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E2-EPF5, a Novel Therapeutic Protein and Target
Screening Methods
FIELD OF THE INVENTION
The present invention pertains to therapeutic methods and pharmaceutical
compositions for
the inhibition of VEGF-dependent vascularisation, and in particular of tumor
related
vascularisation.
BACKGROUND OF THE INVENTION
Vascular endothelial growth factor (VEGF) encodes a protein responsible for
multiple of the
vascular, responses observed in human tumors, including the stimulation of
outgrowth of new
blood vessels and the permeabilization thereof. VEGF is essential for
establishment of
angiogenesis on most solid tumors and the constitutive upregulation of
expression of VEGF
is seen as a major contributor to tumor angiogenesis.
Expression of VEGF and several other angiogenic growth factors are critically
regulated by
hypoxia involving the transcriptional induction of the VEGF gene by the
transcription factor
HIF-1 (hypoxia-inducible factor-1). Hypoxia is a reduction in the normal
levels of tissue
oxygen tension homeostasis and occurs during acute and vascular disease,
pulmonary
disease and cancer. For instance, tumor cells adapt to hypoxia by inducing HIF-
1. HIF-1
mediates adaptive responses to changes in tissue oxygenation and activates the
transcription of a variety of genes involved in multiple processes including
cell proliferation,
cell survival, invasion and metastasis, apoptosis, angiogenesis, glucose and
iron
metabolism. Hif-1 is overexpressed in numerous human cancers and is associated
with poor
prognosis and increased patient mortality. Recent studies have indicated that
HIF-1
mediates resistance to chemotherapy and radiation due to hypoxia, oncogene
activation and
mutations. Inhibition of HIF-1 activity may therefore represent an important
component of
anti-angiogenesis therapies.
E2-EPF5 is a member of the ubiquitin-conjugating enzyme (E2) family that does
conjugate
ubiquitin to cellular proteins. Ubiquitin and ubiquitin-like modifiers are -
processed and
attached to substrate proteins by a mechanistically conserved enzymatic
cascade, which
includes ubiquitin activating enzymes (El), ubiquitin conjugating enzymes (E2
or UBCs) and
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ubiquitin ligases (E3). Such modifications have different functions, the best
characterized of
them being ubiquitin-dependent protein degradation through the 26S proteasome
pathway.
Both E2s and E3s co-operate to transfer ubiquitin to the target protein
thereby determining
substrate specificity. Although, the availability of the human genome sequence
has opened
the possibility to complete the identification of ubiquitin family members
including E2s, E3s
and deubiquitinating proteases only a few substrates of ubiquitination have
been identified
and the understanding of the ubiquitin system and its implications in human
diseases is still
in its infancy. There is a growing body of evidence however, that modification
of cellular
proteins by ubiquitin and ubiquitin-like molecules is an essential regulatory
mechanism in
multiple biological processes, especially in cell cycle progression, cell -
differentiation and
DNA repair.
The present invention now provides a new role for ubiquitin-conjugating enzyme
E2-EPF5 in
hypoxia, and, in particular, in the building up of proteins regulated by
transcription factor HIF-
1 in response to hypoxia.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a method for the treatment of
diseases related to
aberrant neo-vascularisation comprising administering an effective amount of
an agent
inhibiting the expression of the gene encoding ubiquitin conjugating enzyme E2-
EPF5 or
inhibiting an activity of E2-EPF5 gene product. In a preferred embodiment, the
neo-
vascularisation is angiogenesis in tumors, synovial angiogenesis in rheumatoid
arthritis,
ocular neo-vascularisation as observed in diabetic retinopathy, skin
angiogenesis in
psoriasis, or hypoxia-induced angiogenesis in liver cirrhosis. In a
particularly preferred
embodiment, the neo-vascularisation is VEGF-dependent tumor angiogenesis.
In one aspect of the invention, the agent is an inhibitory nucleic acid
capable of specifically
inhibiting ubiquitin conjugating enzyme E2-EPF5, preferably an antisense
oligonucleotide
compound, more preferably an siRNA compound. In another aspect of the
invention the
agent is an antibody specifically binding to ubiquitin conjugating enzyme E2-
EPF5.
In another aspect, the present invention relates to a pharmaceutical
composition comprising
an effective amount of an agent inhibiting the expression of the gene encoding
ubiquitin
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conjugating enzyme E2-EPF5 or inhibiting the an activity of E2-EPF5 gene
product.
Preferably, the E2-EPF5 inhibitor is an antisense oligonucleotide or an siRNA
or an antibody
specifically binding E2-EPF5.
In a further aspect, the present invention relates to methods for identifying
compounds
useful for treatment of VEGF-dependent vascularisation comprising: (a)
contacting a E2-
EPF5 polypeptide with a test compound (b) detecting modulation of E2-EPF5
biological
activity. Preferably, E2-EPF5 biological activity is reduced.
In yet another aspect, the present invention relates to a method for reducing
the amount or
to inhibiting the activity of one or more polypeptides regulated by HIF-1 in
response to
hypoxia comprising inhibiting the expression of ubiquitin conjugating relating
enzyme E2-
EPF5. Preferably, the HIF-I regulated genes or proteins are selected from the
group
consisting of GLUT-1, GLUT-3, HK2, EPO, NOS2, VEGF, TGF-alpha, TGF-beta, VEGFR-
2,
C-Met, UPAR, CXCR4, carbonic anhydrase IX (CAIX).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the surprising discovery that inhibition of
ubiquitin
conjugating enzyme E2-EPF5 interferes with the HIF-1 dependent protein
expression in
response to hypoxia. In particular, inhibiting E2-EPF5 was found to interfere
with the
building-up of VEGF in response to hypoxia. Proangiogenic VEGF is required for
the
proliferation and migration of the endothelial cells that constitute the first
blood vessels. VEGF
is a 34-45 kDa glycoprotein with a wide range of activities that besides
promotion of
angiogenesis also includes enhancement of vascular permeability, a crucial
feature of
inflammations. VEGF not only acts as a growth factor for endothelial cells,
but also on
several other cells including HIV-associated Kaposi's sarcoma cells and other
tumor and
leukemia cells. Neoplastic cells frequently express elevated levels of VEGF,
which not only is
thought to be related to stimulation of angiogenesis but also to activation of
proliferation-
stimulating autocrine signalling pathways. In this contrast it is important to
consider that
besides a direct effect via the signal transduction pathways initiated at the
VEGF receptors,
VEGF also induces indirect effects. For example, the receptor Notch1 is up-
regulated by VEGF allowing also an enhanced response to the ligands of this
cell-surface
receptor too. VEGF expression has been associated with several pathological
states such as
tumor angiogenesis, hypoxia-related angiogenesis, several forms of blindness
(e.g. diabetic
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retinopathy, proliferative diabetic retinopathy, age related macular
degeneration), rheumatoid
arthritis, psoriasis and wound healing and others. A number of studies have
demonstrated
that elevated levels of VEGF alone are sufficient to induce
neovascularization. For instance,
it has been demonstrated that a single injection of VEGF augmented collateral
vessel
development in a rabbit model of ischemia (Takashita et al., 1995 J; Clin.
Invest. 93, 662).
VEGF also can induce neovascularization when injected into the cornea.
The present invention now provides methods for reducing the amount or activity
of one or
more HIF-1 regulated polypeptides, and in particular of VEGF, which is built
up in response
to hypoxia. A number genes encoding a proteins with a variety of functions are
regulated by
HIF-1 under hypoxia is known, including but not limited to the following
genes: GLUT-1,
GLUT-3, HK2, CAIX (involved e.g. in metabolic adaptation); EPO, NOS2 (involved
e.g. in
Apoptosis resistance); VEGF, TGF-alpha, TGF-beta, VEGFR-2 (involved e.g. in
Angiogenesis), C-Met, UPAR, CXCR4 (involved e.g. in tumor invasion,
metastasis). The
term "HIF-1 regulated polypeptide" in the context of the present invention
includes any
protein or polypeptide, the expression of which is controlled or influenced by
the transcription
factor HIF-1, e.g. by binding of HIF-1 to a transcription regulatory element
of the gene
encoding said protein or polypeptide. Preferably, the protein expression is
activated by the
activity of HIF-1 transcription factor, i.e. more protein is expressed in
response to HIF-1
activation. HIF-1 is a heterodimer consisting of one of three alpha subunits
(HIF-la, HIF-2a
or HIF-3a) and a beta subunit (HIF-1 R, also known as the Aryl Hydrocarbon
Nuclear
Translocator, or ARNT). HIF-1 b is constitutively expressed, whereas the
expression of the
alpha subunits is highly regulated. As for any other protein, the level of the
alpha subunits is
determined by the rates of protein synthesis and protein degradation.
Synthesis of the HIF1
alpha subunits is regulated via oxygen-independent mechanisms, whereas
degradation is
regulated primarily via 02-dependent mechanisms. Thus, although the genes for
the alpha
subunits are mostly continuously transcribed and translated, the alpha subunit
proteins is
maintained at very low levels due to rapid destruction via proteasomal
degradation. This
destruction is inhibited under hypoxic conditions and this is the major
mechanism of
induction of HIF1a and the genes dependent on this transcription factor.
E2-EPF5 encodes a 25 kDa class II ubiquitin-conjugating enzyme with a 65aa-
long basic C-
terminal extension of low sequence complexity. E2-EPF5 was first discovered in
a patient
suffering from a skin disease called endemic pemphigus foliaceus (EPF) (Liu et
al., 1992,
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JBC 267, 15829). It was later postulated that E2-EPF5 is functionally distinct
from other
characterized E2 isoenzymes since it catalyzes multiubiquitin chain formation
through lysine
residue K11 and not through the K48 residue, which is the mechanism that is
considered to
mediate proteolytic events (Bach and Ostendorff, Trends in Biochemical
Sciences (2003),
28(4), 189-195). E2-EPF5 was also found to support auto-ubiquitination
suggesting a
possible autoregulatory model for E2-EPF5. Substrates for E2-EPF5 have not
been reported
so far but its highly basic carboxy-terminal extension domain, which is unique
within the E2
family members may indicate specificity to acidic proteins (Liu et al., J Biol
Chem. 271, 2817-
2822). The E2-EPF5 term (biological) activity includes, within the context of
the present
invention, besides its ubiquitin related activities also interference with HIF-
1 mediated
induction of hypoxia-regulated genes.
