U.S. patent application number 12/030558 was filed with the patent office on 2008-10-09 for identification of novel factors that block programmed cell death or apoptosis by targeting jnk.
This patent application is currently assigned to THE UNIVERSITY OF CHICAGO. Invention is credited to Concetta Bubici, Guido Franzoso, Salvatore Papa, Francesca Zazzeroni.
Application Number | 20080247956 12/030558 |
Document ID | / |
Family ID | 46303679 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080247956 |
Kind Code |
A1 |
Franzoso; Guido ; et
al. |
October 9, 2008 |
IDENTIFICATION OF NOVEL FACTORS THAT BLOCK PROGRAMMED CELL DEATH OR
APOPTOSIS BY TARGETING JNK
Abstract
Methods and compositions for modulating apoptosis by acting on
the c-Jun-N-terminal kinase (JNK) pathway and assays for the
isolation of agents capable of modulating apoptosis, including
modulators of the JNK pathway are disclosed. A method of modulating
JNK pathway independent of Gadd46.beta. is disclosed. Methods and
compositions are presented for the preparation and use of novel
therapeutic compositions for modulating diseases and conditions
associated with elevated or decreased apoptosis.
Inventors: |
Franzoso; Guido; (London,
GB) ; Papa; Salvatore; (Chicago, IL) ; Bubici;
Concetta; (Chicago, IL) ; Zazzeroni; Francesca;
(Montesilvano Colle, IT) |
Correspondence
Address: |
BARNES & THORNBURG LLP
P.O. BOX 2786
CHICAGO
IL
60690-2786
US
|
Assignee: |
THE UNIVERSITY OF CHICAGO
Chicago
IL
|
Family ID: |
46303679 |
Appl. No.: |
12/030558 |
Filed: |
February 13, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11032794 |
Jan 10, 2005 |
7354898 |
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12030558 |
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11000365 |
Nov 29, 2004 |
7326418 |
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11032794 |
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10626905 |
Jul 25, 2003 |
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11000365 |
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10263330 |
Oct 2, 2002 |
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10626905 |
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60526231 |
Dec 2, 2003 |
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60328811 |
Oct 12, 2001 |
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60326492 |
Oct 2, 2001 |
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Current U.S.
Class: |
424/9.2 ;
435/7.1 |
Current CPC
Class: |
A61K 31/00 20130101;
C07K 14/47 20130101; C12N 9/1205 20130101; A61K 49/0008 20130101;
A61K 31/711 20130101; C07K 14/4747 20130101; G01N 33/573 20130101;
G01N 33/5041 20130101; A61K 38/1709 20130101; A61K 48/00 20130101;
A61K 31/704 20130101; G01N 2510/00 20130101; A61K 31/7105
20130101 |
Class at
Publication: |
424/9.2 ;
435/7.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; G01N 33/68 20060101 G01N033/68 |
Claims
1. A method for screening and identifying an agent that modulates
activity of the JNK pathway in vivo, the method comprising: (a)
obtaining a candidate agent that binds to a factor which binds to a
molecule with an amino acid sequence consisting essentially of
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); (b)
administering the agent to an animal; and (a) determining whether
the level of JNK activity or programmed cell death in the animal is
increased compared to JNK activity or programmed cell death in
animals not receiving the agent.
2. A method to identify inhibitors of Gadd45.beta., the method
comprising: (a) screening for a candidate compound that binds to
peptidic regions consisting essentially of amino acid sequences
from positions 60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND, SEQ ID NO: 2)
and 69-86 (IALQIHFTLIQSFCCDND, SEQ ID NO: 3) of Gadd45.beta.; and
(b) determining the ability of the candidate compound to bind to
Gadd45.beta. or interfere with Gadd45.beta.-mediated inhibition of
JNKK2.
3. A method to identify agents that interfere with binding of JNKK2
to a molecule capable of binding to positions 142-166
(TGHVIAVKQMRRSGNKEENKRILMD, SEQ ID NO: 1) of the full length JNKK2,
the method comprising: (a) obtaining an agent that interferes with
the binding of the molecule to positions 142-166
(TGHVIAVKQMRRSGNKEENKRILMD) of the full length JNKK2; (b)
contacting a cell with the agent under conditions that would induce
JNK activation or programmed cell death; and (c) comparing cells
contacted with the agent to cells not contacted with the agent to
determine if the JNK pathway is upregulated.
Description
CONTINUITY INFORMATION
[0001] This application is a continuation of U.S. Ser. No.
11/032,794, filed Jan. 10, 2005, which is a continuation-in-part of
U.S. Ser. No. 11/000,365, filed Nov. 29, 2004, which claims
priority to 60/526,231, filed Dec. 2, 2003, and is a
continuation-in-part of U.S. Ser. No. 10/626,905, filed Jul. 25,
2003, which is a continuation-in-part of U.S. Ser. No. 10/263,330,
which claims priority to 60/328,811, filed Oct. 12, 2001, and
60/326,492, filed Oct. 2, 2001, the disclosures of which are hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] Methods and compositions that modulate apoptosis are based
on blocking or stimulating components of cell survival or death
pathways from NF-.kappa.B/I.kappa.B through gene activation, to
Gadd45.beta. interacting with components of the JNK pathway such as
MKK7. Gadd45 .beta.-independent JNK modulation exists in certain
cell types to regulate apoptosis or cell survival. The JNK pathway
is a focus for control of a cell's progress towards survival or
death.
[0003] Apoptosis or programmed cell death is a physiologic process
that plays a central role in normal development and tissue
homeostasis. Many factors interact in complex pathways to lead to
cell death or cell survival.
[0004] A. NF-.kappa.B
[0005] 1. NF-.kappa.B in Immune and Inflammatory Responses
[0006] NF-.kappa.B transcription factors are coordinating
regulators of innate and adaptive immune responses. A
characteristic of NF-.kappa.B is its rapid translocation from
cytoplasm to nucleus in response to a large array of extra-cellular
signals, among which is tumor necrosis factor (TNF.alpha.).
NF-.kappa.B dimers generally lie dormant in the cytoplasm of
unstimulated cells, retained there by inhibitory proteins known as
I.kappa.Bs, and can be activated rapidly by signals that induce the
sequential phosphorylation and proteolytic degradation of
I.kappa.Bs. Removal of the inhibitor allows NF-.kappa.B to migrate
into the cell nucleus and rapidly induce coordinate sets of
defense-related genes, such as those encoding numerous cytokines,
growth factors, chemokines, adhesion molecules and immune
receptors. In evolutionary terms, the association between cellular
defense genes and NF-.kappa.B dates as far back as half a billion
years ago, because it is found in both vertebrates and
invertebrates. While in the latter organisms, NF-.kappa.B factors
are mainly activated by Toll receptors to induce innate defense
mechanisms. In vertebrates, these factors are also widely utilized
by B and T lymphocytes to mount cellular and tumoral responses to
antigens.
[0007] Evidence exists for roles of NF-.kappa.B in immune and
inflammatory responses. This transcription factor also plays a role
in widespread human diseases, including autoimmune and chronic
inflammatory conditions such as asthma, rheumatoid arthritis, and
inflammatory bowel disease. Indeed, the anti-inflammatory and
immunosuppressive agents that are most widely used to treat these
conditions such as glucocorticoids, aspirin, and gold salts, work
primarily by suppressing NF-.kappa.B.
[0008] TNF.alpha. is arguably the most potent pro-inflammatory
cytokine and one of the strongest activators of NF-.kappa.B. In
turn, NF-.kappa.B is a potent inducer of TNF.alpha., and this
mutual regulation between the cytokine and the transcription factor
is the basis for the establishment of a positive feedback loop,
which plays a central role in the pathogenesis of septic shock and
chronic inflammatory conditions such as rheumatoid arthritis (RA)
and inflammatory bowel disease (IBD). Indeed, the standard
therapeutic approach in the treatment of these latter disorders
consists of the administration of high doses of NF-.kappa.B
blockers such as aspirin and glucocorticoids, and the inhibition of
TNF.alpha. by the use of neutralizing antibodies represents an
effective tool in the treatment of these conditions. However,
chronic treatment with NF-.kappa.B inhibitors has considerable side
effects, including immunosuppressive effects, and due to the onset
of the host immune response, patients rapidly become refractory to
the beneficial effects of anti-TNF.alpha. neutralizing
antibodies.
[0009] 2. NF-.kappa.B and the Control of Apoptosis
[0010] In addition to coordinating immune and inflammatory
responses, the NF-.kappa.B/Rel group of transcription factors
controls apoptosis. Apoptosis, that is, programmed cell death
(PCD), is a physiologic process that plays a central role in normal
development and tissue homeostasis. The hallmark of apoptosis is
the active participation of the cell in its own destruction through
the execution of an intrinsic suicide program. The key event in
this process is the activation by proteolytic cleavage of caspases,
a family of evolutionarily conserved proteases. One pathway of
caspase activation, or "intrinsic" pathway, is triggered by Bcl-2
family members such as Bax and Bak in response to developmental or
environmental cues such as genotoxic agents. The other pathway is
initiated by the triggering of "death receptors" (DRs) such as
TNF-receptor 1 (TNF-R1), Fas (CD95), and TRAIL-R1 and R2, and
depends on the ligand-induced recruitment of adaptor molecules such
as TRADD and FADD to these receptors, resulting in caspase
activation.
[0011] The deregulation of the delicate mechanisms that control
cell death can cause serious diseases in humans, including
autoimmune disorders and cancer. Indeed, disturbances of apoptosis
are just as important to the pathogenesis of cancer as
abnormalities in the regulation of the cell cycle. The inactivation
of the physiologic apoptotic mechanism also allows tumor cells to
escape anti-cancer treatment. This is because chemotherapeutic
agents, as well as radiation, ultimately use the apoptotic pathways
to kill cancer cells.
[0012] Evidence including analyses of various knockout
models--suggests that activation of NF-.kappa.B is required to
antagonize killing cells by numerous apoptotic triggers, including
TNF.alpha. and TRAIL. Indeed, most cells are completely refractory
to TNF.alpha. cytotoxicity, unless NF-.kappa.B activation or
protein synthesis is blocked. Remarkably, the potent pro-survival
effects of NF-.kappa.B serve a wide range of physiologic processes,
including B lymphopoiesis, B- and T-cell co stimulation, bone
morphogenesis, and mitogenic responses. The anti-apoptotic function
of NF-.kappa.B is also crucial to ontogenesis and chemo- and
radio-resistance in cancer, as well as to several other
pathological conditions.
[0013] There is evidence to suggest that JNK is involved in the
apoptotic response to TRAIL. First, the apoptotic mechanisms
triggered by TRAIL-Rs are similar to those activated by TNF-R1.
Second, as with TNF-R1, ligand engagement of TRAIL-Rs leads to
potent activation of both JNK and NF-.kappa.B. Thirdly, killing by
TRAIL is blocked by this activation of NF-.kappa.B. Nevertheless,
the role of JNK in apoptosis by TRAIL has not been yet
demonstrated.
[0014] The triggering of TRAIL-Rs has received wide attention as a
powerful tool for the treatment of certain cancers, and there are
clinical trials involving the administration of TRAIL. This is
largely because, unlike normal cells, tumor cells are highly
susceptible to TRAIL-induced killing. The selectivity of the
cytotoxic effects of TRAIL for tumor cells is due, at least in
part, to the presence on normal cells of so-called "decoy
receptors", inactive receptors that effectively associate with
TRAIL, thereby preventing it from binding to the signal-transuding
DRs, TRAIL-R1 and R2. Decoy receptors are instead expressed at low
levels on most cancer cells. Moreover, unlike with FasL and
TNF.alpha., systemic administration of TRAIL induces only minor
side effects, and overall, is well-tolerated by patients.
[0015] Cytoprotection by NF-.kappa.B involves activation of
pro-survival genes. However, despite investigation, the bases for
the NF-.kappa.B protective function during oncogenic
transformation, cancer chemotherapy, and TNF.alpha. stimulation
remain poorly understood. With regard to TNF-Rs, protection by
NF-.kappa.B has been linked to the induction of Bcl-2 family
members, BCl-X.sub.L and A1/Bfl-1, XIAP, and the simultaneous
upregulation of TRAF1/2 and c-IAP1/2. However, TRAF2, c-IAP1,
BCl-X.sub.L, and XIAP are not significantly induced by TNF.alpha.
in various cell types and are found at near-normal levels in
several NF-.kappa.B deficient cells. Moreover, Bcl-2 family
members, XIAP, or the combination of TRAFs and c-IAPs can only
partly inhibit PCD in NF-.kappa.B null cells. In addition,
expression of TRAF1 and A1/Bfl-1 is restricted to certain tissues,
and many cell types express TRAF1 in the absence of TRAF2, a factor
needed to recruit TRAF1 to TNF-R1. Other putative NF-.kappa.B
targets, including A20 and IEX-1L, are unable to protect
NF-.kappa.B deficient cells or were questioned to have
anti-apoptotic activity. Hence, these genes cannot fully explain
the protective activity of NF-.kappa.B.
[0016] 3. NF-.kappa.B in Oncogenesis and Cancer Therapy
Resistance
[0017] NF-.kappa.B plays a role in oncogenesis. Genes encoding
members of the NF-.kappa.B group, such as p52/p100, Rel, and RelA
and the I.kappa.B-like protein Bcl-3, are frequently rearranged or
amplified in human lymphomas and leukemias. Inactivating mutations
of I.kappa.B.alpha. are found in Hodgkin's lymphoma (HL).
NF-.kappa.B is also linked to cancer independently of mutations or
chromosomal translocation events. Indeed, NF-.kappa.B is activated
by most viral and cellular oncogene products, including HTLV-I Tax,
EBV EBNA2 and LMP-1, SV40 large-T, adenovirus E1A, Bcr-Abl,
Her-2/Neu, and oncogenic variants of Ras. Although NF-.kappa.B
participates in several aspects of oncogenesis, including cancer
cell proliferation, the suppression of differentiation, and tumor
invasiveness, direct evidence from both in vivo and in vitro models
suggests that its control of apoptosis is important to cancer
development. In the early stages of cancer, NF-.kappa.B suppresses
apoptosis associated with transformation by oncogenes. For
instance, upon expression of Bcr-Abl or oncogenic variants of
Ras--one of the most frequently mutated oncogenes in human
tumors--inhibition of NF-.kappa.B leads to an apoptotic response
rather than to cellular transformation. Tumorigenesis driven by EBV
is also inhibited by I.kappa.B.alpha.M--a super-active form of the
NF-.kappa.B inhibitor, I.kappa.B.alpha.. In addition, NF-.kappa.B
is essential for maintaining survival of a growing list of late
stage tumors, including HL, diffuse large B cell lymphoma (DLBCL),
multiple myeloma, and a highly invasive, estrogen receptor (ER) in
breast cancer. Both primary tissues and cell line models of these
malignancies exhibit constitutively high NF-.kappa.B activity.
Inhibition of this aberrant activity by I.kappa.B.alpha.M or
various other means induces death of these cancerous cells. In ER
breast tumors, NF-.kappa.B activity is often sustained by PI-3K and
Akt1 kinases, activated by over-expression of Her-2/Neu receptors.
Constitutive activation of this Her-2/Neu/PI-3K/Akt1/NF-.kappa.B
pathway has been associated with the hormone-independent growth and
survival of these tumors, as well as with their well-known
resistance to anti-cancer treatment and their poor prognosis. Due
to activation of this pathway cancer cells also become resistant to
TNF-R and Fas triggering, which helps them to evade immune
surveillance.
[0018] Indeed, even in those cancers that do not contain
constitutively active NF-.kappa.B, activation of the transcription
factors by ionizing radiation or chemotherapeutic drugs (e.g.
daunorubicin and etoposide) can blunt the ability of cancer therapy
to kill tumor cells. In fact, certain tumors can be eliminated in
mice with CPT-11 systemic treatment and adenoviral delivery of
I.kappa.B.alpha.M.
[0019] B. JNK
[0020] 1. Roles of JNK in Apoptosis
[0021] The c-Jun-N-terminal kinases (JNK1/2/3) are the downstream
components of one of the three major groups of mitogen-activated
protein kinase (MAPK) cascades found in mammalian cells, with the
other two consisting of the extracellular signal-regulated kinases
(ERK1/2) and the p38 protein kinases
(p38.alpha./.beta./.gamma./.delta.). Each group of kinases is part
of a three-module cascade that include a MAPK (JNKs, ERKs, and
p38s), which is activated by phosphorylation by a MAPK kinase
(MAPKK), which in turn is activated by phosphorylation by a MAPKK
kinase (MAPKKK). Whereas activation of ERK has been primarily
associated with cell growth and survival, by and large, activation
of JNK and p38 have been linked to the induction of apoptosis.
Using many cell types, it was shown that persistent activation of
JNK induces cell death, and that the blockade of JNK activation by
dominant-negative (DN) inhibitors prevents killing by an array of
apoptotic stimuli. The role of JNK in apoptosis is also documented
by the analyses of mice with targeted disruptions of jnk genes.
