98
all have mutated, non-functional p53 genes (Alai, et al. Negative
effects of wild-type p53 and s-Myc on cellular growth and
tumorigenicity of glioma cells. Implication of the tumor suppressor
genes for gene therapy. J. Neurooncol., 19: 259-268, 1994;
Yamagishi, et al. Modification of the radiosensitivity of human
cells to which simian virus 40 T-antigen was transfected. J.
Radiat. Res. (Tokyo), 36: 239-247, 1995; Badie, et al. Combined
radiation and p53 gene therapy of malignant glioma cells. Cancer
Gene Ther., 6: 155-162, 1999; Vogelbaum, et al. Overexpression of
bax in human glioma cell lines. J. Neurosurg., 91: 483-489, 1999).
In addition, co-transfection with d/n p53 failed to rescue C6 cells
from the apoptotic effects of d/n ATF5 (data not shown).
[0238] We next tested the ATF5 d/n and siRNA constructs for their
capacity to trigger death of cultured astrocytes. In contrast with
the cultured glioma cells, interfering with ATF5 function or
expression had no significant effect on survival of either first
passage rat astrocytes (data not shown) or on rat astrocytes that
had undergone 5 passages (FIG. 13). As noted above, a majority of
the latter cells express ATF5 (FIG. 11). In addition, two cell
lines that express ATF5, HEK293 cells (Aiello, et al. Adenovirus 5
DNA sequences present and RNA sequences transcribed in transformed
human embryo kidney cells (HEK-Ad-5 or 293). Virology 94: 460-469,
1979) and CAD cells (Qi, et al. Characterization of a CNS cell
line, CAD, in which morphological differentiation is initiated by
serum deprivation. J. Neurosci., 17: 1217-1225, 1997) showed no
excess cell death when transfected with d/n ATF5 (data not shown).
PC12 pheochromocytoma cells and embryonic neural progenitor cells
also express high levels of ATF5 and their capacity to
differentiate is accelerated by ATF5 din and siRNA constructs
(Angelastro, et al. Regulated expression of ATF5 is required for
the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003). However, in neither case did we
observe promotion of death (data not shown). Taken together, these
findings indicate that interfering with ATF5 function or expression
causes death of cultured glioma cells but not of non-neoplastic
astrocytes, or of several additional ATF5+ cell types.
ATF5 Loss-of-Function Promotes Selective Death of GBM Cells In
Vivo
[0239] To extend our in vitro findings to an in vivo model and to
further examine the specificity of the death evoked by ATF5
loss-of-function, we infected cells in rat glioma with a retrovirus
expressing d/n ATF5. Tumors were created by stereotactic injection
of C6 cells into the striatum of adult rats (1.times.10.sup.4 cells
in 5 .mu.l). Ten days later, retroviruses encoding eGFP or
eGFP-NTAzip-ATF5 (1.25.times.10.sup.4 CFU in 5 .mu.l) were
stereotactically introduced into the tumors. Under these
conditions, the tumors were large enough to inject, but had not
formed large areas of internal necrosis that might interfere with
detection of induced cell death. On day 13, the animals were
sacrificed and the brains were analyzed by immunohistochemistry for
retroviral infection (presence of eGFP) and for cell death (TUNEL
staining) in the tumor and surrounding tissue.
[0240] For many of the animals, infected cells were detected not
only within the tumors, but also in cells clearly outside the tumor
margins. Although one cannot rule out with certainty that none of
the infected cells outside the tumors were infiltrating tumor
cells, it appears more likely that these were mainly generated from
reactive astrocytes, or other endogenous proliferating cells that
were infected by the viruses. When injected into adult rat brains,
C6 cells form a well-circumscribed tumor with little infiltration.
Moreover, the few cells in C6 tumors that do infiltrate, do so
along blood vessels (Canoll et. al., unpublished data) and we did
not observe that infected cells outside the tumors were associated
with the vasculature. Rather, the cells mostly distributed
throughout corpus callosum and had distribution and morphology most
consistent with reactive astrocytes. For these reasons, infected
cells that were within and outside of the tumors were separately
scored for TUNEL staining.