The sequence of the human E2-EPF5 is available from public databases (GenBank
Accession M91670, GI:181915, SwissProt entry Q16763). The cDNA sequence is set
forth
as SEQ ID NO:1. The amino acid sequence is set forth as SEQ ID NO:2. However,
the
term E2-EPF5 also includes any homologous or orthologous sequences, variants
and
fragments as long as they keep the biological activity of E2-EPF5 herein
described. The
percentage of homology between the homologous sequence and the reference
sequence
desirably is at least 80%, more desirably at least 85%, preferably at least
90%, more
preferably at least 95%, still more preferably at least 99%. Sequence
comparisons are
carried out using a Smith-Waterman sequence alignment algorithm (see e.g.
http://www-
to.usc.edu/software/seqaln/index.html). A "fragment" means any polypeptide
molecule
having at least 5, 10, 15 or optionally at least 25,35, or 45 contiguous amino
acids of E2-
EPF5. Further possible fragments include the catalytic site or domain
including the
recognition sites, ubiquitin binding sites, sites important for subunit
interaction, and sites
important for carrying out the other functions of the ubiquitin conjugating
enzyme. Such
domains or motifs can be identified by means of routine computerized homology
searching
procedures. Fragments, for example, can extend in one or both directions from
the
functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100 amino
acids. Also
encompassed in the term fragment are for instance E2-EPF5 epitopes. An E2-EPF5
epitope
represents a site on the polypeptide against which an antibody may be produced
and to
which the antibody binds. Therefore, polypeptides comprising the amino acid
sequence of a
E2-EPF5 epitope are useful for making antibodies to E2-EPF5 polypeptide.
Preferably, an
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epitope comprises a sequence of at least 5, more preferably at least 10, 15,
20, 25, or 50
amino acid residues in length.
In one aspect the invention provides methods for the treatment of aberrant neo-
vascularisation comprising administering an effective amount of an agent
inhibiting ubiquitin
conjugating relating enzyme E2-EPF5 activity. In a related aspect, the present
invention
provides the use of agent inhibiting E2-EPF5 activity for the manufacture of a
medicament
for the treatment of a pathological state related to VEGF-dependent
vascularisation, in
particular to tumor angiogenesis. The term "aberrant neo-vascularisation" as
used herein
means a vascularisation which does not normally occur in a healthy organism
and is related
to a abnormal or disease state. The aberrant neo-vascularisation is preferably
controlled or
influenced by the activity of VEGF, i.e. "VEGF-dependent vascularisation". In
a particular
preferred embodiment the aberrant neo-vascularisation is VEGF-dependent tumor
angiogenesis.
The term "VEGF-dependent vascularisation" as used herein refers to any
generation of new
blood vessels upon stimulation by VEGF and includes without limitation
angiogenesis in
tumors, synovial angiogenesis in rheumatoid arthritis, ocular neo-
vascularisation as
observed in diabetic retinopathy and some other eye diseases, skin
angiogenesis in
psoriasis, or hypoxia-induced angiogenesis in liver cirrhosis.
In another aspect the present invention provides a method for inhibiting a HIF-
1 regulated
gene in a cell comprising inhibiting the expression or activity of ubiquitin
conjugating relating
enzyme E2-EPF5. The inhibition of the HIF-1 regulated gene may for instance be
achieved
by lowering the amount of HIF-1 by ubiquitin dependent protein degradation
e.g. by
interference with the synthesis or stabilization of HIF-la protein or the HIF-
1a transactivation.
In one embodiment the present invention provides a method for reducing the
amount of a
HIF-1 regulated polypeptide comprising inhibiting the expression or activity
of ubiquitin
conjugating relating enzyme E2-EPF5. In a particular preferred embodiment, the
HIF-1
regulated gene is VEGF. Accordingly, the present invention further provides
anti-angiogenic
methods. Thus, methods are provided for the inhibition of angiogenesis,
including tumor
angiogenesis, comprising inhibiting the expression or activity of ubiquitin
conjugating relating
enzyme E2-EPF5.
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In the context of gene expression or protein activity, the term "inhibition"
means a reduction
of the gene expression or protein activity. Preferably, such a reduction is at
least 20%, more
preferably at least 50%, 60%, 70%, 80%, 90% or 95% as compared to the level of
expression or activity without inhibition. Gene or protein inhibition may be
achieved by any
suitable technique. The skilled person knows a variety of methods and
techniques how to
inhibit gene expression or protein activity. For example, E2-EPF5 can be
inhibited by RNA
interference or antisense technologies or using LMW compounds that interfere
with the
function of E2-EPF5 or by any agent that lowers ubiquitin conjugating relating
enzyme E2-
EPF5.
An agent inhibiting the ubiquitin conjugating relating enzyme E2-EPF5 activity
can be any
substance that reduces the biological activity of E2-EPF5. The agent may, for
instance,
inhibit the expression of an E2-EPF5 gene or an enzymatic activity of E2-EPF5,
may induce
degradation of E2-EPF5 polypeptides or may interfere with the biological
activity of E2-EPF5
in any other way. In a preferred embodiment, the inhibitory agent is a low
molecular weight
compound or an inhibitory nucleic acid or an antibody.
As contemplated herein, the term "inhibitory nucleic acid" refers to nucleic
acid compounds
capable of producing gene-specific inhibition of gene expression. Typical
inhibitory nucleic
acids include, but are not limited to, antisense oligonucleotides, triple
helix DNA, RNA
aptamers, ribozymes and siRNAs. For example, knowledge of a nucleotide
sequence may
be used to design siRNA or an antisense molecules which potently inhibit the
expression of
ubiquitin conjugating relating enzyme E2-EPF5. Similarly, ribozymes can be
synthesized to
recognize specific nucleotide sequences of a gene and cleave it. Techniques
for the design
of such molecules for use in targeted inhibition of gene expression is well
known to one of
skill in the art.
Inhibitory nucleic acid compounds of the present invention may be synthesized
by
conventional means on a commercially available automated DNA synthesizer, e.g.
an
Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA
synthesizer, or like
instrument. Phosphoramidite chemistry may be employed. The inhibitory nucleic
acid
compounds of the present invention may also be modified, for instance,
nuclease resistant
backbones such as e.g. phosphorothioate, phosphorodithioate, phosphoramidate,
or the like,
described in many references may be used. The length of the inhibitory nucleic
acid has to
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be sufficient to ensure that the biological activity is inhibited. Thus, for
instance in case of
antisense oligonucleotides, has to be sufficiently large to ensure that
specific binding will
take place only at the desired target polynucleotide and not at other
fortuitous sites. The
upper range of the length is determined by several factors, including the
inconvenience and
expense of synthesizing and purifying oligomers greater than about 30-40
nucleotides in
length, the greater tolerance of longer oligonucleotides for mismatches than
shorter
oligonucleotides, and the like. Preferably, the antisense oligonucleotides of
the invention
have lengths in the range of about 15 to 40 nucleotides. More preferably, the
oligonucleotide moieties have lengths in the range of about 18 to 25
nucleotides.
Double-stranded RNA, i.e. sense-antisense RNA, also termed small interfering
RNA (siRNA)
molecules, can also be used to inhibit the expression of nucleic acids for E2-
EPF5. RNA
interference is a method in which exogenous, short RNA duplexes are
administered where
one strand corresponds to the coding region of the target mRNA (Elbashir et
al., Nature
2001, 411: 494-498). Upon entry into cells, siRNA molecules cause not only
degradation of
the exogenous RNA duplexes, but also of single-stranded RNAs having identical
sequences,
including endogenous messenger RNAs. Accordingly, siRNA may be more potent and
effective than traditional antisense RNA methodologies since the technique is
believed to act
through a catalytic mechanism. Preferred siRNA molecules are typically from 19
to 25
nucleotides long, preferably about 21 nucleotides in length and comprise the
sequence of a
nucleic acid for E2-EPF5. Effective strategies for delivering siRNA to target
cells include, for
example, transduction using physical or chemical transfection. Alternatively
siRNAs may be
expressed in cells using, e.g., various Pollll promoter expression cassettes
that allow
transcription of functional siRNA or precursors thereof. See, for example,
Scherr et al., Curr.
Med. Chem. 2003, 10(3):245-256; Turki et al., Hum. Gene Ther. 2002,
13(18):2197-2201;
Cornell et al., Nat. Struct. Biol. 2003, 10(2):91-92. The invention also
covers other small
RNAs capable of mediating RNA interference (RNAi) such as for instance micro-
RNA
(miRNA) and short hairpin RNA (shRNA).
In another preferred embodiment, the agent inhibiting the ubiquitin
conjugating relating
enzyme E2-EPF5 activity is an antibody. Such antibodies may include, but are
not limited to
polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric
antibodies,
single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced
by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding
fragments of any of
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the above. Various procedures known in the art may be used for the production
of polyclonal
antibodies.
Various procedures known in the art may be used for the production of
antibodies.
Polyclonal antibodies are heterogeneous populations of antibody molecules
derived from the
sera of animals immunized with an antigen, such as target gene product, or an
antigenic
functional derivative thereof. For the production of polyclonal antibodies,
host animals, may
be immunized by injection with E2-EPF5 polypeptides, derivatives or fragments,
supplemented with suitable adjuvants. Monoclonal antibodies, which are
homogeneous
populations of antibodies to a particular antigen, may be obtained by any
technique which
provides for the production of antibody molecules by continuous cell lines in
culture. These
include, but are not limited to the hybridoma technique of Kohler and
Milstein, (1975, Nature
256:495-497), the human B-cell hybridoma technique, and the EBV-hybridoma
technique.
Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE,
IgA, IgD and
any subclass thereof. The hybridoma producing the mAb of this invention may be
cultivated
in vitro or in vivo. Production of high titers of mAbs in vivo makes this the
presently preferred
method of production.
In addition, techniques developed for the production of "chimeric antibodies"
(i.e. a molecule
in which different portions are derived from different animal species, such as
those having a
variable or hypervariable region derived from a murine mAb and a human
immunoglobulin
constant region) by splicing the genes from a mouse antibody molecule of
appropriate
antigen specificity together with genes from a human antibody molecule of
appropriate
biological activity can be used. Alternatively, techniques described for the
production of
single chain antibodies can be adapted to produce E2-EPF5 antibodies. Single
chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region via an
amino acid bridge, resulting in a single chain polypeptide. Such techniques
are known in the
art.
In a preferred embodiment, the E2-EPF5 antibody is a "humanized antibody."