Mouse embryonic fibroblasts (MEFs) lacking both JNK1 and JNK2 are
completely resistant to apoptosis by various stress stimuli,
including genotoxic agents, UV radiation, and anisomycin, and
jnk3-/- neurons exhibit a severe defect in the apoptotic response
to excitotoxins. Moreover, JNK2 was shown to be required for
anti-CD3-induced apoptosis in immature thymocytes.
[0022] However, while the role of JNK in stress-induced apoptosis
is well established, its role in killing by DRs such as TNF-R1,
Fas, and TRAIL-Rs has remained elusive. Some initial studies have
suggested that JNK is not a critical mediator of DR-induced
killing. This was largely based on the observation that, during
challenge with TNF.alpha., inhibition of JNK activation by DN
mutants of MEKK1--an upstream activator of JNK had no effect on
cell survival. In support of this view, it was also noted that
despite their resistance to stress-induced apoptosis, JNK null
fibroblasts remain sensitive to killing by Fas. In contrast,
another early study using DN variants of the JNK kinase, MKK4/SEK1,
had instead indicated an important role for JNK in pro-apoptotic
signaling by TNF-R.
[0023] 2. Roles of JNK in Cancer
[0024] JNK is potently activated by several chemotherapy drugs and
oncogene products such as Bcr-Abl, Her-2/Neu, Src, and oncogenic
Ras. Hence, cancer cells must adopt mechanisms to suppress
JNK-mediated apoptosis induced by these agents. Indeed,
non-redundant components of the JNK pathway (e.g. JNKK1/MKK4) have
been identified as candidate tumor suppressors, and the
well-characterized tumor suppressor BRCA1 is a potent activator of
JNK and depends on JNK to induce death. Some of the biologic
functions of JNK are mediated by phosphorylation of the c-Jun
oncoprotein at S63 and S73, which stimulates c-Jun transcriptional
activity. However, the effects of c-Jun on cellular transformation
appear to be largely independent of its activation by JNK. Indeed,
knock-in studies have shown that the JNK phospho-acceptor sites of
c-Jun are dispensable for transformation by oncogenes, in vitro.
Likewise, some of the activities of JNK in transformation and
apoptosis, as well as in cell proliferation, are not mediated by
c-Jun phosphorylation. For instance, while mutations of the JNK
phosphorylation sites of c-Jun can recapitulate the effects of JNK3
ablation in neuronal apoptosis--which is dependent on
transcriptional events--JNK-mediated apoptosis in MEFs does not
require new gene induction by c-Jun. Moreover, JNK also activates
JunB and JunD, which act as tumor suppressors, both in vitro and in
vivo. Inhibition of JNK in Ras-transformed cells is reported to
have no effect on anchorage-independent growth or tissue
invasiveness. Hence, JNK and c-Jun likely have independent
functions in apoptosis and oncogenesis, and JNK is not required for
transformation by oncogenes in some circumstances, but may instead
contribute to suppress tumorigenesis. Indeed, the inhibition of JNK
might represent a mechanism by which NF-.kappa.B promotes
oncogenesis and cancer chemoresistance.
[0025] C. Biologic Functions of Gadd45 Proteins
[0026] gadd45.beta. (also known as Myd118) is one of three members
of the gadd45 family of inducible genes, also including
gadd45.alpha. (gadd45) and gadd45.gamma. (oig37/cr6/grp17). Gadd45
proteins are regulated primarily at the transcriptional level and
have been implicated in several biological functions, including
G2/M cell cycle checkpoints and DNA repair. These functions were
characterized with Gadd45.alpha. and were linked to the ability of
this factor to bind to PCNA, core histones, Cdc2 kinase, and p21.
Despite sequence similarity to Gadd45.alpha., Gadd45.beta. exhibits
somewhat distinct biologic activities, as for instance, it does not
appear to participate in negative growth control in most cells.
Over-expression of Gadd45 proteins has also been linked to
apoptosis in some systems. However, it is not clear that this is a
physiologic activity, because in many other systems induction of
endogenous Gadd45 proteins is associated with cytoprotection, and
expression of exogenous polypeptides does not induce death.
Finally, Gadd45 proteins have been shown to associate with
MEKK4/MTK1 and have been proposed to be initiators of JNK and p38
signaling. Other reports have concluded that expression of these
proteins does not induce JNK or p38 in various cell lines, and that
the endogenous products make no contribution to the activation of
these kinases by stress. The ability of Gadd45 proteins to bind to
MEKK4 supports the existence of a link between these proteins and
kinases in the MAPK pathways. Studies using T cell systems, have
implicated Gadd45.gamma. in the activation of both JNK and p38, and
Gadd45.beta. in the regulation of p38 during cytokines
responses.
[0027] D. Summary
[0028] Although many important cellular processes have been
investigated, much is unproven, particularly with respect to the
cellular pathways responsible for controlling apoptosis. For
example, the manner in which NF-.kappa.B controls apoptosis is
unclear. Elucidation of the critical pathways responsible for
modulation of apoptosis is necessary in order Gadd45.beta. in to
develop new therapeutics capable of treating a variety of diseases
that are associated with aberrant levels of apoptosis.
[0029] Inhibitors of NF-.kappa.B are used in combination with
standard anti-cancer agents to treat cancer patients, such as
patients with HL or multiple myeloma. Yet, therapeutic inhibitors
(e.g. glucocorticoids) only achieve partial inhibition of
NF-.kappa.B and exhibit considerable side effects, which limits
their use in humans. A better therapeutic approach might be to
employ agents that block, rather than NF-.kappa.B, its downstream
anti-apoptotic effectors in cancer cells. However, despite
investigation, these effectors remain unknown.
SUMMARY
[0030] Gadd45.beta. independent inactivation of JNKK2/MKK7 is
disclosed. Specific Gadd45.beta. derived peptides bind to and
inactivate JNKK2/MKK7.
[0031] The JNK pathway is a focus for control of pathways leading
to programmed cell death: 1) in addition to playing a role in
stress-induced apoptosis, JNK activation is necessary for efficient
cell killing by TNF-R1, as well as by other DRs such as Fas and
TRAIL-Rs; 2) the inhibition of the JNK cascade represents a
protective mechanism by NF-.kappa.B against TNF.alpha.-induced
cytotoxicity; 3) suppression of JNK activation might represent a
general protective mechanism by NF-.kappa.B and is likely to
mediate the potent effects of NF-.kappa.B during oncogenesis and
cancer chemoresistance; 4) inhibition of JNK activation and
cytoprotection by NF-.kappa.B involve the transcriptional
activation of gadd45.beta.; 5) Gadd45.beta. protein blocks JNK
signaling by binding to and inhibiting JNKK2/MKK7--a specific and
non-redundant activator of JNK. JNKK2 and MKK7 are used
interchangably.
[0032] Gadd45.beta.is required to block apoptosis induced by
TNF.alpha.-and, at least in fibroblasts, there is an additional
factor binding to "peptide 2" described herein, required for this
function. The Gadd45.beta.-interaction domains of JNKK2 and the
JNKK2-binding surface of Gadd45.beta. were identified. This
facilitated the isolation of cell-permeable peptides and small
molecules that are able to interfere with the ability of
Gadd45.beta., and thereby of NF-.kappa.B, to block JNK activation
and prevent apoptosis. The 69-86 amino acidic region of
Gadd45.beta. is sufficient to bind to MKK7 and a slightly longer
region of Gadd45.beta. (i.e. amino acids 60-86) is sufficient to
also inhibit MKK7 activity. This information is very useful for
modulating MKK7 activity and thereby apoptosis in vivo.
Cell-permeable peptides containing this peptidic portion of
Gadd45.beta. can be used in vivo to block TNF.alpha.-induced
apoptosis in cells. This provides a means for blocking apoptosis in
diseases such as neurodegenerative disorders, stroke, myocardial
infraction.
[0033] A method for enhancing programmed cell death induced by
TNF.alpha., the method includes the steps of: [0034] (a) obtaining
a JNKK2 derived peptide that has an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); and [0035] (b)
upregulating the JNK pathway by use of the peptide or a composition
developed from knowledge of the amino acid sequence of the peptide
or a factor binding to the peptide.
[0036] A method for enhancing programmed cell death induced by
TNF.alpha. includes developing an inhibitor to a factor, the
inhibitor capable of disrupting the binding of the factor to JNKK2.
The factor is capable of binding to the peptide that has an amino
acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO:
1).
[0037] A method for enhancing programmed cell death induced by
TNF.alpha. is activated in cells selected from the group consisting
of self-reactive/pro-inflammatory cells or cancer cells.
[0038] A method for screening and identifying an agent that
modulates activity of the JNK pathway in vivo, the method includes
the steps of: [0039] (a) obtaining a candidate agent that binds to
a factor that binds to a factor which binds to a molecule with an
amino acid sequence consisting essentially of
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); [0040] (b)
administering the agent to an animal; and [0041] (c) determining
whether the level of JNK activity or programmed cell death in the
animal is increased compared to JNK activity or programmed cell
death in animals not receiving the agent.
[0042] A method for screening for a modulator of the JNK pathway
different from Gadd45.beta., the method includes the steps of:
[0043] (a) obtaining a candidate modulator of the JNK pathway,
[0044] wherein the candidate modulator is capable of binding to a
peptide that has an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); and [0045] (b)
determining the ability of the candidate modulator to modulate the
JNK pathway by assaying for the level of JNK activity or programmed
cell death.
[0046] A method of treating a subject with a TNF.alpha. or
NF-.kappa.B-dependent disorder, the method includes the steps of:
[0047] (a) obtaining a molecule that inhibits a factor binding to
the peptide consisting essentially of an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1) and interferes
with the inhibition of the JNK pathway by the factor; and [0048]
(b) contacting affected cells of the subject with the molecule.
[0049] A method of treating a subject with a TNF.alpha. or
NF-.kappa.B-dependent disorder, wherein the disorder is selected
from the group consisting of rheumatoid arthritis, inflammatory
bowel disease, stroke, myocardial infarction, psoriasis,
neurodegenerative disorders, and cancer.
[0050] A method of treating a subject with a TNF.alpha. or
NF-.kappa.B-dependent disorder, wherein the molecule is a peptide
mimetic that has the binding properties of a peptide consisting
essentially of an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1).
[0051] A method of treating a subject with a TNF.alpha. or
NF-.kappa.B-dependent disorder, wherein the molecule is an
inhibitor of a cellular factor that binds to a peptide comprising
an amino acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID
NO: 1).
[0052] A method of treating a subject with a TNF.alpha. or
NF-.kappa.B-dependent disorder, wherein the molecule interferes
with an inhibitor of the activation of JNKK2 different from
Gadd45.beta..
[0053] A method of aiding the host immune system to kill cancer
cells by augmenting JNK signaling in cancer cells, the method
includes the steps of: [0054] (a) obtaining an inhibitor that
blocks a cellular inhibitor of JNKK2, wherein the cellular
inhibitor binds to a peptide consisting essentially of an amino
acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1);
and [0055] (b) contacting the cancer cells with the inhibitor.
[0056] A method of identifying JNKK2-interacting cellular factors,
the method includes the steps of: [0057] (a) providing a peptide
consisting essentially of an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); and [0058] (b)
identifying cellular factors that bind to the peptide. A
pharmaceutical composition includes a peptide consisting
essentially of an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1) and a
pharmaceutically acceptable carrier. The peptide in the
pharmaceutical composition is cell permeable.
[0059] A peptide consisting essentially of a contiguous amino acid
sequence identical to the amino acid sequence of Gadd45.beta.,
selected from the group consisting of a peptide whose amino acid
sequences are from positions 60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND,
SEQ ID NO: 2) and 69-86 (IALQIHFTLIQSFCCDND, SEQ ID NO: 3), which
are capable of binding to JNKK2.
[0060] A pharmaceutical composition includes a peptide whose amino
acid sequences are from positions 60-86
(AIDEEEEDDIALQIHFTLIQSFCCDND, SEQ ID NO: 2) and 69-86
(IALQIHFTLIQSFCCDND, SEQ ID NO: 3) of Gadd45.beta., which are
capable of binding to JNKK2. The peptide is cell permeable. A cell
permeable peptide includes an amino acid sequence functionally
equivalent to that of positions 60-86 of Gadd45.beta. protein.
[0061] A method to block JNK pathway or apoptosis, the method
includes the steps of: [0062] (a) obtaining a peptide whose amino
acid sequence is selected from the group consisting of peptides
whose amino acid sequences are from positions 60-86
(AIDEEEEDDIALQIHFTLIQSFCCDND, SEQ ID NO: 2) and 69-86
(IALQIHFTLIQSFCCDND, SEQ ID NO: 3) of Gadd45 .beta.; and [0063] (b)
administering the peptide to block the JNK pathway and apoptosis by
selective inactivation of JNKK2.
[0064] The apoptosis is blocked in inflammatory diseases,
neurodegenerative disorders, stroke, and myocardial infarction. The
peptide useful block JNK pathway or apoptosis is cell permeable.
The peptide is functionally equivalent and structurally similar to
amino acid sequences from positions 60-86
(AIDEEEEDDIALQIHFTLIQSFCCDND, SEQ ID NO: 2) and 69-86
(IALQIHFTLIQSFCCDND, (SEQ ID NO: 3) of Gadd45.beta..
[0065] A method to identify inhibitors of Gadd45.beta., the method
includes the steps of: [0066] (a) screening for a candidate
compound that binds to peptidic regions consisting essentially of
amino acid sequences from positions 60-86
(AIDEEEEDDIALQIHFTLIQSFCCDND, SEQ ID NO: 2) and 69-86
(IALQIHFTLIQSFCCDND, SEQ ID NO: 3) of Gadd45.beta.; and [0067] (b)
determining the ability of the candidate compound to bind to
Gadd45.beta. or interfere with Gadd45.beta.-mediated inhibition of
JNKK2.
[0068] A method to identify agents that interfere with binding of
JNKK2 to a molecule capable of binding to positions 142-166
(TGHVIAVKQMRRSGNKEENKRILMD, SEQ ID NO: 1) of the full length JNKK2,
the method includes the steps of: [0069] (a) obtaining an agent
that interferes with the binding of the molecule to positions
142-166 (TGHVIAVKQMRRSGNKEENKRILMD, SEQ ID NO: 1) of the full
length JNKK2; [0070] (b) contacting a cell with the agent under
conditions that would induce JNK activation or programmed cell
death; and [0071] (c) comparing cells contacted with the agent to
cells not contacted with the agent to determine if the JNK pathway
is upregulated.
[0072] A molecule includes a binding region of JNKK2 characterized
by the amino acid sequence from positions 132-156
(GPVWKMRFRKTGHVIAVKQMRRSGN, SEQ ID NO: 4) of the full length
JNKK2.
[0073] A molecule includes a binding region of JNKK2 characterized
by the amino acid sequence from positions 220-234 (GKMTVAIVKALYYLK,
SEQ ID NO: 5) of the full length JNKK2.
[0074] A molecule includes a binding region of JNKK2 characterized
by the amino acid sequence from positions 142-166
(TGHVIAVKQMRRSGNKEENKRILMD, SEQ ID NO: 1) of the full length
JNKK2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 shows Gadd45.beta. antagonizes TNFR-induced apoptosis
in NF-.kappa.B null cells. FIG. 1A: Gadd45.beta. as well as
Gadd45.alpha. and Gadd45.gamma. (left) rescue RelA-/- MEFs,
TNF.alpha.-induced killing. Plasmids were used as indicated. Cells
were treated with CHS (0.1 ug/ml or CHX plus TNF.alpha. (100
units/ml) and harvested at the indicated time points. Each column
represents the percentage of GHP+ live cells in TNF.alpha. treated
cultures relative to the cultures treated with CHX alone. Values
are the means of three independent experiments. The Figure
indicates that Gadd45.alpha., Gadd45.beta. and Gadd45.gamma. have
anti-apoptotic activity against TNF.alpha.. FIG. 1B: NF-.kappa.B
null 3DO cells are sensitive to TNF.alpha.. Cell lines harboring
I.kappa..beta..alpha.M or neo plasmids were treated with TNF.alpha.