[0241] In the case of animals receiving the control virus, less
than 1% (2/252) of the infected cells within the tumors were
TUNEL+. Likewise, there were no TUNEL+ infected cells outside these
tumors (0/194). In contrast, 96% (215/225) of the cells infected
with the d/n ATF5-expressing virus and that were within the tumors
were TUNEL+ (FIGS. 14 and 15). Inspection of the nuclei of such
cells revealed that many were pyknotic or in various states of
degeneration. By comparison, only 2% (1/63) of the infected cells
outside the tumors were positive for TUNEL staining (FIGS. 14 and
15). Thus, interference with ATF5 function causes death of glioma
cells in vivo, but spares cells outside the tumors.
[0242] These particular studies revealed that ATF5 was expressed by
all 29 human GBMs we surveyed as well as by both rat and human
glioma cell lines. Although ATF5 expression thus presently appears
to be universal among glioblastomas, not all cells in the tumors
were positive for ATF5 staining. This may reflect the findings of
prior reports that ATF5 expression is largely limited to the G1 and
S-phases of the cell cycle Pati, et al. Human Cdc34 and Rad6B
ubiquitin-conjugating enzymes target repressors of cyclic
AMP-induced transcription for proteolysis. Mol. Cell. Biol., 19:
5001-5013, 1999; Persengiev, et al. Inhibition of apoptosis by
ATFx: a novel role for a member of the ATF/CREB family of mammalian
bZIP transcription factors. Genes Dev., 16:1806-1814, 2002).
[0243] The expression of ATF5 in glioma cells contrasts with mature
neurons, astrocytes and oligodendroglia in brain, which do not
express detectable levels of ATF5 (Angelastro, et al. Regulated
expression of ATF5 is required for the progression of neural
progenitor cells to neurons. J. Neurosc., 23: 4590-4600, 2003). On
the other hand, ATF5 is highly expressed by brain neural
progenitor/stem cells (Angelastro, et al. Regulated expression of
ATF5 is required for the progression of neural progenitor cells to
neurons. J. Neurosc., 23: 4590-4600, 2003), as well as by reactive
astrocytes. When constitutively expressed in neural progenitor
cells, ATF5 blocks their differentiation into neurons and
astrocytes and maintains them in a proliferative state, even in the
presence of differentiation-promoting growth factors, such as NGF,
NT3, and CNTF (Angelastro, et al. Regulated expression of ATF5 is
required for the progression of neural progenitor cells to neurons.
J. Neurosci., 23: 4590-4600, 2003). This raises the possibility
that ATF5 contributes to the relatively undifferentiated state of
GBMs and to their capacity for uncontrolled growth. However, ATF5
expression alone does not appear to be sufficient for neoplastic
transformation. When ATF5 was constitutively expressed in SVZ
progenitors in vivo, these cells formed a non-invasive
multi-layered hyperplastic mass by 31/2 months post infection that
exhibited the morphologic features of neural progenitors, but not
of glioblastoma cells (Angelastro et al., unpublished data).
[0244] A somewhat unanticipated finding here was that ATF5
loss-of-function induced death of glioma cells both in culture and
in vivo. This effect was independent of the delivery method
employed in that both transient transfection and retroviral
infection using the same d/n ATF5 construct produced similar
results. Moreover, death was also induced by an ATF5 siRNA
transfected either as an oligoduplex or hair-pin loop.
Significantly, these destructive actions appeared to be selective
for glioma cells. The d/n ATF5 had no effect on survival of
ATF5-expressing astrocytes in culture or of retrovirally-infected
and therefore proliferating) cells in brain that were found outside
the margins of experimental tumors. There were also no apoptotic
effects on cultured CAD neuroblast cells or human embryonic kidney
293 cells, both of which express detectable ATF5. We have also
noted that ATF5 loss-of-function does not compromise survival of
ATF5 positive PC12 rat pheochromocytoma cells (Angelastro, et al.