Techniques
useful for the production of "humanized antibodies" can be adapted to produce
antibodies to
theE2-EPF5 polypeptides, fragments, derivatives, and functional equivalents
disclosed
herein. Such techniques are disclosed in U.S. Patent Nos: 5,932, 448;
5,693,762;
5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126; 5,633,425;
5,789,650;
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5,545,580; 5,661,016; and 5,770,429, the disclosures of all of which are
incorporated by
reference herein in their entirety.
Antibody fragments which recognize specific E2-EPF5 epitopes may be generated
by known
techniques. For example, such fragments include but are not limited to: the
F(ab')2
fragments which can be produced by pepsin digestion of the antibody molecule
and the Fab
fragments which can be generated by reducing the disulfide bridges of the
F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
Suitable antibodies to E2-EPF5 proteins may be obtained from a commercial
source or
produced according to conventional methods. Such antibodies may include, but
are not
limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or
chimeric
antibodies, single chain aritibodies, single domain antibodies, Fv fragments,
Fab fragments,
F(ab')2 fragments, fragments produced by a Fab expression library, anti-
idiotypic (anti-Id)
antibodies, and epitope-binding fragments of any of the above.
A wide variety of antibody/ immunoglobulin frameworks or scaffolds can be
employed so
long as the resulting polypeptide includes one or more binding region which is
specific for
the E2-EPF5 protein. Such frameworks or scaffolds include the 5 main idiotypes
of human
immunoglobulins, or fragments thereof (such as those disclosed elsewhere
herein), and
include immunoglobulins of other animal species, preferably having humanized
aspects.
Single heavy-chain antibodies such as those identified from camelids are of
particular
interest in this regard. Novel frameworks, scaffolds and fragments continue to
be
discovered and developed by those skilled in the art.
Alternatively, non-immunoglobulin frameworks and scaffolds may be employed, as
long as
they comprise a binding region specific for the E2-EPF5 protein. Known non-
immunoglobulin frameworks or scaffolds include Adnectins (fibronectin)
(Compound
Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich,
Switzerland),
domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde,
Belgium)),
lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), Small modular
immuno-
pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies
(Avidia, Inc.
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(Mountain View, CA)), Protein A (Affibody AG, Sweden) and affilin (gamma-
crystallin or
ubiquitin) (Scil Proteins GmbH, Halle, Germany).
According to the instant invention, the anti-E2-EPF5 antibody or fragment
thereof, or the
polypeptide comprising a E2-EPF5-specific binding region, regardless of the
framework or
scaffold employed, may be bound, either covalently or non-covalently, to an
additional
moiety. The additional moiety may be a polypeptide, an inert polymer such as
PEG, small
molecule, radioisotope, metal, ion, nucleic acid or other type of biologically
relevant
molecule. Such a construct, which may be known as an immunoconjugate,
immunotoxin, or
the like, is also included in the meaning of antibody, antibody fragment or
polypeptide
comprising a E2-EPF5-specific binding region, as used herein.
In another embodiment of the present invention, the inhibitory agent is a
small molecule
(e.g., organic or inorganic molecules which are less than about 2 kDa in
molecular weight,
are more preferably less than about 1 kDa in molecular weight, and/or are able
to cross the
blood-brain barrier or gain entry into an appropriate cell) which affect E2-
EPF5 expression or
the activity of E2-EPF5 polypeptide. Such a small molecule compound may be
identified by
the screening methods as described below.
One aspect of the present invention provides E2-EPF5 gene and gene product as
drug
target for the development of therapeutics for use in treatment of individuals
suffering from a
disease as described above. Provided herein, therefore, as part of the present
invention, are
screening methods for identifying compounds that may be used to treat diseases
as
described herein. These methods comprise, in preferred embodiments, contacting
a test
compound to a reaction mixture that contains an E2-EPF5 polypeptide. In
preferred
embodiments, E2-EPF5 is a polypeptide having an amino acid sequence which may
comprise the sequence set forth in SwissProt entry Q16763. However, any E2-
EPF5
homolog, ortholog, variant, etc. may be used in these methods, as can fusion
constructs of
those polypeptides (for example a fusion construct as described in the
Examples, infra). For
instance, the E2-EPF5 polypeptide may have an amino acid sequence that is
substantially
homologous (e.g., at least 75%, 80%, 85%, 90%, 95% or 99% identical) to
SwissProt entry
Q16763.
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In a particularly preferred embodiment, E2-EPF5 is provided as novel target
for the
screening for therapeutics useful in the treatment of diseases in which VEGF-
dependent
angiogenesis plays a role and, in particular, in tumor angiogenesis or ocular
vascularisation
or hypoxia-induced angiogenesis. The present invention provides methods for
identifying a
compound useful for the inhibition of VEGF-dependent vascularisation
comprising (a)
contacting a E2-EPF5 polypeptide with a test compound (b) detecting a
modulation of E2-
EPF5 biological activity. The modulation is usually detected with respect to a
control reaction
lacking the test compound. Modulation as used herein refers to an increase or
reduction of
the biological activity, preferably by at least 10%, at least 20%, at least
30%, at least 50% or
at least 100%.
In another embodiment, the present invention provides a method of identifying
a compound
useful for treatment of a disease related to VEGF-dependent vascularisation,
e.g. tumor
vascularisation, comprising
i) contacting a test compound with a E2-EPF5 polypeptide under sample
conditions
permissive for E2-EPF5 biological activity;
ii) determining the level of said at least one E2-EPF5 biological activity;
iii) comparing said level to that of a control sample lacking said test
compound; and,
optionally,
iv) selecting a test compound which causes said level to change for further
testing as a
compound for the prophylactic and/or therapeutic treatment of a disease
related to VEGF-
dependent vascularisation.
Compound screening assays may include cell-based or cell-free systems. Cell-
based
systems can be native, i.e., cells that normally express the ubiquitin
conjugating enzyme E2-
EPF5, as a biopsy or expanded in cell culture. In one embodiment, however,
cell- based
assays involve recombinant host cells expressing the ubiquitin conjugating
enzyme.
Determining the ability of the test compound to interact with the ubiquitin
conjugating
enzyme E2-EPF5 can also comprise determining the ability of the test compound
to
preferentially bind to the polypeptide as compared to the ability of a known
binding molecule
(e.g., ubiquitin) to bind to the polypeptide. The polypeptides can be used to
identify
compounds that modulate ubiquitin ubiquitin conjugating activity. Such
compounds, for
example, can increase or decrease affinity for ubiquitinated protein
substrate, or
ubiquitinated protein substrate remnants. Such compounds could also, for
example, increase
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or decrease the rate of binding to these components. Such compounds could also
compete
with these components for binding to the ubiquitin conjugating enzyme or
displace these
components bound to the ubiquitin conjugating enzyme. Such compounds could
also affect
interaction with other components, such as ATP, other subunits, such as El
activating
enzymes or E3 ligases.
Ubiquitin conjugating enzyme E2-EPF5, derivatives and fragments can be used in
fast
screening methods e.g. automated high-throughput screens (HTS) to assay
candidate
compounds for the ability to bind to the ubiquitin conjugating enzyme.
Numerous suitable
fast screening assays are known to the skilled person.
These compounds can be further screened against functional ubiquitin
conjugating enzyme
E2-EPF5 to determine the effect of the compound on ubiquitin conjugating
enzyme E2-
EPF5. Compounds can be identified that activate (agonist) or inactivate
(antagonist) E2-
EPF5 to a desired degree. Modulatory methods can be performed in vitro (e.g.,
by culturing
the cell with the agent) or, alternatively, in vivo (e.g., by administering
the agent to a subject.
The E2-EPF5 polypeptides of the present invention can be used to screen a
compound for
the ability to stimulate or inhibit interaction between the E2-EPF5 protein
and a target
molecule that normally interacts with the E2-EPF5 protein. The target can be
ubiquitin,
ubiquitinated substrate, or polyubiquitin or another component of the pathway
with which the
ubiquitin conjugating enzyme protein normally interacts (for example El or E3
proteins). The
assay includes the steps of combining the E2-EPF5 protein with a candidate
compound
under conditions that allow the E2-EPF5 protein or fragment to interact with
the target
molecule, and to detect the formation of a complex between the E2-EPF5 protein
and the
target or to detect the biochemical consequence of the interaction with E2-
EPF5 and the
target. Any of the associated effects of ubiquitin conjugating function can be
assayed. This
includes the production of ubiquinated substrates, proteolysis, decrease of
free polyubiquitin,
stability of the substrate.
Determining the ability of the ubiquitin conjugating enzyme to bind to a
target molecule can
also be accomplished using a technology such as real-time Bimolecular
Interaction Analysis
(BIA). As used herein, "BIA" is a technology for studying biospecific
interactions in real time,
without labeling any of the interactants (e.g., BlAcore ). Changes in the
optical phenomenon
surface plasmon resonance (SPR) can be used as an indication of real-time
reactions
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between biological molecules. The test compounds of the present invention can
be obtained
using any of the numerous approaches in combinatorial library methods known in
the art,
including: biological libraries; spatially addressable parallel solid phase or
solution phase
libraries; synthetic library methods requiring deconvolution; the 'one-bead
one-compound'
library method; and synthetic library methods using affinity chromatography
selection. The
biological library approach is limited to polypeptide libraries, while the
other four approaches
are applicable to polypeptide, non-peptide oligomer or small molecule
libraries of compounds
(Lam, K. S. (1997) Anticancer Drug Des. 12:145). Examples of methods for the
synthesis of
molecular libraries can be found in the art, for example in DeWitt et al.
(1993) Proc. Natl.
Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;
Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science
261:1303;
Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994)
Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of
compounds may be presented in solution (e.g., Houghten (1992) Biotechniques
13:412-
421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature
364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),
plasmids
(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage
(Scott and Smith
(1990) Science 249:386- 390); (Devlin (1990) Science 249:404-406); (Cwirla et
al. (1990)
Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-
310); (Ladner
supra). Candidate compounds include, for example, 1) peptides such as soluble
peptides,
including Ig-tailed fusion peptides and members of random peptide libraries
(see, e.g., Lam
et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial
chemistry-derived molecular libraries made of D- and/or L-configuration amino
acids; 2)
phosphopeptides (e.g., members of random and partially degenerate, directed
phosphopeptide libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778);
3) antibodies
(e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single
chain antibodies
as well as Fab, F(ab') 2, Fab expression library fragments, and epitope-
binding fragments of
antibodies); and 4) small organic and inorganic molecules (e.g., molecules
obtained from
combinatorial and natural product libraries).