(300 units/ml) and harvested at 14 hours. Columns depict
percentages of live cells as determined by PI staining. Western
blots show levels of I.kappa..beta..alpha.M protein (bottom
panels). FIG. 1C: 3DO I.kappa..beta. M-Gadd45.beta. cells are
protected from TNF.alpha. killing. Cells are indicated. Cells were
treated with TNF.alpha. (25 units/ml) or left untreated and
harvested at the indicated time points. Each value represents the
mean of three independent experiments and expresses the percentages
of live cells in treated cultures relatively to controls (left). PI
staining profiles of representative clones after an 8-hour
incubation with or without TNF.alpha. (right panel, TNF.alpha. and
US. respectively). FIG. 1D: Protection correlates with levels of
Gadd45.beta. of the 8-hr. time point experiment shown in (C) with
the addition of two I.kappa.B-Gadd45.beta. lines. Western blots are
as indicated (lower panels),. FIG. 1E: Gadd45.beta. functions
downstream of NF-.kappa.B complexes. EMSA with extracts of
untreated and TNF.alpha.-treated 3DO cells. Composition of the
.kappa.B-binding complexes was assessed by using supershifting
antibodies. FIG. 1F shows Gadd45.beta. is essential to antagonize
TNF.alpha.-induced apoptosis. 3DO lines harboring anti-sense
Gadd45.beta. (AS-Gadd45.beta.) or empty (Hygro) plasmids were
treated with CHX (0.1 .mu.g/ml) plus or minus TNF.alpha. (1000
units/ml) and analyzed at 14 hours by nuclear PI staining. Low
concentration of CHX was used to lower the threshold of apoptosis.
Each column value represents the mean of three independent
experiments and was calculated as described in FIG. 1C.
[0076] FIGS. 2a-2d shows Gadd45.beta. is a transcriptional target
of NF-.kappa.B. FIG. 2a: Northern blots with RNA from untreated and
TNF.alpha. (1000 u/ml) treated RelA-/- and +/+ MEF. Probes are as
indicated. FIG. 2b-2d: 3 DO I.kappa..beta. M cells and controls
were treated with TNF.alpha. (1000 u/ml). PMA (50 g/ml) plus
ionomycin (1 .mu.M) or daunorubicin (0.5 .mu.M), respectively and
analyzed as in FIG. 2a.
[0077] FIGS. 3A-3E shows Gadd45.beta. prevents caspase activation
in NF-.kappa.B null cells. FIG. 3A: Gadd45-dependent blockade of
caspase activity. 3DO lines were treated with TNF.alpha. (50
units/ml) and harvested at the indicated time points for the
measurement of caspase activity by in vitro fluorometric assay.
Values express fluorescence units obtained after subtracting the
background. FIG. 3B: Gadd45.alpha. inhibits TNF.alpha.-induced
processing of Bid and pro-caspases. Cell were treated as described
in FIG. 2A. Closed and open arrowheads indicate unprocessed and
processed proteins, respectively. FIG. 3C: Gadd45.beta. completely
abrogates TNF.alpha.-induced mitochondrial depolarization in
NF.kappa.B-null cells. 3DO lines and the TNF.alpha. treatment were
as described in FIGS. 3A and B. Each value represents the mean of
three independent experiments and expresses the percentage of
JC-1.sup.+ cells in each culture. FIG. 3D-#: Gadd45.beta. inhibits
cisplatinum- and daunorubicin-induced toxicity. Independently
generated I.kappa.B.alpha.M-Gadd45.beta. and -Hygro clones were
treated for 24 hr with (concentration) 0.025 .mu.M cisplatinum
(FIG. 3D) or with 0.025 .mu.M daunorubicin (FIG. 3E) as indicated.
Values represent percentages of live cells as assessed by nuclear
PI staining and were calculated as described in FIG. 1C.
[0078] FIG. 4 shows Gadd45.beta. is a physiologic inhibitor of JNK
signaling. FIG. 4a: Western blots showing kinetics of JNK
activation by TNF.alpha. (1000 U/ml) in I.kappa.B.alpha.M-Hygro and
I.kappa.B.alpha.M-Gadd45.beta. 3DO clones. Similar results were
obtained with four additional I.kappa.B.alpha.M-Gadd45.beta. and
three I.kappa.B.alpha.M-Hygro clones. FIG. 4b: Western blots
showing ERK, p38, and JNK phosphorylation in 3DO clones treated
with TNF.alpha. for 5 minutes. FIG. 4d: Western blots (top and
middle) and kinase assays (bottom) showing JNK activation in
anti-sense-Gadd45.beta. and Hygro clones treated with TNF.alpha. as
in (a). FIG. 4c: JNK activation by hydrogen peroxide
(H.sub.2O.sub.2, 600 .mu.M) and sorbitol (0.3M) in
I.kappa.B.alpha.M-Hygro and I.kappa.B.alpha.M-Gadd45.beta. clones.
Treatments were for 30 minutes.
[0079] FIG. 5a-e shows the inhibition of JNK represents a
protective mechanism by NF-.kappa.B. FIG. 5a: Kinetics of JNK
activation by TNK.alpha. (1000 U/ml) in 3DO-I.kappa.B.alpha.M and
3DO-Neo clones. Western blots with antibodies specific for
phosphorylated (P) or total JNK (top and middle, respectively) and
JNK kinase assays (bottom). Similar results were obtained with two
additional I.kappa.B.alpha.M and five Neo clones. FIG. 5b: Western
blots (top and middle) and kinase assays (bottom) showing JNK
activation in RelA-/- and +/+ MEFs treated as in (a). FIG. 5c:
Western blots (top and middle) and kinase assays (bottom) showing
JNK activation in parental 3DO cells treated with TNF.alpha. (1000
U/ml), TNF.alpha. plus CHX (10 .mu.g/ml), or CHX alone. CHX
treatments were carried out for 30 minutes in addition to the
indicated time. FIG. 5d: Survival of transfected RelA-/- MEFs
following treatment with TNF.alpha. (1000 U/ml) plus CHX (0.1
.mu.g/ml) for 10 hours. Plasmids were transfected as indicated
along with pEGFP (Clontech). FIG. 5e: Survival of
3DO-I.kappa.B.alpha. M cells pretreated with MAPK inhibitors for 30
minutes and then incubated with either TNF.alpha. (25 U/ml) or PBS
for an additional 12 hours. Inhibitors (Calbiochem) and
concentrations are as indicated. In (d) and (e), values represent
the mean of three independent experiments.
[0080] FIG. 6 shows gadd45.beta. expression is strongly induced by
RelA, but not by Rel or p50. Northern blots showing expression of
gadd45.beta. transcripts in HtTA-1 cells and HtTA-p50, HtTA-p50,
HtTA-RelA, and HtTA-CCR43 cell clones maintained in the presence (0
hours) or absence of tetracycline for the times shown. Cell lines,
times after tetracycline withdrawal, and .sup.32P-labeled probes
specific to gadd45.beta., ikba, relA, p50, rel, or control gapdh
cDNAs, are as indicated. The tetracycline-inducible nf-kb
transgenes are boxed. Transcripts from the endogenous p105 gene and
p50 transgene are indicated.
[0081] FIG. 7 shows gadd45.beta. expression correlates with
NF-.kappa.B activity in B cell lines. Northern blots showing
constitutive and inducible expression of gadd45.beta. in 70Z/3
pre-B cells and WEHI-231 B cells (lanes 1-5 and 5-5, respectively).
Cells were either left untreated (lanes 1, 6, and 11) or treated
with LPS (40 .mu.g/ml) or PMA (100 ng/ml) and harvested for RNA
preparation at the indicated time points. Shown are two different
exposures of blots hybridized with a .sup.32P-labeled probe
specific to the mouse gadd45.beta. cDNA (top panel, short exposure;
middle panel, long exposure). As a loading control, blots were
re-probed with gapdh (bottom panel).
[0082] FIG. 8 shows the sequence of the proximal region of the
murine gadd45.beta. promoter (SEQ ID NO: 35). Strong matches for
transcription factor binding sites are underlined and cognate
DNA-binding factors are indicated. Positions where murine and human
sequences are identical, within DNA stretches of high homology, are
highlighted in gray. Within these stretches, gaps introduced for
alignment are marked with dashes. .kappa.B binding sites that are
conserved in the human promoter are boxed. A previously identified
transcription start site is indicated by an asterisk, and
transcribed nucleotides are italicized. Numbers on the left
indicate the base pair position relative to the transcription start
site. It also shows the sequence of the proximal region of the
murine gadd45.beta. promoter. To understand the regulation of
Gadd45.beta. by NF-.kappa.B, the murine gadd45.beta. promoter was
cloned. A BAC library clone containing the gadd45.beta. gene was
isolated, digested with XhoI, and subcloned into pBS. The 7384 b
XhoI fragment containing gadd45.beta. was completely sequenced
(accession number: AF441860), and portions were found to match
sequences previously deposited in GeneBank (accession numbers:
AC073816, AC073701, and AC091518). This fragment harbored the
genomic DNA region spanning from .about.5.4 kb upstream of a
previously identified transcription start site to near the end of
the fourth exon of gadd45.beta.. A TATA box was located at position
-56 to -60 relative to the transcription start site. The
gadd45.beta. promoter also exhibited several NF-.kappa.B-binding
elements. Three strong .kappa.B sites were found in the proximal
promoter region at positions -377/-368, -426/-417, and -447/-438;
whereas a weaker site was located at position -1159/-1150 and four
other matches mapped further upstream at positions -2751/-2742,
-4525/-4516, -4890/-4881, and -5251/-5242 (gene bank accession
number AF441860). Three .kappa.B consensus sites within the first
exon of gadd45.beta. (+27/+36, +71/+80, and +171/+180). The
promoter also contained a Sp1 motif (-890/-881) and several
putative binding sites for other transcription factors, including
heat shock factor (HSF) 1 and 2, Ets, Stat, AP1, N-Myc, MyoD, CREB,
and C/EBP.
[0083] To identify conserved regulatory elements, the 5.4 kb murine
DNA sequence located immediately upstream of the gadd45.beta.
transcription start site was aligned with the corresponding human
sequence, previously deposited by the Joint Genome Initiative
(accession number: AC005624). The -1477/-1197 and -466/-300 regions
of murine gadd45.beta. were highly similar to portions of the human
promoter, suggesting that these regions contain important
regulatory elements (highlighted in gray are identical nucleotides
within regions of high homology). A less well-conserved region was
identified downstream of position -183 to the beginning of the
first intron. Additional shorter stretches of homology were also
identified. No significant similarity was found upstream of
position -2285. The homology region at -466/-300 contained three
.kappa.B sites (referred to as .kappa.B-1, .kappa.B-2, and
.kappa.B-3), which unlike the other .kappa.B sites present
throughout the gadd45.beta. promoter, were conserved among the two
species. These findings suggest that these .kappa.B sites may play
an important role in the regulation of gadd45.beta., perhaps
accounting for the induction of gadd45.beta. by NF-.kappa.B.
[0084] FIG. 9 shows the murine gadd45.beta. promoter is strongly
transactivated by RelA. (A) Schematic representation of CAT
reporter gene constructs driven by various portions of the murine
gadd45.beta. promoter. Numbers indicate the nucleotide position at
the ends of the promoter fragment contained in each CAT construct.
The conserved .kappa.B-1, .kappa.B-2, and .kappa.B-3 sites are
shown as empty boxes, whereas the TATA box and the CAT coding
sequence are depicted as filled and gray boxes, whereas the TATA
box and the CAT coding sequence are depicted as filled and gray
boxes, respectively. (B) Rel-A-dependent transactivation of the
gadd45.beta. promoter. NTera-2 cells were cotransfected with
individual gadd45.beta.-CAT reporter plasmids (6 .mu.g) alone or
together with 0.3, 1, or 3 .mu.g of Pmt2t-RelA, as indicated. Shown
in the absolute CAT activity detected in each cellular extract and
expressed as counts per minute (c.p.m.). Each column represents the
mean of three independent experiments after normalization to the
protein concentration of the cellular extracts. The total amount of
transfected DNA was kept constant throughout by adding appropriate
amounts of insert-less pMT2T. Each reporter construct transfected
into Ntera-2 cells with comparable efficiency, as determined by the
cotransfection of 1 .mu.g of pEGFP (encoding green fluorescent
protein; GFP; Contech), and flow cytometric analysis aimed to
assess percentages of GFP.sup.+ cells and GFP expression
levels.
[0085] FIG. 10 shows the gadd45.beta. promoter contains three
functional .kappa.B elements. (A) Schematic representation of
wild-type and mutated -592/+23-gadd45.beta.-CAT reporter
constructs. The .kappa.B-1, .kappa.B-2, and .kappa.B-3 binding
sites, the TATA box, and the CAT gene are indicated as in FIG. 9A.
Mutated .kappa.B sites are crossed. (B) .kappa.B-1, .kappa.B-2, and
.kappa.B-3 are each required for the efficient transactivation of
the gadd45.beta. promoter by RelA. Ntera-2 cells were cotransfected
with wild-type or mutated -592/+23-gadd45.beta.-CAT reporter
constructs alone or together with 0.3, 1, or 3 .mu.g pMT2T-RelA, as
indicated. Shown is the relative CAT activity (fold induction) over
the activity observed with transfection of the reporter plasmid
alone. Each column represents the mean of three independent
experiments after normalization to the protein concentration of the
cellular extracts. Empty pMT2T vectors were used to keep the amount
of transfected DNA constant throughout. pEGFP was used to control
the transfection efficiencies of CAT plasmids, as described in FIG.
9B.
[0086] FIG. 11 shows .kappa.B elements from the gadd45.beta.
promoter are sufficient for RelA-dependent transactivation. Ntera
cells were cotransfected with .DELTA.56-.kappa.B-1/2-CAT,
.DELTA.56-.kappa.B-3-CAT, or .DELTA.56-.kappa.B-M-CAT reporter
constructs alone or together with 0.3 or 1 .mu.g of RelA expression
plasmids, as indicated. As in FIG. 10B, columns show the relative
CAT activity (fold induction) observed after normalization to the
protein concentration of the cellular extracts and represent the
mean of three independent experiments. Insert-less pMT2T plasmids
were used to adjust for total amount of transfected DNA.
[0087] FIG. 12 shows gadd45.beta. promoter .kappa.B sites bind to
NF-.kappa.B complexes in vitro. (A) EMSA showing binding of p/50p5
and p50/RelA complexes to .kappa.B-1, .kappa.B-2, and .kappa.B-3
(lanes 9-12, 5-8, and 1-4, respectively). Whole cell extracts were
prepared from NTera-2 cells transfected with pMT2T-p50 (9.mu.;
lanes 1-3, 5-7, and 11-12) or pMT2T-p50 (3 .mu.g) plus pMT2T-RelA
(6 .mu.g; lanes 4, 8, and 12). Various amounts of cell extracts
(0.1 .mu.l, lanes 3, 7, and 11; 0.3 .mu.l, lanes 2, 6, and 10; or 1
.mu.l, lanes 1, 4, 5, 8, 9, and 12) were incubated in vitro with
.sup.32P-labeled .kappa.B-1, .kappa.B-2, or .kappa.B-3 probes, as
indicated, and the protein-DNA complexes were separated by EMSA.
NF-.kappa.B-DNA binding complexes are indicated. (B) Supershift
analysis of DNA-binding NF-.kappa.B complexes. .kappa.B sites were
incubated with 1 .mu.l of the same extracts used in (A) or of
extracts from NTera-2 cells transfected with insert-less pMT2T
(lanes 1-3, 10-12, and 19-21). Samples were loaded into gels either
directly or after preincubation with antibodies directed against
human p50 or RelA, as indicated. Transfected plasmids and
antibodies were as shown. DNA-binding NF-.kappa.B complexes,
supershifted complexes, and non-specific (n.s.) bands are labeled.
(C) shows gadd45.beta. .kappa.B sites bind to endogenous
NF-.kappa.B complexes in vitro. To determine whether
gadd45.beta.-.kappa.B elements can bind to endogenous NF-.kappa.B
complexes, whole cell extracts were obtained from untreated and
lypopolysaccharide (LPS)-treated WEHI-231 cells. Cells were treated
with 40 .mu.g/ml LPS (Escherichia coli serotype 0111:B4) for 2
hours, and 2111 of whole cell extracts were incubated, in vitro,
with .sup.32P-labeled gadd45.beta.-.kappa.B probes. Probes,
antibodies against individual NF-.kappa.B subunits, predominant
DNA-binding complexes, supershifted complexes, and non-specific
(n.s.) bands are as labeled. All three gadd45.beta.-.kappa.B sites
bound to both constitutively active and LPS-induced NF-.kappa.B
complexes (lanes 1-3, 9-11, and 17-19). .kappa.B-3 bound avidly to
a slowly-migrating NF-.kappa.B complex, which was supershifted only
by the anti-Rel antibody (lanes 4-8). This antibody also retarded
the migration of the slower dimers binding to .kappa.B-2 and, much
more loosely, to .kappa.B-1, but had no effect on the
faster-migrating complex detected with these probes (lanes 15 and
23, respectively). The slower complex interacting with .kappa.B-1
and .kappa.B-2 also contained large amounts of p50 and smaller
quantities of p52 and RelA (lanes 12-14 and 20-22, RelA was barely
detectable with .kappa.B-1). The faster complex was instead almost
completely supershifted by the anti-p50 antibody (lanes 12 and 20),
and the residual DNA-binding activity reacted with the anti-p52
antibody (lanes 13 and 21; bottom band). With each probe, RelB
dimers contributed to the .kappa.B-binding activity only
marginally. Specificity of the DNA-binding complexes was confirmed
by competitive binding reactions using unlabeled competitor
oligonucleotides. Thus, the faster complex binding to .kappa.B-1
and .kappa.B-2 was predominantly composed of p50 homodimers and
contained significant amounts of p52/p52 dimers, whereas the slower
one was made up of p50/Rel heterodimers and, to a lesser extent,
p52/Rel, Rel/Rel, and RelA-containing dimers. Conversely,
.kappa.B-3 only bound to Rel homodimers. Consistent with
observations made with transfected NTera-2 cells, .kappa.B-1
exhibited a clear preference for p50 and p52 homodimers, while
.kappa.B-2 preferentially bound to Rel- and RelA-containing
complexes. Overall, .kappa.B-3 yielded the strongest
NF-.kappa.B-specific signal, whereas .kappa.B-1 yielded the weakest
one. Interestingly, the in vitro binding properties of the DNA
probes did not seem to reflect the relative importance of
individual .kappa.B sites to promoter transactivation in vivo.