Regulated expression of ATF5 is required for the progression of
neural progenitor cells to neurons. J. Neurosci., 23: 4590-4600,
2003), of brain neural progenitor/stem cells either in culture
(Angelastro, et al. Regulated expression of ATF5 is required for
the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003) or in vivo (Angelastro et. al.,
unpublished data) or of proliferating O4+ oligodendroglial
progenitor cells in vitro or in developing brain (Mason, et. al.,
unpublished data).
[0245] The mechanisms by which loss of ATF5 function or expression
lead to death of glioma cells remain to be fully explored. A
p53-dependent mechanism appears to be ruled out in that a number of
the susceptible glioma lines we used are deficient in p53
expression or activity and because we were unable to protect one
line with normal p53 function from d/n-ATF5-promoted death by
co-transfection with d/n p53. Moreover, over 70% of human GBMs are
reported to be deficient in p53 expression, either due to direct
mutations of this gene or of others that regulate p53 expression
(Collins, V. P. Brain tumours: classification and genes. J. Neurol.
Neurosurg. Psychiatry, 75 Suppl 2: ii2-11, 2004).
[0246] Impaired cell cycle control appears to be another major
feature of glioblastomas (Collins, V. P. Brain tumours:
classification and genes. J. Neural. Neurosurg. Psychiatry, 75
Suppl 2: ii2-11, 2004) and it is in this context that ATF5 and ATF5
loss-of-function may act. As noted, ATF5 appears to play a role in
regulating neural progenitor cell proliferation; constitutive
expression of ATF5 maintains such cells in the cycle (even in
presence of growth factors that would otherwise promote cell cycle
exit), while ATF5 loss-of-function causes such cells to leave the
cycle (Angelastro, et al. Regulated expression of ATF5 is required
for the progression of neural progenitor cells to neurons. J.
Neurosci., 23: 4590-4600, 2003). One possibility is that ATF5
facilitates passage through critical check points during the cell
cycle. In cells such as neural progenitors, loss of ATF5 expression
or function may lead to withdrawal from the cycle, whereas in
glioblastomas, with abnormal cell cycle control, even with ATF5
loss-of-function, the cells may attempt to continue cycling and
pass through check points. Such inappropriate passage through check
points could, in turn, lead to "mitotic catastrophe" and the
triggering of cell death pathways (Canman, C. E. Replication
checkpoint: preventing mitotic catastrophe. Curr. Biol., 11:
R121-124, 2001; Castedo, et al. Cell death by mitotic catastrophe:
a molecular definition. Oncogene, 23: 2825-2837, 2004). Of
potential relevance, inhibition of chk1, a kinase involved in
enforcing the G2/M checkpoint, potentiated the capacity of the
chemotherapeutic methylating agent temozolomide to promote mitotic
catastrophe and death of cultured glioblastoma cell (Sonoda, et al.
Formation of intracranial tumors by genetically modified human
astrocytes defines four pathways critical in the development of
human anaplastic astrocytoma. Cancer Res., 61: 4956-4960, 2001;
Hirose, et al. Abrogation of the Chk1-mediated G(2) checkpoint
pathway potentiates temozolomide-induced toxicity in a
p53-independent manner in human glioblastoma cells. Cancer Res.,
61: 5843-5849, 2001).