Suitable assays for measuring activity of ubiquitin conjugating relating
enzyme activity are
well known in the art. These assays include, but are not limited to, the
appearance of
substrate, including increase in the amount of polyubiquitin or ubiquitinated
substrate protein
or protein remnant, appearance of intermediate and end products, such as
disappearance of
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free ubiquitin monomers, general protein turnover, specific protein turnover,
ubiquitin
binding, binding to ubiquitinated substrate protein, subunit interaction,
interaction with ATP,
interaction with cellular components such as trans- acting regulatory factors,
stabilization of
specific proteins, and the like.
In one aspects, compounds identified by the screening methods in accordance
with the
present invention are provided. Such compounds are preferably low molecular
weight
compounds or antibodies, in particular monoclonal antibodies, or inhibitory
nucleic acids.
The compounds have preferably anti-angiogenic activity, i.e. they inhibit the
growth of blood
vessel in general and in particular aberrant neo-vascularisation. The anti-
angiogenic activity
of such a can be determined by methods known in the art. Such methods include
for
instance microscopic assessment of angiogenesis in tumors, in vivo matrigel
migration and
angiogenesis assays, alginate microbead release assay of angiogenesis, disc
angiogenesis
assay, sponge implant model of angiogenesis, hollow fiber assay for tumor
angiogenesis or
corneal assay for angiogenesis. Such assays are for instance described in
"Angiogenesis
Protocols", March 2001, ISBN: 0-89603-698-7, Series: Methods in Molecular
Medicine,
Volume #: 46.
In order to develop angiogenic and antiangiogenic strategies, concerted
efforts have been
made to provide animal models for more quantitative analysisof in vivo
angiogenesis. In vivo
techniques consist of the cornea pocket andiris implant in the eye, the rabbit
ear chamber,
the dorsal skinfold chamber, the cranial window, the hamster cheek pouch
window, the
sponge implant assay,the fibrin clots, the sodium alginate beads and the
Matrigel plugs, the
ratmesenteric window, the chick embryo chorioallantoic membrane and the airsac
in mice
and rats (1). In this chapter we will discuss the avascular cornea assay, and
the advantages
and disadvantages of using this assay in different species. The cornea assay
is based on
the placement of an angiogenic inducer(tumor tissue, cell suspension, growth
factor) into a
corneal pocket in order toevoke vascular outgrowth from the peripherally
located limbal
vasculature. Incomparison to other in vivo assays, this assay has the
advantage of
measuringonly new blood vessels, because the cornea is initially avascular.
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An additional aspect of the invention relates to the administration of a
pharmaceutical
composition, in conjunction with a pharmaceutically acceptable carrier, for
any of the
therapeutic effects discussed above. Such pharmaceutical compositions comprise
an
effective amount of an agent inhibiting the expression of the gene encoding
ubiquitin
conjugating enzyme E2-EPF5 or inhibiting the an activity of E2-EPF5 gene
product. They
may for instance comprise antibodies, mimetics, agonists, antagonists, or
inhibitory nucleic
acids of ubiquitin conjugating enzyme E2-EPF5 in accordance with the present
invention.
The compositions may be administered alone or in combination with at least one
other
agent, such as stabilizing compound, which may be administered in any sterile,
biocompatible pharmaceutical carrier, including, but not limited to, saline,
buffered saline,
dextrose, and water. The compositions may be administered to a patient alone,
or in
combination with other agents, drugs or hormones.
The pharmaceutical compositions encompassed by the invention may be
administered by
any number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-
articular, intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or
rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate
processing of the active compounds into preparations which can be used
pharmaceutically.
Further details on techniques for formulation and administration may be found
in the latest
edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton,
Pa.).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for
ingestion by the patient.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic solvents than
are the
corresponding free base forms. In other cases, the preferred preparation may
be a
lyophilized powder which may contain any or all of the following: 1-50 mM
histidine, 0. 1%-
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2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer
prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose.
The determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models, usually mice,
rabbits, dogs, or
pigs. The animal model may also be used to determine the appropriate
concentration range
and route of administration. Such information can then be used to determine
useful doses
and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
fragments thereof,
antibodies, agonists, antagonists or inhibitors of the ubiquitin conjugating
enzyme E2-EPF5.
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be
determined by standard pharmaceutical procedures in cell cultures or
experimental animals,
e.g., ED50 (the dose therapeutically effective in 50% of the population) and
LD50 (the dose
lethal to 50% of the population). The dose ratio between toxic and therapeutic
effects is the
therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from
cell culture assays and animal studies is used in formulating a range of
dosage for human
use. The dosage contained in such compositions is preferably within a range of
circulating
concentrations that include the ED50 with little or no toxicity. The dosage
varies within this
range depending upon the dosage form employed, sensitivity of the patient, and
the route of
administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject that requires treatment. Dosage and administration are adjusted to
provide sufficient
levels of the active moiety or to maintain the desired effect. Factors which
may be taken into
account include the severity of the disease state, general health of the
subject, age, weight,
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and gender of the subject, diet, time and frequency of administration, drug
combination(s),
reaction sensitivities, and tolerance/response to therapy. Long-acting
pharmaceutical
compositions may be administered every 3 to 4 days, every week, or once every
two weeks
depending on half-life and clearance rate of the particular formulation.
Normal dosage
amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1
g,
depending upon the route of administration. Guidance as to particular dosages
and methods
of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or
their inhibitors. Similarly, delivery of polynucleotides or polypeptides will
be specific to
particular cells, conditions, locations, etc. Pharmaceutical formulations
suitable for oral
administration of proteins are described, e.g., in U.S. Patents 5,008,114;
5,505,962;
5,641,515; 5,681,811; 5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387;
5,976,569;
and 6,051,561.
Having generally described this invention, a further understanding can be
obtained by
reference to certain specific examples which are provided herein for purposes
of illustration
only, and are not intended to be limiting unless otherwise specified.
EXAMPLES
1. Antisense (ASO) and siRNA oligonucleotides
siRNAs are available from a number of commercial suppliers such as e.g.
Qiagen,
Dharmacon, Proligo.
Oligoribonucleotides can be synthesized using TOM-phosphoramidite chemistry,
as
described by the manufacturer (Xeragon) and purified by RP-HPLC. Purity is
assessed by
capillary gel electrophoresis. Quantification is carried out by UV according
to the extinction
coefficient at 260 nM. Annealing of double-stranded RNA (dsRNA) is performed
as
described elsewhere (Elbashir et al., 2001).
Modified antisense oligodeoxyribonucleotides are synthesized using
phosphoramidite
chemistry purified by HPLC, analyzed and characterized by electrospray mass.
spectrometry
and capillary gel electrophoresis. Quantification is carried out by UV
according to the
extinction coefficient at 260 nM.
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AII oligonucleotides are designed against the human E2-EPF5 sequence (GenBank
Accession M91670, GI number, GI:181915, SwissProt entry Q16763) and checked by
BLAST against the Refseq and UNIGENE databases to maximize the level of
specificity. In
order to identify the most potent oligonucleotides, a series of ASOs and
siRNAs targeting
E2-EPF5 are screened in a dual-color reporter assay as described previously
(Hiasken, D. et
al, 2003). A vector containing the coding sequence of E2-EPF5 is recombined
with a
destination vector that contains the CFP- and YFP reporter sequences. The
resulting dual
reporter plasmid pNAS-X generates a fusion mRNA where the E2-EPF5 target
sequence is
inserted into the 3'-UTR region of the expressed YFP- reporter gene. E2-EPF5
ASO and
siRNA activity is measured by the degree of inhibition of the YFP reporter
protein. The
expression of eCFP protein serves as a means to normalize for plasmid
transfection
efficiency. The list of E2-EPF5 oligonucleotides screened is shown in Table
1(ASOs
targeting E2-EPF5; N = DNA, n= DNA with 2' O-methoxyethyl, s= phosphorothioate
internucleotidic linkage, c=2' methoxyethyl 5-methyl cytidine) and Table 2
(siRNAs targeting
E2-EPF5), respectively (Only the antisense sequence is shown. N(G, C,A, U) =
ribonucleoside, n(g,a,t) = deoxyribonucleoside). Most active oligonucleotides
(8162, 17828,
11723) and corresponding controls are used in follow-up studies.
Table 1
# Annotation Sequence SEQ ID NO:
8162 Match tgt cgTs CsAsCs CsTsCs Cstt gta SEQ ID NO: 3
9679 Mismatch-8162 cgGs CsCsCs AsTsCs Cstt tta SEQ ID NO: 4
8169 Match cca ttGs GsCsGs CsCsCs Ascg ttc SEQ ID NO: 5
8213 Mismatch-8213 cca gtTs GsCsTs CsCsCs Ascg gtc SEQ ID NO: 6
8189 Match tcg cgCs TsCsGs CsCsAs Gsca tgc SEQ ID NO: 7
8233 Mismatch-8189 tct ctCs GsCsGs CsCsAs Gsca ggc SEQ ID NO: 8
8201 Match gca gcGs CsCsCs GsCsTs Tsct tgt SEQ ID NO: 9
8245 Mismatch-8201 gca tcTs CsCsCs GsCsGs Tscg tgt SEQ ID NO: 10
5596 Unrelated Control cct taCs CsTsGs CsTsAs Gsct ggc SEQ ID NO: 11
Table 2
NAS# Annotation Seguence SEQ ID NO:
(antisense strand, 5' -> 3')
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17828 Match E2-EPF5 TTC TTA TCG CGC TCG CCA Gca SEQ ID NO: 12
25296 Mismatch-17828 TTC TGA TCG AGC TCG CCA Gca SEQ ID NO: 13
11723 Match E2-EPF5 TAG GTT CTC CAC GTT GGA Gtt SEQ ID NO: 14
11769 Mismatch 11723 TAT GGT CGC CAC TTT GGA Gtt SEQ ID NO: 15
17906 Match E2-EPF5 TGC GGT CAG TGT CGT CAC Ctc SEQ ID NO: 16
25297 Mismatch-17906 TGC GTT CAG GGT CGT CAC Ctc SEQ ID NO: 17
17866 Match E2-EPF5 ATG GTC AGC AGT ACG TGT Cgg SEQ ID NO: 18
17871 Match GTT GAC GCA GAT CTC GCC Att SEQ ID NO: 19
17876 Match TCT TGG TCA GGA AGT AGC Cct SEQ ID NO: 20
17877 Match AGG AAG TAG CCC TTG GGT Ggg SEQ ID NO: 21
29985 Match UUC GGG UGG AAG AUC UUG Gtc SEQ ID NO: 22
29986 Match UCA UGC GGA ACA GAC CUC Cag SEQ ID NO: 23
25560 HIF-1a match AUA CCU UCU AGA UAU AUG Cat SEQ ID NO: 24
25564 HIF-1a mismatch AUA CAU UCU CGA UAU AUG Cat SEQ ID NO: 25
8549 Unrelated control UCG AAG UAC UCA GCG UAA Gtt SEQ ID NO: 26
2. Cell culture
All cell culture reagents are from Gibco-BRL-invitrogen AG (Basel.