Nevertheless, the findings do demonstrate that each of the
functionally relevant .kappa.B elements of the gadd45.beta.
promoter can bind to NF-.kappa.B complexes, thereby providing the
basis for the dependence of gadd45.beta. expression on
NF-.kappa.B.
[0088] FIG. 13 shows Gadd45.beta. expression protects BJAB cells
against Fas- and TRAIL-R-induced apoptosis. To determine whether
Gadd45.beta. activity extended to DRs other than TNF-Rs, stable
HA-Gadd45.beta. and Neo control clones were generated in BJAB B
cell lymphomas, which are highly sensitive to killing by both Fas
and TRAIL-Rs. As shown by propidium iodide (PI) staining assays,
unlike Neo clones, BJAB clones expressing Gadd45.beta. were
dramatically protected against apoptosis induced either (B) by
agonistic anti-Fas antibodies (APO-1; 1 .mu.g/ml, 16 hours) or (A)
by recombinant (r)TRAIL (100 ng/ml, 16 hours). In each case, cell
survival correlated with high levels of HA-Gadd45.beta. proteins,
as shown by Western blots with anti-HA antibodies (bottom panels).
Interestingly, with Fas, protection by Gadd45.beta. was nearly
complete, even at 24 hours.
[0089] FIG. 14 shows the inhibition of JNK activation protects BJAB
cells from Fas induced apoptosis. Parental BJAB cells were treated
for 16 hours with anti-APO1 antibodies (1 .mu.g/ml), in the
presence or absence of increasing concentrations of the specific
JNK blocker SP600125 (Calbiochem), and apoptosis was monitored by
PI staining assays. While BJAB cells were highly sensitive to
apoptosis induced by Fas triggering, the suppression of JNK
activation dramatically rescued these cells from death, and the
extent of cytoprotection correlated with the concentration of
SP600125. The data indicate that, unlike what was previously
reported with MEFs (i.e. with ASK1- and JNK-deficient MEFs), in B
cell lymphomas, and perhaps in other cells, JNK signaling plays a
pivotal role in the apoptotic response to Fas ligation. This is
consistent with findings that, in these cells, killing by Fas is
also blocked by expression of Gadd45.beta. (FIG. 13B). Thus, JNK
might be required for Fas-induced apoptosis in type 2 cells (such
as BJAB cells), which unlike type 1 cells (e.g. MEFs), require
mitochondrial amplification of the apoptotic signal to activate
caspases.
[0090] FIG. 15 shows JNK is required for efficient killing by
TNF.alpha.. In FIGS. 5d and 5e, the inhibition of JNK by either
expression of DN-MKK7 or high doses of the pharmacological blocker
SB202190 rescues NF-.kappa.B null cells from TNF.alpha.-induced
killing. Together with the data shown in FIG. 5a-c, these findings
indicate that the inhibition of the JNK cascade represents a
protective mechanism by NF-.kappa.B. They also suggest that the JNK
cascade plays an important role in the apoptotic response to the
cytokine. Thus, to directly link JNK activation to killing by
TNF-R1, the sensitivity of JNK1 and JNK2 was tested in double
knockout fibroblasts to apoptosis by TNF.alpha.. Indeed, as shown
in FIG. 15A, mutant cells were dramatically protected against
combined cytotoxic treatment with TNF.alpha. (1,000 U/ml) and CHX
(filled columns) for 18 hours, whereas wild-type fibroblasts
remained susceptible to this treatment (empty columns). JNK kinase
assays confirmed the inability of knockout cells to activate JNK
following TNF.alpha. stimulation (left panels). The defect in the
apoptotic response of JNK null cells to TNF.alpha. plus CHX was not
a developmental defect, because cytokine sensitivity was promptly
restored by viral transduction of MIGR1-JNKK2-JNK1, expressing
constitutively active JNK1 (FIG. 15B; see also left panel, JNK
kinase assays). Thus, together with the data shown in FIG. 5a-e,
these latter findings with JNK null cells indicate that JNK (but
not p38 or ERK) is essential for PCD by TNF-R, and confirm that a
mechanism by which NF-.kappa.B protects cells is the
down-regulation of the JNK cascade by means of Gadd45.beta..
[0091] FIG. 16 shows Gadd45.beta. is a potential effector of
NF-.kappa.B functions in oncogenesis. Constitutive NF-.kappa.B
activation is crucial to maintain viability of certain late stage
tumors such as ER.sup.- breast tumors. Remarkably, as shown by
Northern blots, gadd45.beta. was expressed at constitutively high
levels in ER.sup.- breast cancer cell lines--which depend on
NF-.kappa.B for their survival--but not in control lines or in less
invasive, ER.sup.+ breast cancer cells. Of interest, in these
cells, gadd45.beta. expression correlated with NF-.kappa.B
activity. Hence, as with the control of TNF.alpha.-induced
apoptosis, the induction of gadd45.beta. likely represents a
mechanism by which NF-.kappa.B promotes cancer cell survival, and
thereby oncogenesis. Thus, Gadd45.beta. is a novel target for
anti-cancer therapy.
[0092] FIG. 17 shows the suppression of JNK represents a mechanism
by which NF-.kappa.B promotes oncogenesis. The ER.sup.- breast
cancer cell lines, BT-20 and MDA-MD-231, are well-characterized
model systems of NF-.kappa.B-dependent tumorigenesis, as these
lines contain constitutively nuclear NF-.kappa.B activity and
depend on this activity for their survival. In these cells the
inhibition of NF-.kappa.B activity by well-characterized
pharmacological blockers such as prostaglandin A1 (PGA1, 100
.mu.M), CAPE (50 .mu.g/ml), or parthenolide (2.5 .mu.g/ml) induced
apoptosis rapidly, as judged by light microscopy. All NF-.kappa.B
blockers were purchased from Biomol and concentrations were as
indicated. Treatments were carried out for 20 (PGA 1), 4
(parthenolide), or 17 hours (CAPE). Apoptosis was scored
morphologically and is graphically represented as follows: ++++,
76-100% live cells; +++, 51-75% live cells; ++, 26-50% live cells;
+, 1-25% live cells; -, 0% live cells. Remarkably, concomitant
treatment with the JNK inhibitor SP600125 dramatically rescued
breast tumor cells from the cytotoxicity induced by the inhibition
of NF-.kappa.B, indicating that the suppression of JNK by
NF-.kappa.B plays an important role in oncogenesis.
[0093] FIG. 18 is a schematic representation of TNF-R1-induced
pathways modulating apoptosis. The blocking of the
NF-.kappa.B-dependent pathway by either a RelA knockout mutation,
expression of I.kappa.B.alpha.M proteins or anti-sense gadd45.beta.
plasmids, or treatment with CHX leads to sustained JNK activation
and apoptosis. Conversely, the blocking of TNF.alpha.-induced JNK
activation by either JNK or ASK1 null mutations, expression of
DN-MKK7 proteins, or treatment with well characterized
pharmacological blockers promotes cell survival, even in the
absence of NF-.kappa.B. The blocking of the JNK cascade by
NF-.kappa.B involves the transcriptional activation of
gadd45.beta.. Gadd45.beta. blocks this cascade by direct binding to
and inhibition of MKK7/JNKK2, a specific and non-redundant
activator of JNK. Thus, MKK7 and its physiologic inhibitor
Gadd45.beta., are crucial molecular targets for modulating JNK
activation, and consequently apoptosis.
[0094] FIG. 19 shows physical interaction between Gadd45.beta. and
kinases in the JNK pathway, in vivo. Gadd45.beta. associates with
MEKK4. However, because this MAPKKK is not activated by DRs, no
further examination was made of the functional consequences of this
interaction. Thus, to begin to investigate the mechanisms by which
Gadd45.beta. blunts JNK activation by TNF-R, the ability of
Gadd45.beta. to physically interact with additional kinases in the
JNK pathway was examined, focusing on those MAPKKKs, MAPKKs, and
MAPKs that had been previously reported to be induced by TNF-Rs.
HA-tagged kinases were transiently expressed in 293 cells, in the
presence or absence of FLAG-Gadd45.beta., and cell lysates were
analyzed by co-immunoprecipitation (IP) with anti-FLAG
antibody-coated beads followed by Western blot with anti-HA
antibodies. These assays confirmed the ability of Gadd45.beta. to
bind to MEKK4. These co-IP assays demonstrated that Gadd45.beta.
can also associate with ASK1, but not with other TRAF2-interacting
MAPKKKs such as MEKK1, GCK, and GCKR, or additional MAPKKKs that
were tested (e.g. MEKK3). Notably, Gadd45.beta. also interacted
with JNKK2/MKK7, but not with the other JNK kinase, JNKK1/MKK4, or
with any of the other MAPKKs and MAPKs under examination, including
the two p38-specific activators MKK3b and MKK6, and the ERK kinase
MEK1. Similar findings were obtained using anti-HA antibodies for
IPs and anti-FLAG antibodies for Western blots. Indeed, the ability
to bind to JNKK2, the dominant JNK kinase induced by TNF-R, as well
as to ASK1, a kinase required for sustained JNK activation and
apoptosis by TNF.alpha., may represent the basis for the control of
JNK signaling by Gadd45.beta.. The interaction with JNKK2 might
also explain the specificity of the inhibitory effects of
Gadd45.beta.on the JNK pathway.
[0095] FIG. 20 shows physical interaction between Gadd45.beta. and
kinases in the JNK pathway, in vitro. To confirm the above
interactions, in vitro, GST pull-down experiments were performed.
pBluescript (pBS) plasmids encoding full-length (FL) human ASK1,
MEKK4, JNKK1, and JNKK2, or polypeptides derived from the amino- or
carboxy-terminal portions of ASK1 (i.e. N-ASK1, spanning from amino
acids 1 to 756, and C-ASK1, spanning from amino acids 648 to 1375)
were transcribed and translated in vitro using the TNT coupled
retyculocyte lysate system (Promega) in the presence of
.sup.35S-methionine. 5 .mu.l of each translation mix were
incubated, in vitro, with sepharose-4.beta. beads that had been
coated with either purified glutathione-S-transferase (GST)
polypeptides or GST-Gadd45.beta. proteins. The latter proteins
contained FL murine Gadd45.beta. directly fused to GST. Binding
assays were performed according to standard procedures, and
.sup.35S-labeled proteins that bound to beads, as well as 2 .mu.l
of each in vitro translation mix (input), were then resolved by SDS
polyacrylamide gel electrophoresis. Asterisks indicate the intact
translated products. As shown in FIG. 20, FL-JNKK2 strongly
associated with GST-Gadd45.beta., but not with GST, indicating that
JNKK2 and Gadd45.beta. also interacted in vitro, and that their
interaction was specific. Additional experiments using recombinant
JNKK2 and Gadd45.beta. have demonstrated that this interaction is
mediated by direct protein-protein contact. Consistent with in vivo
findings, GST-Gadd45.beta. also associated with ASK1, N-ASK1,
C-ASK1, and MEKK4--albeit less avidly than with JNKK2--and weakly
with JNKK1. Thus, GST pull-down experiments confirmed the strong
interaction between Gadd45.beta. and JNKK2 observed in vivo, as
well as the weaker interactions of Gadd45.beta. with other kinases
in the JNK pathway. These assays also uncovered a weak association
between Gadd45.beta. and JNKK1.
[0096] FIG. 21 shows Gadd45.beta. inhibits JNKK2 activity in vitro.
Next, the functional consequences, in vitro, of the physical
interactions of Gadd45.beta. with kinases in the JNK pathway were
assessed. Murine and human, full-length Gadd45.beta. proteins were
purified from E. coli as GST-Gadd45.beta. and His.sub.6-tagged
Gadd45.beta., respectively, according to standard procedures. Prior
to employing these proteins in in vitro assays, purity of all
recombinant polypeptides was assured by >98%, by performing
Coomassie blue staining of SDS polyacrylamide gels. Then, the
ability of these proteins, as well as of control GST and
His.sub.6-EF3 proteins, to inhibit kinases in the JNK pathways was
monitored in vitro. FLAG-tagged JNKK2, JNKK1, MKK3, and ASK1 were
immunoprecipitated from transiently transfected 293 cells using
anti-FLAG antibodies and pre-incubated for 10 minutes with
increasing concentrations of recombinant proteins, prior to the
addition of specific kinase substrates (i.e. GST-JNK1 with JNKK1
and JNKK2; GST-p38.gamma. with MKK3; GST-JNNK1 or GST-JNKK2 with
ASK1). Remarkably, both GST-Gadd45.beta. and His.sub.6-Gadd45.beta.
effectively suppressed JNKK2 activity, in vitro, even at the lowest
concentrations that were tested, whereas control polypeptides had
no effect on kinase activity (FIG. 21A). In the presence of the
highest concentrations of Gadd45.beta. proteins, JNKK2 activity was
virtually completely blocked. These findings indicate that, upon
binding to Gadd45.beta., JNKK2 is effectively inactivated.
Conversely, neither GST-Gadd45.beta. nor His.sub.6-Gadd45.beta. had
significant effects on the ability of the other kinases (i.e.
JNKK1, MKK3, and ASK1) to phosphorylate their physiologic
substrates, in vitro, indicating that Gadd45.beta. is a specific
inhibitor of JNKK2. Gadd45.beta. also inhibited JNKK2
auto-phosphorylation (6.times.His tag disclosed as SEQ ID NO:
46.)
[0097] FIG. 22A-B shows Gadd45.beta. inhibits JNKK2 activity in
vivo. The ability of Gadd45.beta. to inhibit JNKK2 was confirmed in
vivo, in 3DO cells. In these cells, over-expression of Gadd45.beta.
blocks JNK activation by various stimuli, and the blocking of this
activation is specific, because Gadd45.beta. does not affect either
the p38 or the ERK pathway. These findings suggest that
Gadd45.beta. inhibits JNK signaling downstream of the MAPKKK
module.
[0098] Kinase assays were performed according to procedures known
to those of skill in the art using extracts from unstimulated and
TNF.alpha.-stimulated 3DO cells, commercial antibodies that
specifically recognize endogenous kinases, and GST-JNK1 (with
JNKK2) or myelin basic protein (MBP; with ASK1) substrates (FIG.
22A). Activity of JNKK1 and MKK3/6 was instead assayed by using
antibodies directed against phosphorylated (P) JNKK1 or MKK3/6
(FIG. 22B)--the active forms of these kinases. In agreement with
the in vitro data, these assays demonstrated that, in 3DO cells,
Gadd45.beta. expression is able to completely block JNKK2
activation by TNF.alpha. (FIG. 22A). This expression also partly
suppressed JNKK1 activation, but did not have significant
inhibitory effects on MKK3/6--the specific activators of p38--or
ASK1 (FIG. 22A-B).
[0099] Hence, Gadd45.beta. is a potent blocker of JNKK2--a specific
activator of JNK and an essential component of the TNF-R pathway of
JNK activation. This inhibition of JNKK2 is sufficient to account
for the effects of Gadd45.beta. on MAPK signaling, and explains the
specificity of these effects for the JNK pathway. Together, the
data indicate that Gadd45.beta. suppresses JNK activation, and
thereby apoptosis, induced by TNF.alpha. and stress stimuli by
direct targeting of JNKK2. Since Gadd45.beta. is able to bind to
and inhibit JNKK2 activity in vitro (FIGS. 20 and 21), Gadd45.beta.
likely blocks this kinase directly, either by inducing
conformational changes or steric hindrances that impede kinase
activity. These findings identify JNKK2/MKK7 as an important
molecular target of Gadd45.beta. in the JNK cascade. Under certain
circumstances, Gadd45.beta. may also inhibit JNKK1, albeit more
weakly than JNKK2. Because ASK1 is essential for sustained
activation of JNK and apoptosis by TNF-Rs, it is possible that the
interaction between Gadd45.beta. and this MAPKKK is also relevant
to JNK induction by these receptors.