[0247] Yet another potential mechanism to account for our findings
is that ATF5 acts as a survival factor and that interference with
its function or expression therefore triggers death. Persengiev et
al. (Persengiev, et al. Inhibition of apoptosis by ATFx: a novel
role for a member of the ATF/CREB family of mammalian bZIP
transcription factors. Genes Dev., 16:1806-1814, 2002) reported
that ATF5 levels fall in several cell lines undergoing death evoked
by trophic factor deprivation and that such death was suppressed by
constitutive ATF5 expression. These authors also found that a d/n
ATF5 lacking a transcriptional regulatory domain promoted death of
HeLa and FL5.12 cells in presence of trophic support. It was
further observed that constitutive expression of ATF5 does not
affect FL5.12 cell proliferation and on this basis it was concluded
that the activity of ATF5 is purely anti-apoptotic (Persengiev, et
al. Inhibition of apoptosis by ATFx: a novel role for a member of
the ATF/CREB family of mammalian bZIP transcription factors. Genes
Dev., 16: 1806-1814, 2002). Although our findings indicate that
ATF5 can affect cell proliferation and differentiation and that its
loss or absence (as in the case of mature neurons and glia) does
not necessarily result in cell death, it remains possible that it
acts as a survival factor for glioma cells independently from its
role in cell growth.
[0248] In summary, these findings indicate that ATF5 is universally
expressed by glioma cells and that interference with its function
or expression leads to their death, both in vitro and in vivo. In
contrast, ATF5 is undetectable in mature neurons and glia and
abrogation of its expression or function does not cause death of
brain cells that express this protein, including developing neural
progenitor cells or activated astrocytes. These observations raise
ATF5 as a potential therapeutic target for treatment of
glioblastomas, either by direct intervention in its expression or
activity such as achieved here, or by indirectly manipulating other
molecules involved in its regulation or function.
Example 3
ATF5 is Widely Expressed in an Array of Different Tumor Types
[0249] The inventors have screened various tumor types for
expression of ATF5. The screen was conducted using micro tissue
array with anti-ATF5 antiserum, and the resulting data was
interpreted by a pathologist. The results demonstrate that ATF5 is
widely expressed by various tumor. The following is a list of
specific tumor types that tested positive for ATF5 (number positive
for ATF5/total number assessed): breast (20/28); ovary (18/26);
endometrium (17/25); gastric (20/22); colon (20/24); liver (10/14);
pancrease (26/28); kidney (16/22); bladder (24/26); prostate
(20/22); testis (6/10); skin (8/10); esophagus (8/14); tongue
(16/20); mouth (8/8); parotid (4/6); larynx (9/11); pharynx (2/4);
lymph node (4/12); lung (22/24); and brain (8/12).
Example 4
[0250] Selective Interference with ATF5 Function in Breast
Carcinoma Triggers Cell Death
[0251] The inventors have provided the first study of ATF5
expression in human breast tissue. ATF5 expression was evaluated
using immunohistochemistry, and cell culture experiments were
performed to assess the effect of interfering with its
function.
[0252] ATF5 antiserum was used to immunostain a cancer tissue
microarray and additional paraffin-embedded sections of human
breast tissue (10 ductal carcinomas, 7 lobular carcinomas, and 5
normal). Staining was quantified by determining the number of ATF5
positive nuclei (per 200 total nuclei in duct epithelium). ATF5
expression and apoptotic cell death were also evaluated in cell
lines (5 breast cancer and 3 normal) transfected with a control or
ATF5 dominant negative construct.
[0253] Immunostaining of all sections showed 93% of invasive breast
carcinomas stained strongly for ATF5. The proportion of
ATF5-positive nuclei in paraffin-embedded sections was 45.+-.4%
(controls), 80.+-.4% (in-situ ductal), 73+7% (in-sutu lobular),
82+2% (invasive ductal), and 83+3% (invasive lobular). The breast
stroma was consistently negative. Apoptosis in neoplastic cells
transfected with the dominant negative ATF5 was significantly
greater than in cells transfected with the control construct. In
contrast, cell death in non-neoplastic cells was not significantly
altered.
[0254] The results indicate that ATF5 is highly expressed in breast
carcinoma and, to a lesser extent, in normal breast tissue cells.
Interference with ATF5 function triggers increased cell death in
neoplastic, but not normal breast cells. The tumor specific effect
of interference with ATF5 function likely has important
implications for therapeutic approaches to breast carcinoma.