Switzerland). Basal
culture medium used are Dulbecco's Modified Eagle Medium (DMEM) for HeLa cells
and
RPMI1640 for NCI-H1299 cells. Media are supplemented with 10% fetal bovine
serum (FBS)
and 1% L-Glutamine plus optionally 50 microgram/mI Gentamycine antibiotic. For
routine
maintenance cells are split 1: 4 twice a week. On the day before the
transfection, cells are
trypsinized, resuspended in medium with FBS but without antibiotics, and
plated either into
24-well culture plates at a density of 1.5-2 x 104 cells per 0.5 ml per well
or into 6-well plates
at a density of 5-10 x 104 cells per 2 ml per well. The plates are incubated
for 24 hours at
37 C in 5% CO2 at high humidity.
3. Transfection of siRNAs and desferrioxamine (DFO) and cobalt chloride
treatment
Transfection of siRNAs is performed using Oligofectamine according to the
protocols
provided by the manufacturer (Invitrogen). Briefly, siRNAs are diluted in
Optimem to 1.2 pM
siRNA per pl. Separately, Oligofectamine is diluted to 0.25 pl Oligofectamine
per pl
Optimem. Equal amounts of these siRNA and Oligofectamine solutions are mixed
and
incubated 20-25 minutes at room temperature to allow complex formation to
proceed. Next,
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the siRNA/oligofectamine complexes are diluted in standard medium with 10 %
FBS without
antibiotics to the final desired concentration, typically 20-80 nM siRNA.
Subsequently, the old
medium is removed from the cells and replaced with the siRNA/Oligofectamine
suspension:
for 24-well plates 0.5 ml per well and for 6-well plates 2 ml per well. After
48-72 hr incubation
in a humidified C02 incubator, the media with the siRNAs are replaced with
fresh media with
% FBS either without inducer or containing 150 M deferrioxamine (abbreviated:
DFO) or
CoC12 (both from Sigma; from freshly prepared aqueous stock solutions). After
6 h at 37 C
the medium of the cells is collected for determination of secreted VEGF
whereas of the cell
monolayer whole cell extracts are prepared for Western blot or total RNA
analysis
4. Transfection of ASOs and DFO and cobalt chloride treatment:
ASOs are stored at 1 mM concentration in TE (10 mM Tris pH 8.0, 1 mM EDTA).
Prior to the
experiment, a 10 pM working solution is prepared in Optimem (Life Sciences
Inc.). ASOs are
diluted to 2 x the final concentration in Optimem, mixed with Lipofectin or
Effectene also
diluted at 2 x the final concentration and left at room temperature for 30
minutes. The
standard medium is removed from a 30-50 % confluent T24 cell culture and the
cell
monolayer is once with serum-free OptiMEM1 (Gibco BRL). The ASO/Lipofectin mix
is then
added to the cell monolayer which is then incubated 4 h at 37 C in the C02
incubator, after
which the medium is removed and replaced with standard medium with 10 % FBS.
After 24-
72 hr incubation in a humidified C02 incubator, the media are replaced with
fresh media with
10 % FBS either without inducer or containing 150 ~M deferrioxamine
(abbreviated : DFO)
or CoC12 (both from Sigma; from freshly prepared aqueous stock solutions).
After 6 h at
37 C the medium of the cells is collected for determination of secreted VEGF
whereas of the
cell monolayer total RNA is prepared or whole cell protein extract for Western
blot analysis.
5. Real-time reverse transcriptase PCR
Total RNA is prepared using the RNeasy 96 kit (Qiagen #74183) following the
manufacturer's instructions. Primer pairs and FAM-labelled TaqMan probes for
real time
PCR are designed using the Primer Express v1.0 program (ABI PRISM, PE
Biosystems) and
purchased from Microsynth (Switzerland), Qiagen or Applied Biosystems ("Assays-
on-
demand"). The following primer sequences are used:
E2-EPF5 (Acc. Nr GI 181915):
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Reverse primer: 5'-AAAGACCTTGATGCCATCGG-3' (SEQ ID NO: 27)
Forward primer: 5'-TCCGCCTGGTGTACAAGGA-3' (SEQ ID NO: 28)
TaqMan probe: 5'-FAM-TGACGACACTGACCGCAGACCCA-TAMRA-3' (SEQ ID NO: 29)
HIF-la (Acc. Nr): NM_18105
ABI Assay-on-Demand : Hs00153153_m1
VEGF (Acc. Nr): NM_003376
Reverse primer: 5'-CACATTTGTTGTGCTGTAGGAAGC-3' (SEQ ID NO: 30)
Forward primer: 5'-TGAGATCGAGTACATCTTCAAGCC-3' (SEQ ID NO: 31)
TaqMan probe: 5'-FAM-CCATGCAGATTATGCGGATCAACCTCA-TAMRA-3' (SEQ ID NO:
32)
Or ABI Assay-on-Demand : Hs00173626_m1
GLUT-1 (Acc. Nr): NM_006516
ABI Assay-on-Demand : Hs00197884_m1
Hexokinase 2 (Acc. Nr): NM 000189.
Reverse primer: 5'-TGTCTTGAGCCGCTCTGAGAT-3' (SEQ ID NO: 33)
Forward primer: 5'-TGTCCGTAACATTCTCATCGATTT-3' (SEQ ID NO: 34)
TaqMan probe: 5'-FAM-CCAAGCGTGGACTGCTCTTCCGAG-TAMRA-3' (SEQ ID NO: 35)
Beta Actin (Acc. Nr) : X00351
Reverse primer: 5'-TAATGTCACGCACGATTTCCC-3' (SEQ ID NO: 36)
Forward primer: 5'-TCACCGAGCGCGGCT-3' (SEQ ID NO: 37)
TaqMan probe: 5'-FAM-CAGCTTCACCACCACGGCCGA-TAMRA-3' (SEQ ID NO: 38)
BcI-XL (Acc. Nr) : Z23115
Reverse primer: 5'-GGTCGCATTGTGGCCTTT-3' (SEQ ID NO: 39)
Forward primer: 5'-TCCTTGTCTACGCTTTCCACG-3' (SEQ ID NO: 40)
TaqMan probe: 5'-FAM-ACAGTGCCCCGCCGAAGGAGA-TAMRA-3' (SEQ ID NO: 41)
CYPA (Cyclophilin-A, Acc. Nr) : NM_021130
Reverse primer: 5'-TCGAGTTGTCCACAGTCAGCA-3' (SEQ ID NO: 42)
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Forward primer: 5'-GCGCTTTGGGTCCAGGA-3' (SEQ ID NO: 43)
TaqMan probe: 5'-FAM-TGGCAAGACCAGCAAGAAGATCACCA-TAMRA-3' (SEQ ID NO:
44)
For the real time PCR reaction 25 ng total RNA in 6 l water is mixed with
0.33 l 5' and 3'
primers (10 M each), 0.33 l TaqMan probe (5 M), 6.33 l RT PCR Master Mix
Kit
(Eurogentec, # RT-QRT-032X) in a total volume of 13 l following the
manufacturer's
instructions. Reverse transcription and real time PCR is performed in a ABI
PRISM
sequence detector 5700 or 7700 (Applied Biosystems) as follows: 30 minutes
reverse
transcription at 48 C, 10 minutes denaturation at 95 C followed by 50 cycles
of denaturation
for 15 sec. at 95 C and annealing and elongation for 1 min at 60 C. The
relative quantitation
of gene expression is calculated using the delta-Ct method as described in the
ABI PRISM
5700 user bulletin #2.
6. Western Blotting
Cell lysates are prepared in M-PER mammalian protein extraction reagent
according the
protoCol provided by the manufacturer (Pierce, Rockford, IL). 10 pg total
protein extract is
loaded on 8% premade acrylamide gels (NOVEX, San Diego, CA) and after
electrophoretic
separation transferred onto PVDF membranes. After saturation of the membrane
in TBST-5
% milk powder, proteins are immunostained with the relevant primary antibodies
diluted in
TBST-5 % milk powder. Next, the membrane is rinsed 3 x with TBST and for 30 in
incubated
with the appropriate secondary antibody also dissolved in TBST-5 % milk
powder. Next, the
membrane is rinsed 3 x with TBST and ECL Western blotting detection reagents
(Amersham
Biosciences) are used to visualise the immunostaining according to the
manufacturer's
protocol.
Dilutions used for primary antibodies are 1:250 for mouse anti-HIF-1a (BD
Biosciences/Pharmingen 610958), 1: 100.000 for mouse anti-P-actin (Sigma
A5441), 1:
2000 for rabbit anti-ARNT (Aryl hydrocarbon Receptor Nuclear Translocator,
Novus NB 730-
H), 1: 500 for rabbit anti-GLUTI (Glucose Transporter ABCAM ab652), 1: 1'000
for rabbit
anti- HIF2a-EPAS1 (Novus ab199 Cat. No. 730-H).
As secondary antibodies are used : goat anti-mouse IgG (Fab specific)-
Peroxidase antibody
(Sigma A2304) at a dilution of 1: 1: 5'000-20'000 or Goat polyclonal antibody
to Rabbit IgG
(HRP conjugated) Novus Catalog Number NB 730-H at a dilution of 1: 20'000
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7. Quantification of VEGF secretion
Secreted VEGF is determined by using an enzyme linked immunoassay (ELISA) kit
for
human VEGF (R& D Systems) according to the manufacture's instructions.
8. Characterization of E2-EPF5 siRNAs
In order to characterize E2-EPF5 specific gene inhibitors, NCI-H 1299 and HeLa
cells are
exposed to E2-EPF5 siRNAs and the inhibition of E2-EPF5 mRNA is studied by
TaqMan RT-
PCR. E2-EPF5 mRNA is reduced 70 to 90% after 48 h, respectively 72 h siRNA
incubation
in both cell lines. Mismatch and an unrelated control siRNAs do not affect E2-
EPF5 gene
expression. In the same manner, NCI-H1299 cells expressing recombinant E2-EPF5
tagged
with a HIS-FLAG epitope at the N-terminus are exposed to E2-EPF5 siRNAs and
controls.