[0100] FIG. 23A-B shows that two distinct polypeptide regions in
the kinase domain of JNKK2 are essential for the interaction with
Gadd45.beta.. By performing GST pull-down assays with GST- and
GST-Gadd45.beta.-coated beads, the regions of JNKK2 that are
involved in the interaction with Gadd45.beta. were determined. pBS
plasmids encoding various amino-terminal truncations of JNKK2 were
translated in vitro in the presence of .sup.35S-metionine, and
binding of these peptides to GST-Gadd45.beta. was assayed as
described herein (FIG. 23A, Top), JNKK2(1-401; FL), JNKK2 (63-401),
JNKK2 (91-401), and JNKK2 (132-401) polypeptides strongly
interacted with Gadd45.beta., in vitro, indicating that the amino
acid region spanning between residue 1 and 131 is dispensable for
the JNKK2 association with Gadd45.beta.. However, shorter JNKK2
truncations--namely JNKK2 (157-401), JNKK2 (176-401), and JNKK2
(231-401)--interacted with Gadd45.beta. more weakly, indicating
that the amino acid region between 133 and 156 is critical for
strong binding to Gadd45.beta.. Further deletions extending beyond
residue 244 completely abrogated the ability of the kinase to
associate with Gadd45.beta., suggesting that the 231-244 region of
JNKK2 also contributes to binding to Gadd45.beta..
[0101] To provide further support for these findings,
carboxy-terminal deletions of JNKK2 were generated, by programming
retyculo-lysate reactions with pBS-JNKK2 templates that had been
linearized with appropriate restriction enzymes (FIG. 23B, bottom).
Binding assays with these truncations were performed as described
herein. Digestions of pBS-JNKK2 (FL) with SacII (FL), PpuMI, or
NotI did not significantly affect the ability of JNKK2 to interact
with Gadd45.beta., indicating that amino acids 266 to 401 are
dispensable for binding to this factor. Conversely, digestions with
XcmI or BsgI, generating JNKK2 (1-197) and JNKK2 (1-186)
polypeptides, respectively, partly inhibited binding to
Gadd45.beta.. Moreover, cleavage with BspEI, BspHI, or PflMI,
generating shorter amino terminal polypeptides, completely
abrogated this binding. Together these findings indicate that the
polypeptide regions spanning from amino acids 139 to 186 and 198 to
265 and are both responsible for strong association of JNKK2 with
Gadd45.beta.. The interaction of JNKK2 with Gadd45.beta. was mapped
primarily to two polypeptides spanning between JNKK2 residue 132
and 156 and between residue 231 and 244. JNKK2 might also contact
Gadd45.beta. through additional amino acid regions.
[0102] The finding that Gadd45.beta. directly contacts two distinct
amino acid regions within the catalytic domain of JNKK2 provides
mechanistic insights into the basis for the inhibitory effects of
Gadd45.beta. on JNKK2. These regions of JNKK2 shares no homology
within MEKK4, suggesting that Gadd45.beta. contacts these kinases
through distinct surfaces. Since it is not known to have enzymatic
activity (e.g. phosphatase or proteolytic activity), and its
binding to JNKK2 is sufficient to inhibit kinase function, in
vitro, Gadd45.beta. might block JNKK2 through direct interference
with the catalytic domain, either by causing conformational changes
or steric hindrances that inhibit kinase activity or access to
substrates. With regard to this, the 133-156 peptide region
includes amino acid K149--a critical residue for kinase
activity--thereby providing a possible mechanism for the potent
inhibition of JNKK2 by Gadd45.beta..
[0103] FIG. 24A-B shows the Gadd45.beta. amino acid region spanning
from residue 69 to 104 is essential for interaction with JNKK2 (see
also FIGS. 36 and 37). To identify the region of Gadd45.beta. that
mediated the association with JNKK2, GST pull-down experiments were
performed. Assays were performed using standard protocols and
GST-JNKK2- or GST-coated beads. pBS plasmids encoding progressively
shorter amino-terminal deletions of Gadd45.beta. were translated in
vitro and labeled with .sup.35S-metionine (FIG. 24A). Murine
Gadd45.beta. (1-160; FL), Gadd45.beta. (41-160), Gadd45.beta.
(60-160), and Gadd45.beta. (69-160) polypeptides strongly
interacted with JNKK2, whereas Gadd45.beta. (87-160) bound to the
kinase only weakly. In contrast, Gadd45.beta. (114-160) was unable
to associate with JNKK2.
[0104] To confirm these findings, a series of carboxy-terminal
Gadd45.beta. truncations were generated by programming in vitro
transcription/translation reactions with appropriately linearized
pBS-Gadd45.beta. plasmids (FIG. 24B). Although digestion of
pBS-Gadd45.beta. with NgoMI did not affect Gadd45.beta. binding to
JNKK2, digestions with SphI and EcoRV, generating Gadd45.beta.
(1-95) and Gadd45.beta. (1-68), respectively, progressively
impaired Gadd45.beta. affinity for JNKK2. Indeed, the latter
polypeptides were unable to associate with JNKK2. Together the data
indicate that the Gadd45.beta. polypeptide spanning from residue 69
to 104 participates in an interaction with JNKK2.
[0105] FIG. 25 show the amino acid region spanning between residue
69 and 113 is needed for the ability of Gadd45.beta. to suppress
TNF.alpha.-induced apoptosis (but see FIGS. 36-37). By performing
mutational analyses, the domain of Gadd45.beta. that is required
for the blocking of TNF.alpha.-induced killing was mapped to the
69-113 amino acid region. Upon expression in RelA.sup.-/- cells,
GFP-Gadd45.beta. (69-160) and GFP-Gadd45.beta. (1-113) exhibited
anti-apoptotic activity against TNF.alpha. that was comparable to
that of full-length GFP-Gadd45.beta.. In contrast, in these assays,
GFP proteins fused to Gadd45.beta. (87-160) or Gadd45 (1-86) had
only modest protective effects. Shorter truncations had virtually
no effect on cell survival, indicating that the Gadd45.beta. region
spanning between amino acids 69 and 113 provides cytoprotection,
and that the adjacent 60-68 region contributes only modestly to
this activity.
[0106] This amino acid region contains the domain of Gadd that is
also responsible for the interaction with JNKK2. This is consistent
with the notion that the protective activity of Gadd45.beta. is
linked to its ability to bind to JNKK2 and suppress JNK
activation.
[0107] FIG. 26 shows that Gadd45.beta. physically interacts with
kinases in the JNK pathway. a, b, Western blots with anti-FLAG
immunoprecipitates (top) or total lysates (middle and bottom) from
293 cells showing Gadd45.beta. association with ASK1, MEKK4, and
MKK7. c, Pull-down assays using GST- or GST-Gadd45.beta.-coated
beads and .sup.35S-labeled, in vitro translated proteins. Shown is
40% of the inputs.
[0108] FIG. 27 shows that Gadd45.beta. and NF-.kappa.B specifically
inhibit MKK7, in vivo. a-e, Western blots with antibodies against
phosphorylated (P) or total kinases and kinase assays (K.A.)
showing MAPKK and MAPKKK activation by TNF.alpha. or P/I in (a-c)
I.kappa.B.alpha.M-Hygro and I.kappa.B.alpha.M-Gadd45.beta. clones
and in (d, e) Neo and I.kappa.B.alpha. M 3DO clones. a, d, MKK7
phosphorylation (P-MKK7) was monitored by combined
immunoprecipitation (anti-P-MKK7 antibodies) and Western blotting
(anti-total MKK7 antibodies).
[0109] FIG. 28 shows that Gadd45.beta. is a direct inhibitor of
MKK7. a, Immunoprecipitations followed by Western blots showing
physical association of endogenous Gadd45.beta. and MKK7 (top) in
3DO cells treated with P/I (2 hours) or left untreated (US).
Protein levels are shown (bottom). b, g, Coomassie brilliant blue
staining (CS) showing purity of the proteins used in (c) and (d,
e), respectively. c, In vitro pull-down assays with purified
proteins showing direct interaction between
His.sub.6/T7-Gadd45.beta. and GST-MKK7. Precipitated GST proteins
and bound His.sub.6/T7-tagged proteins were visualized by CS and
Western blotting (WB) with anti-T7 antibodies, respectively. Inputs
of His.sub.6/T7-tagged proteins are indicated. The fraction of
His.sub.6/T7-Gadd45.beta. and His.sub.6/T7-JIP1 binding to GST-MKK7
(expressed as arbitrary units [a.u.]; left) was calculated
relatively to a standard curve generated with known protein
concentrations.sup.19. d, e, Kinase assays showing specific
inhibition of active MKK7 by purified GST-Gadd45.beta. and
His.sub.6-Gadd45.beta., in vitro. FLAG-tagged kinases were
immunoprecipitated from 293 cells treated with TNF.alpha. (10
minutes) or left untreated and pre-incubated with the indicated
concentrations of Gadd45.beta. polypeptides. f, Western blots
showing exogenous kinase levels in 293 cells (6.times.His tag
disclosed as SEQ ID NO: 46).
[0110] FIG. 29 shows that MKK7 contacts Gadd45.beta. through two
petidic regions in its catalytic domain. a, c, e, are schematic
representations of the MKK7 N- and C-terminal truncations and
peptides, respectively, used for binding assays. Interaction
regions are shaded in gray. b, d, f, GST are pull-downs showing
GST-Gadd45 .beta. binding to the indicated .sup.35S-labeled, in
vitro translated MKK7 products. Shown is 40% of the inputs. g, is
an amino acid sequence of Gadd45.beta.-interacting peptides 1 (SEQ
ID NO: 4) and 7. K149 (SEQ ID NO: 5) is highlighted.
[0111] FIG. 30 shows that peptide 1 impairs the ability of Gadd45
.beta. (and NF-.kappa.B) to suppress JNK activation and apoptosis
induced by TNF.alpha.. a, Kinase assay (K.A.) showing that binding
to peptidic region 1 is required for MKK7 inactivation by Gadd45
.beta.. FLAG-MKK7 was immunoprecipitated from TNF.alpha.-treated
(10 minutes) 293 cells. b, c, are apoptosis assays showing that
peptide 1 promotes killing by TNF.alpha. in
I.kappa.B.alpha.M-Gadd45 .beta. and Neo clones, respectively.
Values (expressed as arbitrary units) were obtained by subtracting
background values with untreated cells from values with
TNF.alpha.-treated cells, and represent the mean (+/- standard
deviation) of three experiments.
[0112] FIG. 31 (A-D) shows nucleotide and amino acid sequences of
human (SEQ ID NOS 49 and 50) and murine (SEQ ID NOS 51 and 52)
JNKK2.
[0113] FIG. 32 shows that Gadd45.beta. blocks MKK7 by contacting a
peptidic region in its catalytic domain. a, Schematic
representation of the MKK7 peptides used for binding assays.
Interaction regions are in gray. b, d, e, GST pull-down assays
showing GST-Gadd45.beta. binding to the indicated .sup.35S-labeled,
in vitro translated MKK7 products. 40% of the inputs is shown (b,
d,). e, ATP was used as indicated. c, Amino acid sequence of
Gadd45.beta.-interacting, peptides 1 (SEQ ID NO: 4) and 7 (SEQ ID
NO: 5), and peptide 1 mutants used in (SEQ ID NOS 6-12,
respectively in order of appearance) (d). K149 is marked by an
asterisk. Amino acids involved in binding to Gadd45.beta. are in
gray, and darkness correlates with their apparent relevance for
this binding. f, Kinase assay (K.A.) showing that binding to
peptidic region 1 is required for MKK7 inactivation by
Gadd45.beta.. FLAG-MKK7 was immunoprecipitated from
TNF.alpha.-treated (10 minutes) 293 cells. The underlined and bold
amino acids in c represent inserted amino acids that were not
present in the original p1 (132-156).
[0114] FIG. 33 shows that Gadd45.beta.-mediated suppression of MKK7
is required to block TNF.alpha.-induced apoptosis. A-B, Apoptosis
assays showing that peptide 1 effectively promotes killing by
TNF.alpha. in I.kappa.B.alpha.M-Gadd45.beta. and Neo 3DO clones,
respectively. C-D, Apoptosis assays showing that both peptide 1 and
peptide 2 can facilitate TNF.alpha.-induced cytotoxicity in
wild-type MEFs, and that only peptide 2 promotes this killing in
Gadd45.beta. null MEFs, respectively. (C-D), MEFs were from twin
embryos and were used at passage (p)4. A-D, Values (expressed as
arbitrary units) were obtained by subtracting background values
with untreated cells from values with TNF.alpha.-treated cells, and
represent the mean (+/-standard deviation) of three
experiments.
[0115] FIG. 34 shows that synthetic, FITC-labeled TAT peptides
enter cells with comparable efficiencies. a-d, FCM (a, c) and
confocal microscopy (b, d) analyses of 3DO cells after a 20-minute
incubation with DMSO (Ctr) or the indicated peptides (5 .mu.M). a,
c, Depicted in the histograms are the overlaid profiles of
DMSO-(gray) and peptide-treated (black) cells. e, Amino acid
sequence of the peptide 1 mutants that were fused to TAT for in
vivo studies (SEQ ID NOS 4, 60, 9, and 61, respectively in order of
appearance). Note that Ala-II* contains the R140 mutation, not
present in Ala-II, and that in Ala-V*, mutations are shifted of 1
amino acid to the C-terminus as compared to Ala-V (see FIG. 32c).
Ala-IV* is identical, in its MKK7-mimicking portion, to Ala-IV.
[0116] FIG. 35 shows that peptides that interfere with Gadd45.beta.
binding to MKK7 blunt the Gadd45.beta. protective activity against
TNF.alpha..
[0117] FIG. 36 shows that the 69-86 amino acid region of
Gadd45.beta. is sufficient to bind to MKK7 in vitro.
[0118] FIG. 37 shows that the Gadd45.beta.-mediated inhibition of
MKK7 requires a polypeptide region of Gadd45.beta., including the
section between amino acids 60 and 86 (SEQ ID NOS 36-44,
respectively in order of appearance).
DETAILED DESCRIPTION
[0119] The JNK pathway is a focus for control of pathways leading
to programmed cell death.
[0120] The present invention facilitates development of new methods
and compositions for ameliorating of diseases. Indeed, the
observation that the suppression of JNK represents a protective
mechanism by NF-.kappa.B suggests that apoptosis of unwanted
self-reactive lymphocytes and other pro-inflammatory cells (e.g.
macrophages) at the site of inflammation--where there are high
levels of TNF.alpha.--may be augmented by interfering with the
ability of NF-.kappa.B to shut down JNK activation. Potential means
for achieving this interference include, for instance, using
blockers of Gadd45.beta. and agents that interfere
JNKK2-interacting factors. One such agent is a peptide
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1).
[0121] Like Fas, TNF-R1 is also involved in host immune
surveillance mechanisms. Thus, in another aspect of the invention,
the agents might provide a powerful new adjuvant in cancer
therapy.
[0122] Conversely, an enhancement of cell survival by the
down-modulation of JNK will have beneficial effects in degenerative
disorders and immunodeficiencies, conditions that are generally
characterized by exaggerated cell death.
[0123] The invention allows design of agents to modulate the JNK
pathway e.g. cell permeable, fusion peptides (such as TAT-fusion
peptides) encompassing the amino acid regions of JNKK2 that come
into direct contact with Gadd45.beta.. The sequence GRKKRRQRRR (SEQ
ID NO: 53) is found in the TAT protein of HIV-1 virus and renders
the peptides cell permeable. Synthetic fusion peptides such as, for
example PTD4 having a sequence GGYARAAARQARA (SEQ ID NO: 54) can
also be used to render the desired peptides cell permeable. These
peptides will effectively compete with endogenous Gadd45.beta.
proteins for binding to JNKK2. In addition, these findings allow
design of biochemical assays for the screening of libraries of
small molecules and the identification of compounds that are
capable to interfere with the ability of Gadd45.beta. to associate
with JNKK2. Both these peptides and these small molecules are able
to prevent the ability of Gadd45.beta., and thereby of NF-.kappa.B,
to shut down JNK activation, and ultimately, to block apoptosis.
These compounds are useful in the treatment of human diseases,
including chronic inflammatory and autoimmune conditions and
certain types of cancer.
[0124] The new molecular targets for modulating the anti-apoptotic
activity of NF-.kappa.B, are useful in the treatment of certain
human diseases. The application of these findings appears to
pertain to the treatment of two broadly-defined classes of human
pathologies: a) immunological disorders such as autoimmune and
chronic inflammatory conditions, as well as immunodeficiencies; b)
certain malignancies, in particular those that depend on
NF-.kappa.B for their survival--such as breast cancer, HL, multiple
myeloma, and DLBCL.
[0125] A question was whether JNK played a role in TNF-R-induced
apoptosis. Confirming findings in NF-.kappa.B-deficient cells,
evidence presented herein now conclusively demonstrated that JNK
activation is obligatory not only for stress-induced apoptosis, but
also for efficient killing by TNF.alpha.. It was shown that
fibroblasts lacking ASK1--an essential component of the TNF-R
pathway signaling to JNK (and p38)--are resistant to killing by
TNF.alpha.. Foremost, JNK1 and JNK2 double knockout MEFs exhibit a
profound--albeit not absolute--defect in the apoptotic response to
combined cytotoxic treatment with TNF.alpha. and cycloheximide.