[0255] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
Sequence CWU 1
1
1911034DNARattus norvegicus 1gcacctgtgc ctcagccatg tcactcctgg
cgaccctggg actggagctg gacagggccc 60tgctcccagc tagcgggctg ggctggctcg
tagactatgg gaaactcccc ctggcccctg 120cccccctggg cccctatgag
gtccttgggg gtgccctgga gggcgggctt ccaggggggg 180gagagcccct
ggcaggtgac ggcttctctg attggatgac cgagcgggtg gacttcacag
240ccctccttcc tctggaggcc cctctgcccc caggcactct ccccccaccc
tcccctgccc 300cccctgacct ggaagccatg gcatccctac tcaagaagga
gctagaacag atggaagact 360tcttccttga tgccccactc cttccaccgc
cctccccacc tccaccccca cccccagcac 420cctctctgcc cctgccctta
cccttgccca cctttgatct cccgcagcct cctaccctgg 480ataccctgga
cttgctagct gtttactgcc gcagtgaggc tgggccaggg gattcaggct
540tgacaaccct gcctgtcccc cagcagcctc ctcctctggc ccctctgcct
tcaccctccc 600gaccagcccc ctatcctagt cctgccagca cccgagggga
ccgcaagcaa aagaagagag 660accagaataa gtcagctgct ctcaggtacc
gccagaggaa gcgggcagag ggcgaggccc 720tggagggcga gtgccaaggg
ctagaggcgc ggaatcggga gctgagggag agggcagagt 780cagtggaacg
ggagatccag tatgtgaagg atctgctaat tgaggtgtat aaggcacgaa
840gccagaggac ccgcagtgcc tagggtacag gaggaggcag ttctggtgta
cctgtgcctc 900cagcttcacc ctgtccctcc atttcacttc cctgtgcatc
cgtgtctagg tctcccctct 960gcctatcccc attatgggtt atttggcata
gtcagtttct gtaccccttc agtgcaactg 1020agaaccaagc tcga
10342281PRTRattus norvegicus 2Met Ser Leu Leu Ala Thr Leu Gly Leu
Glu Leu Asp Arg Ala Leu Leu1 5 10 15Pro Ala Ser Gly Leu Gly Trp Leu
Val Asp Tyr Gly Lys Leu Pro Leu 20 25 30Ala Pro Ala Pro Leu Gly Pro
Tyr Glu Val Leu Gly Gly Ala Leu Glu 35 40 45Gly Gly Leu Pro Gly Gly
Gly Glu Pro Leu Ala Gly Asp Gly Phe Ser 50 55 60Asp Trp Met Thr Glu
Arg Val Asp Phe Thr Ala Leu Leu Pro Leu Glu65 70 75 80Ala Pro Leu
Pro Pro Gly Thr Leu Pro Pro Pro Ser Pro Ala Pro Pro 85 90 95Asp Leu
Glu Ala Met Ala Ser Leu Leu Lys Lys Glu Leu Glu Gln Met 100 105
110Glu Asp Phe Phe Leu Asp Ala Pro Leu Leu Pro Pro Pro Ser Pro Pro
115 120 125Pro Pro Pro Pro Pro Ala Pro Ser Leu Pro Leu Pro Leu Pro
Leu Pro 130 135 140Thr Phe Asp Leu Pro Gln Pro Pro Thr Leu Asp Thr
Leu Asp Leu Leu145 150 155 160Ala Val Tyr Cys Arg Ser Glu Ala Gly
Pro Gly Asp Ser Gly Leu Thr 165 170 175Thr Leu Pro Val Pro Gln Gln
Pro Pro Pro Leu Ala Pro Leu Pro Ser 180 185 190Pro Ser Arg Pro Ala
Pro Tyr Pro Ser Pro Ala Ser Thr Arg Gly Asp 195 200 205Arg Lys Gln
Lys Lys Arg Asp Gln Asn Lys Ser Ala Ala Leu Arg Tyr 210 215 220Arg
Gln Arg Lys Arg Ala Glu Gly Glu Ala Leu Glu Gly Glu Cys Gln225 230