All match siRNAs decrease E2-EPF5 protein levels specifically compared to the
controls with
17828 showing the strongest reduction.
9. Inhibition of E2-EPF5 suppresses VEGF expression in hypoxia
NCI-H1299, HeLa and DU-145 cell lines, which have been reported to be amenable
to
transfection of oligonucleotides are exposed to hypoxia-simulating compounds:
desferrioxamine (DFO), cobalt chloride (CoC12) and N-Oxalylglycine. The
results
demonstrate that in NCI-H1299 and Hela cells, DFO consistently induced VEGF
mRNA 4-7-
fold and VEGF protein 2-3-fold after 6 and 24 h stimulation. The extent of
induction by CoC12
is similar, but less consistent, declining after prolonged stimulation. N-
Oxalylglycine
significantly induces VEGF expression especially after 6 h. The basal level of
expression of
VEGF mRNA and protein (2 ng secreted VEGF protein per h per million cells) is
found to be
about 10-fold higher (2160 120 pg VEGF/h, 106 cells) in DU-145 than in the
two other cell
lines tested (NCI-H1299: 318 40 pg VEGF/h,106 cells; HeLa: 301 60 pg
VEGF/h,106
cells). Significant VEGF induction 2-3-fold at the RNA and 1.3-1.5-fold at the
protein level
can only be demonstrated with all inducers but only after 6 not after 24 h
stimulation.
Notably, under the applied conditions E2-EPF5 mRNA is not found to be
significantly
regulated in neither of the cell lines investigated.
To confirm the results of the HRE-reporter gene assay, the effect of siRNA-
mediated
inhibition of E2-EPF5 on the expression of endogenous HRE-driven target genes,
especially
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vascular endothelial growth factor (VEGF) is examined. NCI-H1299 cells are
exposed 3 days
days to E2-EPF5 siRNAs followed by a 6 h exposure with DFO and CoCI2,
respectively. As
shown in Table 4, all E2-EPF5 siRNAs induce a marked reduction of VEGF
secretion as
compared with cells transfected with corresponding mismatch and unrelated
control
oligonucleotides. In alignment with the observed activities on suppression of
E2-EPF5
siRNA 17828 leads to the most significant suppression of secreted VEGF. Same
results are
obtained using CoC12 instead of DFO as hypoxia inducing agent.
60nM STDEV
11723 38 7
11769 50 10
17828 27 2
25296 97 2
17906 71 4
25297 112 2
8548 100 7
8548-no DFO 71 2
Table 4: Effect of siRNA-mediated E2-EPF5 inhibition on the secretion of VEGF.
NCI-H1299
cells are transfected with 60 nM siRNAs against E2-EPF5, corresponding
mismatch controls
and an unrelated control siRNA 8548. 72 h later, cells are incubated with 150
M DFO for 6
h in comparison with the untreated 8548 control. VEGF protein concentrations
in conditioned
media are determined by ELISA and expressed as pg/ml per the total amount of
protein in
each well.
A further analysis applying the most potent E2-EPF5 siRNA 17828 reveals that
in both NCI-
H1299 and HeLa cell lines, inhibition of E2-EPF5 reduces the hypoxia-induced
expression of
VEGF mRNA and the secretion of hypoxia-induced VEGF protein. Reversion of
hypoxic
VEGF mRNA and protein is nearly complete to the levels observed in
normoxically cultured
cells as this is the case by a control siRNA targeting the transcription
factor HIF-1 a.
In addition, as shown in Table 5, E2-EPF5 downregulation inhibits another
endogenous Hif-1
target gene, namely GLUT-1 (Glucose transporter 1) whereas Hif-1 mRNA is not
significantly
changed. This suggests that E2-EPF5 suppression inhibits Hif-1 mediated
transcriptional
activation of HRE-driven genes without affecting the transcription of Hif-1
itself.
siRNA % GLUT-1 mRNA STDEV
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17828 189 7.8
25296 560 2.0
25560 240 0.4
25584 486 7.2
8548 457 17.3
8548-DFO 100 5.1
Table 5: siRNA-mediated inhibition of E2-EPF5 suppresses hypoxia-induced
expression of
GLUT-1 mRNA to a similar extent than an siRNA targeting HIF-1 a. HeLa cells
are
transfected with 40 nM siRNAs against E2-EPF5 (17828), HIF-l a (25560),
corresponding
mismatch controls (E2-EPF5: 25296, Hif-1 a: 25584) and an unrelated control
siRNA 8548.
72 h later, cells are incubated with 150 M DFO for 6 h in comparison with the
untreated
8548 control. GLUT-1 mRNA is assayed by TaqMan RT-PCR.
10. Expression of E2-EPF5 and cell proliferation
E2-EPF5 encodes an ubiquitin-conjugating enzyme (E2). E2s attach ubiquitin to
cellular
proteins thereby targeting them for proteasomal degradation or modulate their
function,
similarly to phosphorylation. Ubiquitin pathways play a key role in the
regulation of cell
growth and proliferation by controlling the abundance of cell cycle proteins
(Bashier et al,
2003) and many components of the ubiquitination machinery have been found to
be
disregulated, mutated or amplified in various cancers and/or correlate with a
poor prognosis.
The expression of E2-EPF5 in various human tissues by TaqMan real-time PCR
using
primers specific to E2-EPF5 is examined. E2-EPF5 is slightly upregulated in
thymus and
testis suggesting a potential relationship between cell proliferation and
expression of E2-
EPF5 (Table 6).
Tissue Arb. Units STDEVP
1 brain 0.33 0.08
2 heart 0.38 0.35
3 kidney 0.04 0.04
4 liver 0.03 0.03
lung 0.10 0.01
6 trachea 0.13 0.03
7 bone marrow 0.94 0.20
8 colon 0.33 0.14
9 intestine 0.33 0.12
spleen 0.44 0.19
11 stomach 0.11 0.00
12 thymus 1.59 0.53
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13 heart 0.71 0.03
14 mammary gland 0.15 0.00
15 prostate 0.84 0.47
16 skeletal muscle 0.03 0.01
17 testis 3.78 0.01
18 uterus 0.27 0.02
19 brain 0.78 0.19
20 cerebellum 0.92 0.12
21 fetal brain 0.71 0.11
22 fetal liver 0.87 0.24
23 spinal cord 0.50 0.05
24 placenta 0.17 0.06
25 adrenal gland 0.32 0.07
26 liver 0.04 0.02
27 pancreas 0.01 0.00
28 prostate 0.39 0.01
29 salivary gland 0.03 0.01
30 thyroid 0.03 0.02
Table 6: Expression of E2-EPF5 mRNA in various tissues (Clontech). The tissue
distribution
is determined by TaqMan real-time PCR using primers specific for E2-EPF5.
11. Cloning of human E2-EPF5 cDNA in vector pDONR201
E2-EPF5 cDNA is cloned by two sequential PCR reactions followed by insertion
in a
GatewayTM donor vector The first DNA amplification is done in the presence of
1 ng of
Quick-CloneTM cDNA from human fetal liver tissue (Clontech) using 10 pmoles
each of the
E2-EPF5-specific PCR primers (forward: ATC GAA GGT CGT ATG AAC TCC AAC GTG
GAG AAC CTA CCC CCG (SEQ ID NO: 45), reverse: TCA CTT GTC GTC GTC GTC CTT
GTA GTC CAG CCG CCG CAG CGC CCG CAG CGC CCG (SEQ ID NO: 46)),10 nmoles
each of the dNTPs, 2 mM MgSO2, 5 U of TaKaRa Ex TaqTM DNA polymerase in 50 pl
of Ex
-----
--T --q- - b-uff--er-overla - -
aid_ with 50 pl mineral oil, in-
-a-ther~mocycler block. The PCR cycling
conditions are as follows: 94 C for 10 min, [94 C for 1 min, 62 C for 1
min, 72 C for 1 min]
30 cycles, 72 C for 10 min, then 10 C on hold. The PCR products were
analysed by PAGE.
DNA is eluted from agarose by the Gene Clean II kit. Weak DNA bands are
reamplified by
the same protocol. A typical yield of amplified PCR product is about 8 pg DNA
in 50 pl H20.
The PCR product is composed of 5' - FXa site-specific E2 sequence-FLAG tag-
3'. The
second DNA amplification is done in the presence of 100 ng template DNA from
the first
PCR reaction, 100 pmoles each of PCR primers ATTB1 FXA2 (GGG ACA AGT TTG TAC
AAA AAA GCA GGC TTA GCT GGT ATC GAA GGT CGT ATG (SEQ ID NO: 47)) and
ATTB2FLAG (GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TCA CTT GTC GTC
GTC GTC CTT GTA GTC (SEQ ID NO: 48)), 20 nmoles each of the dNTPs, 2 mM MgSO4,
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% DMSO, 2.5 U of Pwo DNA polymerase in 100 pl buffer (10 mM Tris-HCI pH 8.8,
25 mM
KCI, 5 mM (NH4)S04), overlaid with 100 pl mineral oil, in a thermocycler
block. The PCR
cycling conditions are as follows: 94 C 2 min, [94 C for 1 min, 65 C for 1
min, 72 C for I
min] 2 cycles, [94 C for 1 min, 60 C for 1 min, 72 C for I min] 2 cycles,
[94 C for 1 min,
55 C for 1 min, 72 C for 1 min] 30 cycles, 72 C for 2 min, then 10 C on
hold.. The att-PCR
products are analysed by PAGE. DNA was purified for cloning with the PCR
Purification Kit
(Qiagen).Typically, the yield of amplified att-PCR products is 7-8 pg DNA in
50 ial H20. The
att-PCR products are composed of 5' - ATTB1-FXa site-specific E2 sequence-FLAG
tag-
ATTB2 - 3'. The att-PCR product is cloned in the GatewayTM donor vector
pDONR201. The
BP ClonaseTM enzyme mix catalyses a site-specific and orientation-specific in
vitro
recombination reaction via the attB1/B2 (PCR product) and attPl/P2 (vector)
sites. The
reaction mix (20 pL) contains BP buffer (as provided), 200 ng att-PCR DNA, 300
ng
pDONR201 vector and 4 pL BP ClonaseTM enzyme mix. After I h at 25 C proteinase
K (2
pg/pL) is added and samples are incubated for 10 min at 37 C. 1 pL is used to
transform
competent DH5a E. coli cells. Transformants are selected on LB plates with
kanamycin (50
mg/L). Clones are characterized using restriction enzymes and by DNA
sequencing. A
suitable clone is designated as pBM2537/NPL002981.