Moreover, it was shown that the TNF.alpha. homolog of Drosophila,
Eiger, completely depends on JNK to induce death, whereas it does
not require the caspase-8 homolog, DREDD. Thus, the connection to
JNK appears to be a vestigial remnant of a primordial apoptotic
mechanism engaged by TNF.alpha., which only later in evolution
begun to exploit the FADD-dependent pathway to activate
caspases.
[0126] How can then the early observations with DN-MEKK1 be
reconciled with these more recent findings? Most likely, the key
lies in the kinetics of JNK induction by TNF-Rs. Indeed, apoptosis
has been associated with persistent, but not transient JNK
activity. This view is supported by the recent discovery that JNK
activation is apoptogenic on its own--elegantly demonstrated by the
use of MKK7-JNK fusion proteins, which result in constitutively
active JNK in the absence of extrinsic cell stimulation. Unlike UV
and other forms of stress, TNF.alpha. causes only transient
induction of JNK, and in fact, this induction normally occurs
without significant cell death, which explains why JNK inhibition
by DN-MEKK1 mutants has no effect on cell survival. JNK
pro-apoptotic activity is instead unmasked when the kinase is
allowed to signal chronically, for instance by the inhibition of
NF-.kappa.B.
[0127] The exact mechanism by which JNK promotes apoptosis is not
known. While in some circumstances JNK-mediated killing involves
modulation of gene expression, during challenge with stress or
TNF.alpha., the targets of JNK pro-apoptotic signaling appear to be
already present in the cell. Killing by MKK7-JNK proteins was shown
to require Bax-like factors of the Bcl-2 group; however, it is not
clear that these factors are direct targets of JNK, or that they
mediate JNK cytotoxicity during TNF-R signaling.
[0128] I. Activation of the JNK Cascade is Required for Efficient
Killing by DRs (TNF-R1, Fas, and TRAIL-Rs), and the Suppression of
this Cascade is Crucial to the Protective Activity of
NF-.kappa.B.
[0129] A. TNF-Rs-Induced Apoptosis.
[0130] The JNK and NF-.kappa.B pathways--almost invariably
co-activated by cytokines and stress--are intimately linked. The
blocking of NF-.kappa.B activation by either the ablation of the
NF-.kappa.B subunit RelA or expression of the I.kappa.B.alpha.M
super-inhibitor hampers the normal shut down of JNK induction by
TNF-R (FIGS. 5a and 5b). Indeed, the down-regulation of the JNK
cascade by NF-.kappa.B is needed for suppression of
TNF.alpha.-induced apoptosis, as shown by the finding that
inhibition of JNK signaling by various means rescues
NF-.kappa.B-deficient cells from TNF.alpha.-induced apoptosis
(FIGS. 5d and 5e). In cells lacking NF-.kappa.B, JNK activation
remains sustained even after protective treatment with caspase
inhibitors, indicating that the effects of NF-.kappa.B on the JNK
pathway are not a secondary consequence of caspase inhibition.
Thus, NF-.kappa.B complexes are true blockers of JNK activation.
These findings define a novel protective mechanism by NF-.kappa.B
and establish a critical role for JNK (and not for p38 or ERK) in
the apoptotic response to TNF.alpha. (see FIG. 18).
[0131] B. Fas-Induced Apoptosis.
[0132] Although ASK1.sup.-/- and JNK null fibroblasts are protected
against the cytotoxic effects of TNF.alpha., these cells retain
normal sensitivity to Fas-induced apoptosis, which highlights a
fundamental difference between the apoptotic mechanisms triggered
by Fas and TNF-R. Nevertheless, in certain cells (e.g. B cell
lymphomas), JNK is also involved in the apoptotic response to Fas
triggering. Indeed, the suppression of JNK by various means,
including the specific pharmacological blocker SP600125, rescues
BJAB cells from Fas-induced cytotoxicity (FIG. 14). Consistent with
this observation, in these cells, killing by Fas is also almost
completely blocked by over-expression of Gadd45.beta. (FIG. 13B).
Together, these findings indicate that JNK is required for
Fas-induced apoptosis in some circumstance, for instance in type 2
cells (e.g. BJAB cells), which require mitochondrial amplification
of the apoptotic signal to activate caspases and undergo death.
[0133] Like TNF-Rs, Fas plays an important role in the host immune
surveillance against cancerous cells. Of interest, due to the
presence of constitutively high NF-.kappa.B activity, certain tumor
cells are able to evade these immune surveillance mechanisms. Thus,
an augmentation of JNK signaling--achieved by blocking the JNK
inhibitory activity of Gadd45.beta., or more broadly of
NF-.kappa.B--aids the immune system to dispose of tumor cells
efficiently.
[0134] Fas is also critical for lymphocyte homeostasis. Indeed,
mutations in this receptor or its ligand, FasL, prevent elimination
of self-reactive lymphocytes, leading to the onset of autoimmune
disease. Thus, for the treatment of certain autoimmune disorders,
the inhibitory activity of Gadd45.beta. on JNK may serve as a
suitable target.
[0135] C. TRAIL-R-Induced Apoptosis.
[0136] Gadd45.beta. also blocks TRAIL-R-involved in apoptosis (FIG.
1A), suggesting that JNK plays an important role in the apoptotic
response to the triggering of this DR. The finding that JNK is
required for apoptosis by DRs may be exploited for cancer therapy.
For example, the sensitivity of cancer cells to TRAIL-induced
killing by adjuvant treatment is enhanced with agents that
up-regulate JNK activation. This can be achieved by interfering
with the ability of Gadd45.beta. or NF-.kappa.B to block
TRAIL-induced JNK activation. This finding may also provide a
mechanism for the synergistic effects of combined anti-cancer
treatment because JNK activation by genotoxic chemotherapeutic
drugs may lower the threshold for DR-induced killing.
[0137] II. The Suppression of JNK Represents a Mechanism by which
NF-.kappa.B Promotes Oncogenesis and Cancer Chemoresistance.
[0138] In addition to antagonizing DR-induced killing, the
protective activity of NF-.kappa.B is crucial to oncogenesis and
chemo- and radio-resistance in cancer. However, the bases for this
protective activity is poorly understood. It is possible that the
targeting of the JNK cascade represents a general anti-apoptotic
mechanism by NF-.kappa.B, and indeed, there is evidence that the
relevance of this targeting by NF-.kappa.B extends to both
tumorigenesis and resistance of tumor cells to anti-cancer therapy.
During malignant transformation, cancer cells must adopt mechanisms
to suppress JNK-mediated apoptosis induced by oncogenes, and at
least in some cases, this suppression of apoptotic JNK signaling
might involve NF-.kappa.B. Indeed, while NF-.kappa.B activation is
required to block transformation-associated apoptosis,
non-redundant components of the JNK cascade such as MKK4 and BRCA1
have been identified as tumor suppressors.
[0139] Well-characterized model systems of NF-.kappa.B-dependent
tumorigenesis, including such as breast cancer cells provide
insight into mechanism of action. Breast cancer cell lines such as
MDA-MD-231 and BT-20, which are known to depend on NF-.kappa.B for
their survival, can be rescued from apoptosis induced by
NF-.kappa.B inhibition by protective treatment with the JNK blocker
SP600125 (FIG. 17). Thus, in these tumor cells, the ablation of JNK
can overcome the requirement for NF-.kappa.B, suggesting that
cytotoxicity by NF-.kappa.B inactivation is associated with an
hyper-activation of the JNK pathway, and indicates a role for this
pathway in tumor suppression. Gadd45.beta. mediates the protective
effects of NF-.kappa.B during oncogenesis and cancer
chemoresistance, and is a novel target for anti-cancer therapy.
[0140] With regard to chemoresistance in cancer, apoptosis by
genotoxic stress--a desirable effect of certain anti-cancer drugs
(e.g. daunorubicin, etopopside, and cisplatinum)--requires JNK
activation, whereas it is antagonized by NF-.kappa.B. Thus, the
inhibition of JNK is a mechanism by which NF-.kappa.B promotes
tumor chemoresistance. Indeed, blockers of NF-.kappa.B are
routinely used to treat cancer patients such as patients with HL
and have been used successfully to treat otherwise recalcitrant
malignancies such as multiple myeloma. However, these blockers
(e.g. glucocorticoids and proteosome inhibitors) can only achieve a
partial inhibition of NF-.kappa.B, and when used chronically,
exhibit considerable side effects, including immune suppressive
effects, which limit their use in humans. Hence, as discussed with
DRs, in the treatment of certain malignancies, it is beneficial to
employ, rather than NF-.kappa.B-targeting agents, therapeutic
agents aimed at blocking the anti-apoptotic activity of
NF-.kappa.B. For instance, a highly effective approach in cancer
therapy may be the use of pharmacological compounds that
specifically interfere with the ability of NF-.kappa.B to suppress
JNK activation. These compounds not only enhance JNK-mediated
killing of tumor cells, but allow uncoupling of the anti-apoptotic
and pro-inflammatory functions of the transcription factor. Thus,
unlike global blockers of NF-.kappa.B, such compounds lack
immunosuppressive effects, and thereby represent a promising new
tool in cancer therapy. A suitable therapeutic target is
Gadd45.beta. itself, because this factor is capable of inhibiting
apoptosis by chemotherapeutic drugs (FIGS. 3D and 3E), and its
induction by these drugs depends on NF-.kappa.B (FIG. 2D). With
regard to this, the identification of the precise mechanisms by
which Gadd45.beta. and NF-.kappa.B block the JNK cascade (i.e. the
testing of JNKK2) opens up new avenues for therapeutic intervention
in certain types of cancer, in particular in those that depend on
NF-.kappa.B, including tumors driven by oncogenic Ras, Bcr-Abl, or
EBV-encoded oncogenes, as well as late stage tumors such as HL,
DLBCL, multiple myeloma, and breast cancers.
[0141] III. Gadd45.beta. Mediates the Inhibition of the JNK Cascade
by NF-.kappa.B.
[0142] A. Gadd45.beta. Mediates the Protective Effects of
NF-.kappa.B Against DR-Induced Apoptosis.
[0143] Cytoprotection by NF-.kappa.B involves activation of a
program of gene expression. Pro-survival genes that mediate this
important function of NF-.kappa.B were isolated. In addition to
gaining a better understanding of the molecular basis for cancer,
the identification of these genes provides new targets for cancer
therapy. Using a functional screen in NF-.kappa.B/RelA null cells,
Gadd45.beta. was identified as a pivotal mediator of the protective
activity of NF-.kappa.B against TNF.alpha.-induced killing.
gadd45.beta. is upregulated rapidly by the cytokines through a
mechanism that requires NF-.kappa.B (FIGS. 2A and 2B), antagonizes
TNF.alpha.-induced killing (FIG. 1F), and blocks apoptosis in
NF-.kappa.B null cells (FIGS. 1A, 1C, 1D, 3A and 3B).
Cytoprotection by Gadd45.beta. involves the inhibition of the JNK
pathway (FIGS. 4A, 4C and 4D), and this inhibition is central to
the control of apoptosis by NF-.kappa.B (FIGS. 5A, 5B, 5D and 5E).
Expression of Gadd45.beta. in cells lacking NF-.kappa.B completely
abrogates the JNK activation response to TNF.alpha., and inhibition
of endogenous proteins by anti-sense gadd45.beta. hinders the
termination of this response (FIG. 4D). Gadd45.beta. also
suppresses the caspase-independent phase of JNK induction by
TNF.alpha., and hence, is a bona fide inhibitor of the JNK cascade
(FIGS. 4A and 4C). There may be additional NF-.kappa.B-inducible
blockers of JNK signaling.
[0144] Activation of gadd45.beta. by NF-.kappa.B was shown to be a
function of three conserved .kappa.B elements located at positions
-447/-438 (.kappa.B-1), -426/-417 (.kappa.B-2), and -377/-368
(.kappa.B-3) of the gadd45.beta. promoter (FIGS. 8, 9A, 9B, 10A,
10B, and 11). Each of these sites binds to NF-.kappa.B complexes in
vitro and is required for optimal promoter transactivation (FIGS.
12A, 12B, and 12C). Together, the data establish that Gadd45.beta.
is a novel anti-apoptotic factor, a physiologic inhibitor of JNK
activation, and a direct transcriptional target of NF-.kappa.B.
Hence, Gadd45.beta. mediates the targeting of the JNK cascade and
cytoprotection by NF-.kappa.B.
[0145] The protective activity of Gadd45.beta. extends to DRs other
than TNF-Rs, including Fas and TRAIL-Rs. Expression of Gadd45.beta.
dramatically protected BJAB cells from apoptosis induced by the
triggering of either one of these DRs, whereas death was
effectively induced in control cells (FIGS. 13B and 13A,
respectively). Remarkably, in the case of Fas, protection by
Gadd45.beta. was nearly complete. Similar to TNF-R1, the protective
activity of Gadd45.beta. against killing by Fas, and perhaps by
TRAIL-Rs, appears to involve the inhibition of the JNK cascade
(FIGS. 13A, 13B and 14). Thus, Gadd45.beta. is a new target for
modulating DR-induced apoptosis in various human disorders.
[0146] B. Gadd45.beta. is a Potential Effector of the Protective
Activity of NF-.kappa.B During Oncogenesis and Cancer
Chemoresistance.
[0147] The protective genes that are activated by NF-.kappa.B
during oncogenesis and cancer chemoresistance are not known.
Because it mediates JNK inhibition and cytoprotection by
NF-.kappa.B, Gadd45.beta. is a candidate. Indeed, as with the
control of DR-induced apoptosis, the induction of gadd45.beta.
represents a means by which NF-.kappa.B promotes cancer cell
survival. In 3DO tumor cells, Gadd45.beta. expression antagonized
killing by cisplatinum and daunorubicin (FIGS. 3D and 3E)--two
genotoxic drugs that are widely-used in anti-cancer therapy. Thus,
Gadd45.beta. blocks both the DR and intrinsic pathways of caspase
activation found in mammalian cells. Since apoptosis by genotoxic
agents requires JNK, this latter protective activity of
Gadd45.beta. might also be explained by the inhibition of the JNK
cascade. In 3DO cells, gadd45.beta. expression was strongly induced
by treatment with either daunorubicin or cisplatinum, and this
induction was almost completely abolished by the I.kappa.B.alpha.M
super-repressor (FIG. 2D), indicating that gadd45.beta. activation
by these drugs depends on NF-.kappa.B. Hence, Gadd45.beta. may
block the efficacy of anti-tumor therapy, suggesting that it
contributes to NF-.kappa.B-dependent chemoresistance in cancer
patients, and that it represents a new therapeutic target.
[0148] Given the role of JNK in tumor suppression and the ability
of Gadd45.beta. to block JNK activation, Gadd45.beta. also is a
candidate to mediate NF-.kappa.B functions in tumorigenesis.
Indeed, expression patterns suggest that Gadd45.beta. may
contribute to NF-.kappa.B-dependent survival in certain late stage
tumors, including ER breast cancer and HL cells. In cancer cells,
but not in control cells such as less invasive, ER.sup.+ breast
cancers, gadd45.beta. is expressed at constitutively high levels
(FIG. 16), and these levels correlate with NF-.kappa.B
activity.
[0149] C. Identification of the Mechanisms by which Gadd45.beta.
Blocks JNK Activation: the Targeting of JNKK2/MKK7
[0150] Neither Gadd45 nor NF-.kappa.B affect the ERK or p38
cascades (FIG. 4C), suggesting that these factors block JNK
signaling downstream of the MAPKKK module. Consistent with this
notion, the MAPKK, JNKK2/MKK7--a specific activator of JNK and an
essential component of the TNF-R pathway of JNK activation were
identified as the molecular target of Gadd45.beta. in the JNK
cascade.
[0151] Gadd45.beta. was previously shown to associate with MEKK4.
However, since this MAPKKK is not activated by DRs, the functional
consequences of this interaction were not further examined. Thus,
to begin to investigate the mechanisms by which Gadd45.beta.
controls JNK induction by TNF-R, Gadd45.beta. was examined for the
ability to physically interact with additional kinases, focusing on
those MAPKKKs, MAPKKs, and MAPKs that have been reported to be
induced by TNF-Rs. Co-immunoprecipitation assays confirmed the
ability of Gadd45.beta. to bind to MEKK4 (FIG. 19). These assays
also showed that Gadd45.beta. is able to associate with ASK1, but
not with other TRAF2-interacting MAPKKKs such as MEKK1, GCK, and
GCKR, or additional MAPKKK that were tested (e.g. MEKK3) (FIG. 19).