235 240Gly Leu Glu Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala Glu Ser
Val 245 250 255Glu Arg Glu Ile Gln Tyr Val Lys Asp Leu Leu Ile Glu
Val Tyr Lys 260 265 270Ala Arg Ser Gln Arg Thr Arg Ser Ala 275
280315DNARattus norvegicus 3catgagaacc tagtc 15419DNAArtificial
Sequencesynthetic primer 4cttggtttct cagttgcac 19523DNAArtificial
Sequencesynthetic primer 5tgcacctgtg cctcagccat gtc
23657DNAArtificial Sequencesynthetic primer 6ctcgagaacc atggactaca
aggacgatga tgacaaagga tcactcctgg cgaccct 57757DNAArtificial
Sequencesynthetic primer 7ctcgagaagc atggactaca aggacgatga
tgacaaagga gcatccctac tcaagaa 57830DNAArtificial Sequencesynthetic
primer 8gaattctcga gcttggtttc tcagttgcac 30987DNAArtificial
Sequencesynthetic primer 9ttcttctgct tctttttcta gtagttcttc
gttttctctt gctagttctt ctgctctttg 60ttcgagggtg ctggcaggac taggata
871083DNAArtificial Sequencesynthetic primer 10gcaagagaaa
acgaagaact actagaaaaa gaagcagaag aactagaaca agaaatgcag 60agctagaggg
cgagtgccaa ggg 8311100DNAArtificial Sequencesynthetic primer
11gaattcaacc atggactaca aggacgatga tgacaaaatg gcatctatga ctggaggaca
60acaaatggga agagacccag acctcgaaca aagagcagaa 1001299PRTArtificial
Sequencesynthetic tag 12Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala
Ser Met Thr Gly Gly1 5 10 15Gln Gln Met Gly Arg Asp Pro Asp Leu Glu
Gln Arg Ala Glu Glu Leu 20 25 30Arg Glu Asn Glu Glu Leu Leu Glu Lys
Glu Ala Glu Glu Leu Glu Gln 35 40 45Glu Asn Ala Glu Leu Glu Gly Glu
Cys Gln Gly Leu Glu Ala Arg Asn 50 55 60Arg Glu Leu Arg Glu Arg Ala
Glu Ser Val Glu Arg Glu Ile Gln Tyr65 70 75 80Val Lys Asp Leu Leu
Ile Glu Val Tyr Lys Ala Arg Ser Gln Arg Thr 85 90 95Arg Ser
Ala1392DNAArtificial Sequencesynthetic oligonucleotide 13tcgagtcatg
gtaaaaatga cgtcatggta attatcatgg taaaaatgac gtcatggtaa 60ttatcatggt
aaaaatgacg tcatggtaat ta 921492DNAArtificial Sequencesynthetic
oligonucleotide 14agcttaatta ccatgacgtc atttttacca tgataattac
catgacgtca tttttaccat 60gataattacc atgacgtcat ttttaccatg ac
921515PRTArtificial Sequencesynthetic peptide 15Cys Thr Arg Gly Asp
Arg Lys Gln Lys Lys Arg Asp Gln Asn Lys1 5 10 151621RNAArtificial
Sequencesynthetic siRNA primer 16aagucagcug cucucaggua c
211765DNAArtificial Sequencesynthetic siRNA primer 17gatccgtcag
ctgctctcag gtacttcaag agagtacctg agagcagctg accttttttc 60tagag
651821RNAArtificial Sequencesynthetic siRNA primer 18aagucagcug
cucucaggua c 211921RNAArtificial Sequencesynthetic siRNA primer
19aagucggcgg cucugaggua c 21
* * * * *