12. Cloning of E2-EPF5 cDNA into the GatewayTM expression vector pDEST12.2
The ClonaseTM LR enzyme mix mediates the GATEWAY LR recombination reaction via
the
attL1/L2 (pBM2537 entry clone) and attR1/R2 (pDEST12.2 vector) sites. The
reaction mix
(20 pL) contains LR buffer (as provided by the manufacturer), 200 ng entry
clone (pBM2537)
DNA, 300 ng expression vector (pDEST12.2, GIBCO-BRL-Invitrogen Corp) and 4 pL
LR
ClonaseTM enzyme mix. After 1 h at 25 C proteinase K (2 pg/pL) was added and
samples
are incubated for 10 min at 37 C. 1pL was used to transform competent DH5a
cells.
Transformants are selected on LB plates with ampicillin (50 mg/L). Clones are
characterized
using restriction enzymes and by DNA sequencing. A suitable clone is
designated as
pDEST-EPF5/ NPL006653.
13. Transfection of NCI-H1299 with pDEST-type expression plasmids
Media, fetal calf serum (FCS), Versene, Lipofectin, Geneticin are purchased
from Life
Sciences Inc.. Cell culture Petri dishes (d = 8 cm) used are Falcon type 3003,
6-well, 24-well
and 96 well multidishes are obtained from Nunc (Life Sciences Inc.). NCI-H1299
cells (CRL-
5803) are obtainable from the American Type Culture Collection (ATCC). The
cells are
maintained at 37 C in humidified atmosphere with 5% C02 in RPMI1640 with 10%
FCS and
601ag/ml Gentamycin. To propagate the culture, cells are split weekly: i.e.
rinsed twice with
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Versene, treated for 5 min. with Trypsin-EDTA solution, diluted in 15 x the
original medium
volume and replated 10 mI/8cm tissue culture dish or 20 ml per 75 cm2 T-
flask). After 3-4
days, 0.5 volume of medium is added to replenish the nutrients. NCI-H1299
cells are
transfected using Lipofectamine Plus reagent (Life Sciences-Invitrogen Corp.)
Briefly, cells
are seeded in 6-well multidishes at 1x105 per 6-well and grown for 1 day. In
two wells of a
96-well plate, 2 pg plasmid DNA (pDEST-12.2 or pDEST-EPF5) is mixed with 100
pl of
Optimem and 15 pl of PLUS reagent, and incubated at room temperature for 15
min. In
parallel, 10 pl of Lipofectamine is mixed with 100 pl of Optimem medium and
also incubated
for 15 min at room temperature. Subsequently, the DNA mixtures are added to
the
Lipofectamine solution, mixed well, and again incubated at room temperature
for 15 min. In
the mean time, the FCS-containing medium is aspired from the cells, the cells
are rinsed
with I mi/well Optimem and then 1 ml of Optimem is added to each well. Next
100 pl of the
plasmid/PLUS reagent/Lipofectamine complex is added to the medium and the
cells (now in
1150 pl of medium) are incubated for 4 h at 37 C in a humidified incubator
under 5% C02.
At this point the DNA-liposome mix is removed from the cells and 2 ml of fresh
RPMI1640
medium (with 10 % FCS and 60pg/ml Gentamycin) is added. The next day the cells
are
trypsinized as described above, resuspended in 3 ml RPMI1640 medium with FCS
and 300,
150, 60 or 30 pl of the cell suspension are plated in 8-cm Petri dishes with
10 ml of
RPMI1640 with 10% FCS and 60pg/ml Gentamycin. The next day 1.0 mg/ml Geneticin
is
added. Twice weekly, the selective medium (RPMI1640 + 10 % FCS + 1 mg/ml
Geneticin) is
replaced by fresh medium. Colonies appear after 2-3 weeks. From a plate with
well-
separated growing colonies transformed by pDEST-EPF5, 24 are scraped off using
a
pipettor with 100 pl tip while simultaneously aspiring the scraped-off cells
into the tip. The
cells from the tip are then transferred to a 24-well plate and 0.5 m[selective
medium is
added. 5-10 days later the cloned cells have formed a confluent monolayer. The
cells from
such a confluent 24-well are trypsinized, divided into two 24-well and one 6-
well "well". A few
days later, total RNA is isolated from one of the 24-wells of each clone and
the level of E2-
EPF5 mRNA is measured by RT-PCR as described in example 5. This RT-PCR
measures
the combined level of the recombinant E2-EPF5 mRNA from the integrated copy
(or copies)
of pDEST-EPF5 and of the native endogenous E2-EPF5 mRNA. In NCI-H1299 cells
successfully transformed by pDEST-EPF5 the combined E2-EPF5 mRNA is more than
2.5 x
higher than in native H1299 cells or cells transformed with pDEST12.2. Three
independent
NCI-H1299/pDEST-EPF5 clones are further amplified (nrs 1, 5, and 15).
Expression of
recombinant E2-EPF5 by NCI-H1299/pDEST-EPF5 cells is confirmed on Western
blots as
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described in example 6 using anti-FLAG epitope antibody (Sigma). As control
several 8 cm
Petridishes with ca. 200 colonies of NCI-H1299 cells transformed by pDEST12.2
are
trypinized and propagated to be used as negative control in experiments with
NCI-
H1299/pDEST-EPF5 cells.
13. Increased VEGF secretion in pDEST-EPF5 NCI-H1299 cell lines .
To determine the effect of forced overexpression of E2-EPF5-FLAG the cell
lines described
in example 12 are plated in 24-well plates at 20'000 cells per well. 20 h
later medium is
replaced by RPM11640 medium + 10 % FCS or RPMI1640 medium + 10 % FCS
containing
in addition 150 M DFO. 6 hours later the medium is harvested and frozen for
determination
of the secreted VEGF protein levels (example 7), whereas from the cells total
RNA is
isolated and the level of a set of mRNA species is measured by RT-PCR (example
5). The
amount of VEGF protein secreted by pDEST-EPF5-transformed NCI-H1299 cells,
which
express an elevated level of E2-EPF5, is significantly greater (2 0.3-fold
more) than the
amount secreted by pDEST12.2-cells, which express normal E2-EPF5 levels (Table
7). In
contrast the induction by DFO is essentially unchanged (Table 7). However, an
equivalent
DFO-induction in combination with elevated basal VEGF levels, implies that the
absolute
levels of VEGF after DFO-induction are 2-fold higher than in NCI-H1299 not
expressing
elevated E2-EPF5 cells.
% VEGF protein level STDEV % DFO induction STDEV
NCI-H1299 parental line 100 4 419 33
NCI-H1299/vector pool 87 3 366 4
NCI-H1299/EPF5#1 191 31 396 13
NCI-H1299/EPF5#5 236 21 309 7
NCI-H1299/EPF5#15 185 7 355 14
NCI-H1299/EPF5 3 lines 204 28 353 44
Table 7: VEGF protein levels in 6 h conditioned medium of pDEST12.2 and pDEST-
EPF5-
transformed NCI-H1299 cells compared to the level in parental NCI-H1299 cells
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14. Messenger RNA levels in pDEST-EPF5 NCI-H1299 cell lines
Using the total RNA isolated from plasmid-transformed NCI-H1299 cells (example
13), the
levels of several mRNAs species are determined by RT-PCR (example 5). The E2-
EPF5
mRNA level (recombinant + native E2-EPF5 mRNA) is in cells transformed with
pDEST-
EPF5 2.3-3-fold higher than in the parental NCI-H1299 cells (Table 8). This is
accompanied
by a significantly elevated level of the mRNA of the mRNA of 3 known HIF-
regulated genes:
VEGF mRNA (38 10 % increased), hexokinase-2 mRNA (HK2, 4.9 2.5 -fold
increased)
and GLUT-1 mRNA (3.8 0.2 -fold increased). In contrast, the basal levels of
the mRNAs of
a number of genes not regulated by HIF are not significantly changed (R-actin,
Bcl-xL, CYPA
and HIF1a). The rate of induction of the HIF-regulated genes by DFO was not
altered in E2-
EPF5 overexpressing cells, but the combined effect of higher basal mRNA levels
and normal
DFO-induction nevertheless results in higher levels of these HIF-responsive
mRNAs than
observed in DFO-stimulated NCI-H1299 cells that do not express extra E2-EPF5.
HIF-regulated Reference genes
EPF5 VEGF HK2 GLUT1 actin Bcl-xL CYPA HIF1a
H1299 parental line 108 89 275 170 114 120 98 131
H1299/vector pool 100 100 100 100 100 100 100 100
H1299/EPF5#1 245 143 334 382 145 102 80 124
H1299/EPF5#5 304 143 362 347 90 51 86 121
H1299/EPF5#15 235 127 771 415 118 154 98 136
H1299/EPF5 aIl 3 261 138 489 381 118 102 88 127
H1299 parental line STDEV 19 12 120 65 8 18 20 14
H1299/vector pool STDEV 9 4 31 38 10 30 14 22
H1299/EPF5#1 STDEV 85 28 117 11 5 20 22 42
H1299/EPF5#5 STDEV 79 6 98 11 18 2 22 12
H1299/EPF5#15 STDEV 40 15 227 42 10 64 1 13
H1299/EPF5 all 3 STDEV 37 10 251 18 27 55 16 22
lo induction b DFO EPF5 VEGF HK2 GLUT1 actin Bcl-xL CYPA HIF1a
H1299 parental line n.d.. 419 479 254 59 53 74 49
H1299/vector pool n.d.. 326 1395 198 58 60 74 31
H1299/EPF5#1 n.d.. 403 701 258 59 81 108 49
H1299/EPF5#5 n.d.. 256 725 292 65 76 78 67
H1299/EPF5#15 n.d.. 249 344 248 63 34 89 90
H1299/EPF5 all 3 n.d.. 303 590 266 63 64 92 69
H1299 parental line STDEV n.d.. 78 278 45 4 7 11 6
H1299/vector pool STDEV n.d.. 33 345 4 12 14 8 8
H1299/EPF5#1 STDEV n.d.. 18 145 60 4 13 18 10
H1299/EPF5#5 STDEV n.d.. 19 143 78 15 6 12 5
H1299/EPF5#15 STDEV n.d.. 7 58 83 5 6 6 5
H1299/EPF5 all 3 STDEV n.d.. 87 169 23 3 18 15 14
Table 7: mRNA expression levels in pDEST12.2 and pDEST-EPF5-transformed NCI-
H1299
cell lines (n. d. = not determined)
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15. Production of recombinant E2-EPF5-FLAG protein in E. coli
The insert from plasmid pBM2537/NPL002981 (example 11) is recombined according
to the
GatewayTM system manual with vector pDEST17 (Invitrogen Corp) to give plasmid
E2-12-
flag-pDEST17/ NPL006782. The encoded amino acid sequence of the recombinant E2-
EPF5 protein is :
MSYYHHHHHHLESTSLYKKAGLIEGRMNSNVENLPPHI IRLVYKEVTTLTADPPDGIKVF
PNEEDLTDLQVTIEGPEGTPYAGGLFRMKLLLGKDFPASPPKGYFLTKIFHPNVGANGEI
CVNVLKRDWTAELGIRHVLLTIKCLLIHPNPESALNEEAGRLLLENYEEYAARARLLTEI
HGGAGGPSGRAEAG RALASGTEASSTDPGAPGGPGGAEGPMAKKHAG ERDKKLAAKKKT
DKKRALRALRRLDYKDDDDK (SEQ ID NO: 49)
This plasmid is used to express recombinant E2-EPF5 in E. coli. Through the N-
terminal 6-
HIS tag it can be purified by standard metal-chelate affinity chromatography
and this results
in a more than 95 % pure preparation of tagged E2-EPF5. It is active in an in
vitro
autoubiquitination assay and can be detected using antibodies to the C-
terminal FLAG-tag
peptide sequence (DYKDDDDK).