Notably, Gadd45.beta. also interacted with JNKK2/MKK7, but not with
the other JNK kinase, JNKK1/MKK4, or with any of the other MAPKKs
and MAPKs under examination, including the two p38-specific
activators MKK3b and MKK6, and the ERK kinase MEK1 (FIG. 19). In
vitro GST pull-down experiments have confirmed a strong and direct
interaction between Gadd45.beta. and JNKK2, as well as a much
weaker interaction with ASK1 (FIG. 20). They also uncovered a very
weak association between Gadd45.beta. and JNKK1 (FIG. 20).
[0152] Gadd45.beta. is a potent inhibitor of JNKK2 activity. This
has been shown both in in vitro assays (FIG. 22A), using
recombinant Gadd45.beta. proteins, and in in vivo assays, using
lysates of 3DO clones (FIG. 22A). The effects of Gadd45.beta. on
JNKK2 activity are specific, because even when used at high
concentrations, this factor is unable to inhibit either JNKK1,
MKK3b, or--despite its ability to bind to it--ASK1 (FIGS. 21B, 21C,
22A and 22B). This inhibition of JNKK2 is sufficient to account for
the effects of Gadd45.beta. on MAPK signaling, and likely explains
the specificity of these effects for the JNK pathway. Together, the
data indicate that Gadd45.beta. suppresses JNK activation, and
thereby apoptosis, induced by TNF.alpha. and stress stimuli by
directly targeting JNKK2 (FIGS. 21A and 22A). Consistent with the
notion that it mediates the effects of NF-.kappa.B on the JNK
cascade, Gadd45.beta. and NF-.kappa.B have similar effects on MAPK
activation by TNF.alpha., in vivo (FIG. 4C). Because ASK1 is
essential for sustained activation of JNK and apoptosis by TNF-Rs,
it is possible that the interaction between Gadd45.beta. and this
MAPKKK is also relevant to JNK induction by these receptors.
[0153] By performing GST pull-down experiments using either
GST-Gadd45.beta. or GST-JNKK2 and several N- and C-terminal
deletion mutants of JNKK2 and Gadd45.beta., respectively, the
kinase-binding surfaces(s) of Gadd45.beta. (FIGS. 24A and 24B) and
the Gadd45.beta.-binding domains of JNKK2 (FIGS. 23A and 23B) were
identified (see also FIGS. 36 and 37). Gadd45.beta. directly
contacts two distinct amino acid regions within the catalytic
domain of JNKK2 (FIGS. 23A and 23B), which provides important
mechanistic insights into the basis for the inhibitory effects of
Gadd45.beta. on JNKK2. These regions of JNKK2 share no homology
within MEKK4, suggesting that Gadd45.beta. contacts these kinases
through distinct surfaces. Since it is not known to have enzymatic
activity (e.g. phosphatase or proteolytic activity), and its
binding to JNKK2 is sufficient to inhibit kinase function, in vitro
(FIG. 21A), Gadd45.beta. might block JNKK2 through direct
interference with the catalytic domain, either by causing
conformational changes or steric hindrances that inhibit kinase
activity or access to substrates.
[0154] By performing mutational analyses, a domain of Gadd45.beta.
that is responsible for the blocking of TNF.alpha.-induced killing
was mapped (FIG. 25). Cytoprotection assays in Rel.sup.-/- cells
have shown that GFP-Gadd45.beta. (69-160) and GFP-Gadd45.beta.
(1-113) exhibit anti-apoptotic activity against TNF.alpha. that is
comparable to that of full-length GFP-Gadd45.beta. while GFP
proteins fused to Gadd45.beta. (87-160) or Gadd45.beta. (1-86) have
only modest protective effects. Shorter truncations have virtually
no effect on cell survival (FIG. 25), indicating that the
Gadd45.beta. region spanning between amino acids 69 and 113
facilitating cytoprotection.
[0155] This same amino acid region containing Gadd45.beta. domain
(69-104) that is essential for the Gadd45.beta. interaction with
JNKK2 (FIGS. 24A and 24B). This is consistent with the notion that
the protective activity of Gadd45.beta. is linked to its ability to
bind to JNKK2 and suppress JNK activation. Of interest, these
findings now allow the design of cell permeable, TAT-fusion
peptides encompassing the amino acid regions of JNKK2 that come
into direct contact with Gadd45.beta.. It is expected that these
peptides can effectively compete with endogenous Gadd45.beta.
proteins for binding to JNKK2. In addition, these findings allow to
design biochemical assays for screening libraries of small
molecules and identifying compounds that are capable of interfering
with the ability of Gadd45.beta. to associate with JNKK2. Both
these peptides and these small molecules prevent the ability of
Gadd45.beta., and thereby of NF-.kappa.B, to shut down JNK
activation, and ultimately, to block apoptosis. As discussed
throughout this summary, these compounds might find useful
application in the treatment of human diseases, including chronic
inflammatory and autoimmune conditions and certain types of
cancer.
EXAMPLES
[0156] The following examples are included to demonstrate
embodiments of the invention. It should be appreciated by those of
skill in the art that techniques disclosed in the examples which
follow represent techniques discovered by the inventor to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments which are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
Example 1
Identification of Gadd45.beta. as Novel Antagonist of TNFR-Induced
Apoptosis
[0157] Functional complementation of RelA-/- fibroblasts which
rapidly undergo apoptosis when treated with TNF.alpha. (Beg and
Baltimore, 1996), was achieved by transfection of cDNA expression
libraries derived from TNF.alpha.-activated, wild-type fibroblasts.
A total of four consecutive cycles of library transfection,
cytotoxic treatment with TNF.alpha., and episomal DNA extraction
were completed, starting from more than 4.times.10.sup.6
independent plasmids.
[0158] After selection, .about.200 random clones were analyzed in
transient transfection assays, with 71 (35%) found to significantly
protect RelA-null cells from TNF.alpha.-induced death. Among these
were cDNAs encoding murine RelA, cFLIP, and dominant negative (DN)
forms of FADD, which had been enriched during the selection
process, with RelA representing 3.6% of the newly-isolated library.
Thus, the library abounded in known regulators of TNFR-triggered
apoptosis (Budihardjo et al., 1999).
[0159] One of the cDNAs that scored positive in cytoprotection
assays encoded full-length Gadd45.beta., a factor that had not been
previously implicated in cellular responses to TNF.alpha..
Gadd45.beta. inserts had been enriched 82 folds after two cycles of
selection, reaching an absolute frequency of 0.41%. The above
experiment shows that Gadd45.beta. is a novel putative
anti-apoptotic factor.
[0160] To confirm the above findings, pEGFP-Gadd45.beta.,
pEGFP-RelA, or insert-less pEGFP constructs were tested in
transient transfection assays in RelA-/- fibroblasts. Whereas cells
expressing control GFP proteins were, as expected, highly
susceptible to TNF.alpha.-induced death, whereas in contrast, cells
that had received pEGFP-Gadd45.beta. were dramatically protected
form apoptosis-exhibiting a survival rate of almost 60% after an
8-hour treatment versus 13% in control cultures (FIG. 1A). As shown
previously, with pEGFP-RelA the cell rescue was virtually complete
(Beg and Baltimore, 1996).
[0161] To determine whether the activity of Gadd45.beta. was cell
type-specific an additional cellular model of NK-.kappa.B
deficiency was generated, where 3DO T cell hybridomas were forced
to stably express I.kappa.B.alpha.M, a variant of the
I.kappa.B.alpha. inhibitor that effectively blocks the nuclear
translocation of NF-.kappa.B (Van Antwerp et al., 1996).
[0162] In the presence of the repressor, 3DO cells became highly
sensitive to TNF.alpha.-induced killing, as shown by nuclear
propidium iodide (PI) staining, with the degree of the toxicity
correlating with I.kappa.B.alpha.M protein levels (FIG. 1B, lower
panels). Neo control cells retained instead, full resistance to the
cytokine. Next, constructs expressing full-length Gadd45.beta., or
empty control vectors (Hygro) were stably introduced into the
3DO-I.kappa.B.alpha.M-25 line, which exhibited the highest levels
of I.kappa.B.alpha.M (FIG. 1B). Although each of 11
I.kappa.B.alpha.M-Hygro clones tested remained highly susceptible
to TNF.alpha., clones expressing Gadd45.beta. became resistant to
apoptosis, with the rates of survival of 31 independent
I.kappa.B.alpha.M-Gadd45.beta. clones correlating with Gadd45.beta.
protein levels (FIGS. 1C and 1D, representative lines expressing
high and low levels of Gadd45.beta. and I.kappa.B.alpha.M-Hygro
controls). The protective effects of Gadd45.beta. were most
dramatic at early time points, when viability of some
I.kappa.B.alpha.M-Gadd45.beta. lines was comparable to that of Neo
clones (FIGS. 1C and 1D, 8 hours). In the
I.kappa.B.alpha.M-Gadd45.beta.-33 line, expressing high amounts of
Gadd45.beta., the frequency of cell death was only .about.15%
higher than in Neo controls even at 24 hours (FIG. 1C). Thus,
Gadd45.beta. is sufficient to temporarily compensate for the lack
of NF-.kappa.B.
[0163] Further, I.kappa.B.alpha.M-Gadd45.beta. cells retained
protein levels of I.kappa.B.alpha.M that were similar or higher
than those detected in sensitive I.kappa.B.alpha.M clones (FIG. 1D,
lower panels) and that were sufficient to completely block
NF-.kappa.B activation by TNF.alpha., as judged by electrophoretic
mobility shift assays (EMSAs; FIG. 1E). Hence, as also seen in
RelA-/- cells, Gadd45.beta. blocks apoptotic pathways by acting
downstream of NF-.kappa.B complexes.
Example 2
Gadd45 is a Physiologic Target of NF-.kappa.B
[0164] Gadd45 can be induced by cytokines such as IL-6, IL-18, and
TGF.beta., as well as by genotoxic stress (Zhang et al., 1999; Yang
et al., 2001; Wang et al., 999b). Because the NF-.kappa.B
anti-apoptotic function involves gene activation, whether
Gadd45.beta. was also modulated by TNF.alpha. was determined. As
shown in FIG. 2A, cytokine treatment determined a strong and rapid
upregulation of Gadd45.beta. transcripts in wild-type mouse embryo
fibroblasts (MEF). In contrast, in cells lacking RelA, gene
induction was severely impaired, particularly at early time points
(FIG. 2A, compare +/+ and -/- lanes at 0.5 hours). In these cells,
induction was also delayed and mirrored the pattern of expression
of I.kappa.B.alpha.M a known target of NH-.kappa.B (Ghosh et al.,
1998), suggesting that the modest induction was likely due to
NF-.kappa.B family members other than RelA (i.e., Rel).
Gadd45.alpha. was not activated by TNF.alpha., while Gadd45.gamma.
was modestly upregulated in both cell types.
[0165] Analogously, Gadd45.beta. was induced by TNF.alpha. in
parental and Neo 3DO cells, but not in the I.kappa.B.alpha. M lines
(FIG. 2B), with modest activation seen only in I.kappa.B.alpha.M-6
cells, which expressed low levels of the repressor (see FIG. 1B).
In Neo clones, Gadd45.beta. was also induced by daunorubicin or PMA
plus ionomycin (P/I; FIGS. 2D and 2C, respectively), treatments
that are known to activate NF-.kappa.B (Wang et al., 1996). Again,
gene induction was virtually abrogated by I.kappa.B.alpha.M.
Gadd45.alpha. was unaffected by TNF.alpha. treatment, but was
upregulated by daunorubicin or P/I, albeit independently of
NF-.kappa.B (FIG. 2B, C, D); whereas Gadd45.gamma. was marginally
induced by the cytokine only in some lines (FIG. 2B). nfkb1 was
used as a positive control of NF-.kappa.B-dependent gene expression
(Ghosh et al., 1998).
[0166] The results establish that gadd45.beta. is a novel
TNF.alpha.-inducible gene and a physiologic target of NF-.kappa.B.
The inspection of the gadd45.beta. promoter revealed the presence
of 3 .kappa.B binding sites. EMSAs and mutational analyses
confirmed that each of these sites was required for optimal
transcriptional activation indicating that gadd45.beta. is also a
direct target of NF-.kappa.B. These finding are consistent with a
role of gadd45.beta. as a physiologic modulator of the cellular
response to TNF.alpha..
Example 3
Endogenous Gadd45.beta.is Required for Survival of TNF.alpha.
[0167] Gadd45.beta. is a downstream target of NF-.kappa.B and
exogenous Gadd45.beta. can partially substitute for the
transcription factor during the response to TNF.alpha.. However, it
could be argued that since experiments were carried out in
overexpression, cytoprotection might not represent a physiologic
function of Gadd45.beta.. To address this issue, 3DO clones stably
expressing Gadd45.beta. in anti-sense orientation were generated.
The inhibition of constitutive Gadd45.beta. expression in these
clone led to a slight redistribution in the cell cycle, reducing
the fraction of cells residing in G.sub.2, which might underline
previously proposed roles of Gadd45 proteins in G.sub.2/M
checkpoints (Wang et al., 1999c). Despite their ability to activate
NF-.kappa.B, cells expressing high levels of anti-sense
Gadd45.beta. (AS-Gadd45.beta.) exhibited a marked susceptibility to
the killing by TNF.alpha. plus sub-optimal concentrations of CHX
(FIG. 1F). In contrast, control lines carrying empty vectors
(AS-Hygro) remained resistant to the treatment (FIG. 1F). As with
the alterations of the cell cycle, cytotoxicity correlated with
high levels of anti-sense mRNA. The data indicate that, under
normal circumstances, endogenous Gadd45.beta. is required to
antagonize TNFR-induced apoptosis, and suggest that the sensitivity
of NF-.kappa.B-null cells to cytokine killing is due, at least in
part, to the inability of these cells to activate its
expression.
Example 4
Gadd45.beta. Effectively Blocks Apoptotic Pathways in
NF-.kappa.B-Null Cells
[0168] A question was whether expression of Gadd45.beta. affected
caspase activation. In NF-.kappa.-deficient cells, caspase-8
activity was detected as early as 4 hours after TNF.alpha.
treatment, as assessed by the ability of 3DO extracts to proteolyze
caspase-8-specific substrates in vitro (FIG. 3A, I.kappa.B.alpha.M
and I.kappa.B.alpha.M-Hygro). This coincided with the marked
activation of downstream caspases such as caspase-9, -2, -6, and
-3/7. As previously reported, this cascade of events, including the
activation of procaspase-8, was completely blocked by NF-.kappa.B
(Neo; Wang et al., 1998). The cytokine-induced activation of both
initiator and executioner caspases was also suppressed in
I.kappa.B.alpha.M-Gadd45.beta.-10 cells expressing high levels of
Gadd45.beta. (FIG. 3A). Although very low caspase-3/7 activity was
detected in these latter cells by 6 hours (bottom, middle panel),
the significance of this finding is not clear since there was no
sign of the processing of either caspase-3 or -7 in Western blots
(FIG. 3B). Indeed, in I.kappa.B.alpha.M-Gadd45.beta. and Neo cells,
the cleavage of other procaspases, as well as of Bid, was also
completely inhibited, despite the presence of normal levels of
protein proforms in these cells (FIG. 3B). Proteolysis was specific
because other proteins, including .beta.-actin, were not degraded
in the cell extracts. Thus, Gadd45.beta. abrogates
TNF.alpha.-induced pathways of caspase activation in
NF-.kappa.B-null cells.
[0169] To further define the Gadd45.beta.-dependent blockade of
apoptotic pathways, mitochondrial functions were analyzed. In
I.kappa.B.alpha. M and I.kappa.B.alpha.M-Hygro clones, TNF.alpha.
induced a drop of the mitochondrial .DELTA..PSI.m, as measured by
the use of the fluorescent dye JC-1. JC-1.sup.+ cells began to
appear in significant numbers 4 hours after cytokine treatment,
reaching .about.80% by 6 hours (FIG. 3C). Thus in NF-.kappa.B-null
3DO cells, the triggering of mitochondrial events and the
activation of initiator and executioner caspases occur with similar
kinetics. The ability of Bcl-2 to protect I.kappa.B.alpha. M cells
against TNF.alpha.-induced killing indicates that, in these cells,
caspase activation depends on mitochondrial amplification
mechanisms (Budihardjo et al, 1999). In
I.kappa.B.alpha.M-Gadd45.beta.-10 as well as in Neo cells,
mitochondrial depolarization was completely blocked (FIG. 3A).
Inhibition was nearly complete also in
I.kappa.B.alpha.M-Gadd45.beta.-5 cells, where low caspase activity
was observed (FIG. 3A). These findings track the protective
activity of Gadd45.beta. to mitochondria, suggesting that the
blockade of caspase activation primarily depends on the ability of
Gadd45.beta. to completely suppress mitochondrial amplification
mechanisms. As shown in FIGS. 3D and 3E, Gadd45.beta. was able to
protect cells against cisplatinum and daunorubicin, suggesting that
it might block apoptotic pathways in mitochondria. Consistent with
this possibility, expression of this factor also protected cells
against apoptosis by the genotoxic agents cisplatinum and
daunorubicin (FIGS. 3D and 3E, respectively). Because Gadd45.beta.
does not appear to localize to mitochondria, it most likely
suppresses mitochondrial events indirectly, by inhibiting pathways
that target the organelle.