16. Production of rabbit antibody to recombinant E2-EPF5-FLAG protein
Rabbits are immunized with E. coli-derived recombinant E2-EPF5 (Example 15) by
subcutaneous injection of 0.2 mg with complete Freund's adjuvans followed at
weekly
intervals by a boost with 0.1 mg protein with incomplete Freund's adjuvans. 20-
30 ml blood
is harvested after 5, 6, 7, 8 and 9 weeks followed by a terminal bleeding of
100-120 ml blood
of after 10 weeks from which serum is prepared. From the terminal bleeding
serum
polyclonal anti-E2-EPF5 antibodies are purified by affinity chromatography on
immobilized
_ _ - -- ----- -- ----- --- ----- --- ,_ . - - recombinant E2-EPF5. Briefly:
Recombinant E.coli-derived (FLAG-tagged) E2EPF5
(example 15) was covalently coupled to Anti-FLAG Sepharose using dimethyl
pimelimidate
in borate buffer as described in: Harlow, E. and Lane, D. Using Antibodies: A
Laboratory
Manual, (1999) Cold Spring Harbor NY Cold Spring Harbor Press, pp. 522-523. To
isolate
E2-EPF5-specific antibodies 1.5 ml crude rabbit anti-E2-EPF5 serum is applied
to 150
microliter packed beads equilibrated with PBS. Unbound antibodies are removed
with an
excess of PBS. Anti-E2-EPF5 antibodies are eluted using Glycine-HCI 0.2M (pH
2.5),
immediately neutralized by addition of 1/10 volume of Tris-HCI 1 M pH 9 and
stored at 4 C.
17. Detection of E2-EPF5 in mouse tumor tissue
Tumor used for staining are from B16BL6 melanomas grown in C57BL/6 mice.
Female
black, C57BL/6 mice, weighing between 17 to 20 g, are obtained from Iffa Credo
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(L'Arbresle, France) animal breeding facility. They are identified via tail
markings and kept
in groups (6-7 animals per cage) under normal conditions and observed daily.
Six mice are
used per treatment group. The melanin producing murine melanoma tumor cell
line
B16/BL6, is derived from a spontaneous tumor of C57BL/6 mice, has been
extensively
characterized and has been obtained from Dr. Isaiah J.Fidler, Texas Medical
Center,
Houston, USA. The cultured tumor cells used in all experiments are free of
Mycoplasma.
They are cultured at 37 C and 5% C02 in MEM (MEM EBS, AMIMED, Allschwil) with
stable
glutamine supplemented with 5% fetal calf serum, 1% sodium pyruvate, 1% non-
essential
amino acids and 2% vitamins and grown until confluency. Subsequently they are
detached
with 0.25% trypsin-0.02% EDTA (2 min at 37 C), and then processed. Viability
is assessed
by trypan blue exclusion, and only suspensions with >90% viability are used.
The tumor cells
are resuspended in Hanks Buffer and a suspension of 5 x 104 cells/pI is
prepared for
intradermal (i.d.) injection into the ears of immuno-competent syngeneic
C57BL/6 female
mice. For tumor cell injection, anesthesia is induced by a 3% Isoflurane
(Forene r, Abbott
AG, Cham, Switzerland) inhalation. The animals are placed on a warmed
operative field
maintained at a temperature of 39 C and their ears are gently extended over a
steel cone
fitted with a double-sided sticker. With the aid of a microscope, a 30G
hypodermic needle is
then inserted at the periphery of the ear and tunneled for 4-5 mm in a
subcutaneous plane to
allow delivery of the tumor cells to a site distal to the needle entry point.
The injection site is
always located on the dorsum of the ear between the first and second
neurovascular bundle.
Using a microliter syringe (250 pl, Hamilton, Bonaduz, Switzerland), 1 pl of
tumor cells
suspension (5 x 104 cells ) are injected into the subcutaneous plane of the
mouse ear
forming a 2 x 2 mm sub-dermal blister. After one week, the primary tumor
starts to develop
and a black dot can be easily seen in the middle of the ear. Primary tumor
size was
monitored at day 7, 14 and 21. After three weeks (Day 21) the animals are
killed by CO2
inhalation, the cervical lymph nodes weighed, fixed' in 4.2 % formaldehyde,
and embedded
in paraffin.
5pm paraffin sections (prepared with a Microtome, MICROM) are placed on
SuperFrost+
(Menzel) glas slides, dried over night at 37oC, and heated for 5 minutes at
59oC on a hot
plate. Sections are dewaxed 2x in Xylene, rehydrated in decreasing ethanol
solutions (100%,
95%,90%, 70%), rinsed in dd water and subsequently subjected to a high
temperature
antigen unmasking technique. Sections are microwaved (14 minutes heat up to
98oC, 10min
hold at 98oC; Milestone #T/TMEGA) in 0.1 m Na-citrate pH 6Ø Sections are
cooled down to
room temperature, rinsed in double-distilled water (ddH2O) and immersed in
PBS. To block
CA 02580883 2007-03-19
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endogenous peroxidase activity, sections are incubated for 30 minutes in 0.3%
H202 in PBS
and rinsed in PBS. To block non-specific antibody binding, sections are
incubated for 20
minutes with PBS containing 1.5% goat serum (Vector Laboratories) at room
temperature in
a humid chamber. The blocking serum is blotted off, and the sections are
incubated for 1
hour with the primary antibody (0.5 -1.Opg/ml polyclonal rabbit anti-EPF5)
diluted in PBS
containing 0.1 % Tween-20. Sections are rinsed 3x 2 minutes in PBS and
subsequently
incubated with the secondary link antibody (biotinylated goat anti-rabbit IgG,
Vector
Laboratories) diluted in PBS containing 1.5% blocking serum. Sections are
rinsed 3x2
minutes in PBS and incubated for 30 minutes at room temperature with avidin
horseradish H
complex (VECTASTAIN Elite ABC kit PK-6101). The sections are washed 3x2
minutes in
PBS and stained for 5 to 10 minutes with Vector NovaRed (substrate kit for
peroxidase,
Vector Laboratories #SK-480) and rinsed 3x2 minutes in ddH2O. Sections are
then
counterstained for 30 seconds in Mayer's Hematoxylin (Fluka #51275), rinsed
for 5 minutes
in running tap water, rinsed in ddH2O and air dried. The sections are mounted
with Eukitt
(Fluka #03989) and analyzed by brightfield microscopy.
Examination of B16BL6 lymph node metastases reveals a strikingly intense
staining of the
tumor cells immediately adjacent to the necrotic core of the tumor. These are
the living
tumor cells furthest away from the supplying blood vessels, which is a
presumably hypoxic
area. An elevated E2-EPF5 protein level in this area is therefore consistent
with a role of E2-
EPF5 in the hypoxia response of the tumor cells. As the hypoxia response is
very important
for the survival of the tumor cells, inhibiting E2-EPF5 is expected to
interfere with the growth
of the tumors an effect that can be exploited therapeutically.
18. Detection of E2-EPF5 in areas of neovascularization in a mouse eye disease
model
Ischemic retinopathy.is produced in C57/BL6J mice (Smith et al.1994, Oxygen-
induced
retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101-111.). Briefly,
seven-day-old
mice and their mothers are placed in an airtight incubator and exposed to an
atmosphere of
75 3% oxygen for 5 days (hyperoxia). Incubator temperature is maintained at
23 2 C,
and oxygen is measured every 8 hours with an oxygen analyzer. After 5 days,
the mice are
removed from the incubator, placed in room air, and after 5 days at P17, the
mice are
sacrificed, eyes are rapidly removed and frozen in optimum cutting temperature
embedding
compound (OCT; Miles Diagnostics, Elkhart, IN) or fixed in 4% phosphate-
buffered
formaldehyde and embedded in paraffin. C67BL6J mice of the same gender and
age, which were
not exposed to hyperoxia, served as controls. They are treated by gavage with
vehicle and after 5
days, they are sacrificed and their eyes are processed for frozen or paraffin
sections. 5pm
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frozen sections are air dried, fixed for 10 minutes at 4oC in dry acetone, and
rinsed in PBS.
Endogenous tissue peroxidase activity is blocked by incubating the sections 30
minutes in
0.3% H202 in PBS. The sections are rinsed and incubated for 20 minutes with
PBS
containing 1.5% goat serum. All subsequent immunostaining steps are performed
as
described in 17. Immunostaining of EPF5 in paraffin sections is performed as
described
in17. Examination of the tissue slides reveals significantly higher levels of
E2-EPF5 staining
in the areas of pathological neovascularization in ROP eyes including the
blood vessel cells
themselves, but not in the corresponding areas of control eyes. The
observation of elevated
levels of E2-EPF5 protein in a tissue reacting to hypoxia is consistent with a
functional role of
E2-EPF5 in the cellular hypoxia response. Conversely, due to the relative
hypoxia
experienced when the mice are transferred from high to normal oxygen levels an
abnormal
vascularisation of the retina occurs. This retinopathy of prematurity model
(ROP) is a widely
used retinal angiogenesis model. It is likely that inhibiting E2-EPF5
interferes with this
hypoxia response, an effect considered beneficial in several disease states.
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