Example 5
Gadd45.beta. is a Specific Inhibitor of JNK Activation
[0170] A question explored was whether Gadd45.beta. affected MAPK
pathways, which play an important role in the control of cell death
(Chang and Karin, 2001). In I.kappa.B.alpha.M-Hygro clones,
TNF.alpha. induced a strong and rapid activation of JNK, as shown
by Western blots with anti-phospho-JNK antibodies and JNK kinase
assays (FIGS. 4A and 5A, left panels). Activation peaked at 5
minutes, to then fade, stabilizing at sustained levels by 40
minutes. The specific signals rose again at 160 minutes due to
caspase activation (FIGS. 4A and 5A). Unlike the early induction,
this effect could be prevented by treating cells with the caspase
inhibitor zVAD-fmk. In I.kappa.B.alpha.M-Gadd45.beta. cells, JNK
activation by TNF.alpha. was dramatically impaired at each time
point, despite the presence of normal levels of JNK proteins in
these cells (FIG. 4A, right panels). Gadd45.beta. also suppressed
the activation of JNK by stimuli other than TNF.alpha., including
sorbitol and hydrogen peroxide (FIG. 4B). The blockade,
nevertheless, was specific, because the presence of Gadd45.beta.
did not affect either ERK or p38 activation (FIG. 4C). The
anti-sense inhibition of endogenous Gadd45.beta. led to a prolonged
activation of JNK following TNFR triggering (FIG. 4D,
AS-Gadd45.beta. and Hygro), indicating that this factor, as well as
other factors (see down-regulation in AS-Gadd45.beta. cells) is
required for the efficient termination of this pathway. The
slightly augmented induction seen at 10 minutes in AS-Gadd45.beta.
cells showed that constitutively expressed Gadd45.beta. may also
contribute to the inhibition of JNK (see FIG. 2, basal levels of
Gadd45.beta.). Gadd45.beta. is a novel physiological inhibitor of
JNK activation. Given the ability of JNK to trigger apoptotic
pathways in mitochondria, these observations may offer a mechanism
for the protective activity of Gadd45.beta..
Example 6
Inhibition of the JNK Pathway as a Novel Protective Mechanism by
NF-.kappa.B
[0171] Down-regulation of JNK represents a physiologic function of
NF-.kappa.B. Whereas in Neo cells, JNK activation returned to
near-basal levels 40 minutes after cytokine treatment, in
I.kappa.B.alpha.M as well as in I.kappa.B.alpha.M-Hygro cells,
despite down-modulation, JNK signaling remained sustained
throughout the time course (FIG. 7A; see also FIG. 5A).
Qualitatively similar results were obtained with RelA-deficient MEF
where, unlike what is seen in wild-type fibroblasts,
TNF.alpha.-induced JNK persisted at detectable levels even at the
latest time points (FIG. 5B). Thus, as with Gadd45.beta.,
NF-.kappa.B complexes are required for the efficient termination of
the JNK pathway following TNFR triggering thus establishing a link
between the NF-.kappa.B and JNK pathways.
[0172] CHX treatment also impaired the down-regulation to
TNF.alpha.-induced JNK (FIG. 5C), indicating that, in 3DO cells,
this process requires newly-induced and/or rapidly turned-over
factors. Although in some systems, CHX has been reported to induce
a modest activation of JNK (Liu et al., 1996), in 3DO cells as well
as in other cells, this agent alone had no effect on this pathway
(FIG. 5C; Guo et al., 1998). The findings indicate that the
NF-.kappa.B-dependent inhibition of JNK is most likely a
transcriptional event. This function indicates the involvement of
the activation of Gadd45.beta., because this factor depends on the
NF-.kappa.B for its expression (FIG. 2) and plays an essential role
in the down-regulation of TNFR-induced JNK (FIG. 4D).
[0173] With two distinct NF-.kappa.B-null systems, CXH-treated
cells, as well as AS-Gadd45.beta. cells, persistent JNK activation
correlated with cytotoxicity, whereas with
I.kappa.B.alpha.M-Gadd45.beta. cells, JNK suppression correlated
with cytoprotection. To directly assess whether MAPK cascades play
a role in the TNF.alpha.-induced apoptotic response of
NF-.kappa.B-null cells, plasmids expressing catalytically inactive
mutants of JNKK1 (MKK4; SEK1) or JNKK2 (MKK7), each of which blocks
JNK activation (Lin et al., 1995), or of MKK3b, which blocks p38
(Huang et al., 1997), or empty vectors were transiently transfected
along with pEGFP into RelA-/- cells. Remarkably, whereas the
inhibition of p38 had no impact on cell survival, the suppression
of JNK by DN-JNKK2 dramatically rescued RelA-null cells from
TNF.alpha.-induced killing (FIG. 5D). JNKK1 is not primarily
activated by proinflammatory cytokines (Davis, 2000), which may
explain why JNKK1 mutants had no effect in this system. Similar
findings were obtained in 3DO-I.kappa.B.alpha.M cells, where MAPK
pathways were inhibited by well-characterized pharmacological
agents. Whereas, PD98059 and low concentrations of SB202190 (5
.mu.M and lower), which specifically inhibit ERK and p38,
respectively, could not antagonize TNF.alpha. cytotoxicity, high
concentrations of SB202190 (50 .mu.M), which blocks both p38 and
JNK (Jacinto et al., 1998), dramatically enhanced cell survival
(FIG. 5E). The data indicate that JNK, but not p38 (or ERK),
transduces critical apoptotic signals triggered by TNFR and that
NF-.kappa.B complexes protect cells, at least in part, by prompting
the down-regulation of JNK pathways.
Example 7
Gadd45.beta. is Induced by the Ectopic Expression of RelA, but not
Rel or p50
[0174] The activation of gadd45.beta. by cytokines or stress
requires NF-.kappa.B, as is disclosed herein because induction in
abolished either by RelA-null mutations or by the expression of
I.kappa.B.alpha. M, a variant of the I.kappa.B.alpha. inhibitor
that blocks that nuclear translocation of NF-.kappa.B (Van Antwerp
et al, 1996). To determine whether NF-.kappa.B is also sufficient
to upregulate gadd45.beta. and, if so, to define which NF-.kappa.B
family members are most relevant to gene regulation, HeLa-derived
HtTA-RelA, HtTA-CCR43, and HtTA-p50 cell lines, which express RelA,
Rel, and p50, respectively, were used under control of a
teracyclin-regulated promoter (FIG. 6). These cell systems were
employed because they allow NF-.kappa.B complexes to localize to
the nucleus independently of extracellular signals, which can
concomitantly activate transcription factors of the
NF-.kappa.B.
[0175] As shown in FIG. 6, the withdrawal of tetracycline prompted
a strong increase of gadd45.beta. mRNA levels in HtTA-RelA cells,
with kinetics of induction mirroring those of relA, as well as
i.kappa.b.alpha.and p105, two known targets of NF-.kappa.B. As
previously reported, RelA expression induced toxicity in these
cells (gadph mRNA levels) lead to underestimation of the extent of
gadd45.beta. induction. Conversely, gadd45.beta. was only
marginally induced in HtTA-CCR43 cells, which conditionally express
high levels of Rel. i.kappa.b.alpha.and p105 were instead
significantly activated in these cells, albeit to a lesser extent
than in the HtTA-RelA line, indicating that tetracycline withdrawal
yielded functional Rel-containing complexes. The induction of p50,
and NF-.kappa.B subunit that lacks a defined activation domain, did
not affect endogenous levels of either gadd45.beta.,
i.kappa.b.alpha., or p105. The withdrawal of tetracycline did not
affect gadd45.beta. (or relA, rel, or p105) levels in HtTA control
cells, indicating the gadd45.beta. induction in HtTA-RelA cells was
due to the activation of NF-.kappa.B complexes.
[0176] Kinetics of induction of NF-.kappa.B subunits were confirmed
by Western blot analyses. Hence gadd45.beta. expression is
dramatically and specifically upregulated upon ectopic expression
of the transcriptionally active NF-.kappa.B subunit RelA, but not
of p50 or Rel (FIG. 6). These findings are consistent with the
observations with RelA-null fibroblasts described above and
underscore the importance of RelA in the activation of
gadd45.beta..
Example 8
Gadd45.beta. Expression Correlates with NF-.kappa.B Activity in B
Cell Lines
[0177] NF-.kappa.B plays a critical role in B lynphopoiesis and is
required for survival of mature B cells. Thus, constitutive and
inducible expression of gadd45.beta. were examined in B cell model
systems that had been well-characterized from the stand point of
NF-.kappa.B. Indeed, gadd45.beta. mRNA levels correlated with
nuclear NF-.kappa.B activity in these cells (FIG. 7). Whereas
gadd45.beta. transcripts could be readily seen in unstimulated
WEHI-231 B cells, which exhibit constitutively nuclear NF-.kappa.B,
mRNA levels were below detection in 70Z/3 pre-B cells, which
contain instead the classical inducible form of the transcription
factor. In both cell types, expression was dramatically augmented
by LPS (see longer exposure for 70Z/3 cells) and, in WEH-231 cells,
also by PMA, two agents that are known to activate NF-.kappa.B in
these cells. Thus gadd45.beta. may mediate some of the important
functions executed by NF-.kappa.B in B lymphocytes.
Example 9
The Gadd45.beta. Promoter Contains Several Putative .kappa.B
Elements
[0178] To investigate the regulation of gadd45.beta. expression by
NF-.kappa.B, the muring gadd45.beta. promoter was cloned. A BAC
clone containing the gadd45.beta. gene was isolated from a 129SV
mouse genomic library, digested with XhoI, and subcloned into pBS
vector. The 7384 bp XhoI fragment containing gadd45.beta. was
completely sequenced, and portions were found to match sequences
previously deposited in GeneBank (accession numbers AC073816,
AC073701, and AC091518) (see also FIG. 8). The fragment harbored
the genomic DNA region spanning from .about.5.4 kbp upstream of a
transcription start site to near the end of the 4.sup.th exon of
gadd45.beta.. Next, the TRANSFAC database was used to identify
putative transcription factor-binding elements. A TATAA box was
found to be located at position -56 to -60 relative to the
transcription start site (FIG. 10). The gadd45.beta. promoter also
exhibited several .kappa.B elements, some of which were recently
noted by others. Three strong .kappa.B sites were found in the
proximal promoter region at positions -377/-368, -426/-417, and
-447/-438 (FIG. 8); whereas a weaker site was located as position
-4516, -4890/-4881, and -5251/-5242 (FIG. 8). Three .kappa.B
consensus sites were also noted with the first exon of gadd45.beta.
(+27/+36, +71/+80, and +171/+180). The promoter also contained an
Sp1 motif (-890/-881) and several putative binding sties for other
transcription factors, including heat shock factor (HSF) 1 and 2,
Ets, Stat, AP1, N-Myc, MyoD, CREB, and C/EBP (FIG. 8).
[0179] To identify conserved regulatory elements, the 5.4 kbp
murine DNA sequence immediately upstream of the gadd45.beta.
transcription start site was aligned with corresponding human
sequence, previously deposited by the Joint Genome Initiative
(accession number AC005624). As shown in FIG. 8, DNA regions
spanning from position -1477 to -1197 and from -466 to -300 of the
murine gadd45.beta. promoter were highly similar to portions of the
human promoter (highlighted in gray are identical nucleotides
within regions of homology), suggesting that these regions contain
important regulatory elements. A less well-conserved regions was
identified downstream of position -183 up to the beginning of the
first intron. Additional shorter stretches of homology were also
identified (see FIG. 8). No significant similarity was found
upstream of position -2285. The -466/-300 homology region contained
three .kappa.B sites (hereafter referred to as .kappa.B1,
.kappa.B2, and .kappa.B3), which unlike the other .kappa.B sites
present throughout the gadd45.beta. promoter, were conserved among
the two species. These findings suggest that these .kappa.B sites
play an important role in the regulation of gadd45.beta., perhaps
accounting for the induction of gadd45.beta. by NF-.kappa.B.
Example 10
NF-.kappa.B Regulates the Gadd45.beta. Promoter Through Three
Proximal .kappa.B Elements
[0180] To determine the functional significance of the .kappa.B
sites present in the gadd45.beta. promoter, a series of CAT
reporter constructs were generated where CAT gene expression is
driven by various portions of this promoter (FIG. 9A). Each CAT
construct was transfected alone or along with increasing amounts of
RelA expression plasmids into NTera-2 embryo carcinoma cells, and
CAT activity measured in cell lysates by liquid scintillation
counting (FIG. 9B). RelA was chosen for these experiments because
of its relevance to the regulation of gadd45.beta. expression as
compared to other NF-.kappa.B subunits (see FIG. 6). As shown in
FIG. 9B, the -5407/+23-gadd45.beta.-CATT reporter vector was
dramatically transactivated by RelA in a dose-dependent manner,
exhibiting an approximately 340-fold induction relative to the
induction seen in the absence of RelA with the highest amount of
pMT2T-RelA. Qualitatively similar, RelA-dependent effects were seen
with the -3465/+23-gadd45.beta.- and -592/+23-gadd45.beta.-CAT
constructs, which contained distal truncations of the gadd45.beta.
promoter. The relatively lower constructs, which contained distal
truncations of the gadd45.beta. promoter. The relatively lower
basal and RelA-dependent CAT activity observed with the
-5407/+23-gadd45.beta.-CAT, may have been due, at least in part, to
the lack of a proximal 329 bp regulatory region, which also
contained the TATA box, in the former constructs (FIGS. 9A and 9B).
Even in the presence of this region, deletions extending proximally
to position -592 completely abolished the ability of RelA to
activate the CAT gene (FIG. 9B, see -265/+23-gadd45.beta.- and
-103/+23-gadd45.beta.-CAT constructs). Similar findings were
obtained with analogous reporter constructs containing an
additional 116 b promoter fragment downstream of position +23.
Whereas analogously to -592/+23-gadd45.beta.-CAT,
-592/+139-gadd45.beta.-CAT was highly response to RelA,
-265/+139-gadd45.beta.-CAT was not transactivated even by the
highest amounts of pMT2T-RelA. It should be noted that this
reporter construct failed to respond to RelA despite retaining two
putative .kappa.B binding elements at position +27/+36 and +71/+80
(see FIG. 8, SEQ ID NO: 35). Together, the findings indicate that
relevant NF-.kappa.B/RelA responsive elements in the murine
gadd45.beta. promoter reside between position -592 and +23. They
also imply that the .kappa.B sites contained in the first exon, as
well as the distal .kappa.B sites, may not significantly contribute
to the regulation of gadd45.beta. by NF-.kappa.B. Similar
conclusions were obtained in experiments employing Jurkat or HeLa
cells where NF-.kappa.B was activated by PMA plus ionomycin
treatment.
[0181] As shown in FIG. 8, the -592/+23 region of the gadd45.beta.
promoter contains three conserved .kappa.B binding sties, namely
.kappa.B1, .kappa.B2, and .kappa.B3. To test the functional
significance of these .kappa.B elements, each of these sites were
mutated in the context of -592/+23-gadd45.beta.-CAT (FIG. 10A),
which contained the minimal promoter region that can be
transactivated by RelA. Mutant reporter constructs were transfected
alone or along with increasing amounts of PMT2T-RelA in NTera-2
cells and CAT activity measured as described for FIG. 9B. As shown
in FIG. 10B, the deletion of each .kappa.B site significantly
impaired the ability of RelA to transactivate the
-592/+23-gadd45.beta.-CAT construct, with the most dramatic effect
seen with the mutation of .kappa.B1, resulting in a .about.70%
inhibition of CAT activity (compare -592/+23-gadd45.beta.-CAT and
.kappa.B-1M-gadd45.beta.-CAT). Of interest, the simultaneous
mutation of .kappa.B1 and .kappa.B2 impaired CAT induction by
approximately 90%, in the presence of the highest amount of
transfected RelA plasmids (FIG. 10B) (see
.kappa.B-1/2M-gadd45.beta.-CAT). Dramatic effects were also seen
when the input levels of RelA were reduced to 1 .mu.g or 0.3 .mu.g
(.about.eight- and .about.five-fold reduction, respectively, as
compared to the wild-type promoter). The residual CAT activity
observed with the latter mutant construct was most likely due to
the presence of an intact .kappa.B3 site. Qualitatively similar
results were obtained with the transfection of RelA plus p50, or
Rel expression constructs. It was concluded that the gadd45.beta.
promoter contains three functional .kappa.B elements in its
proximal region and that each is required for optimal
transcriptional activation of NF-.kappa.B.
[0182] To determine whether these sites were sufficient to drive
NF-.kappa.B-dependent transcription the .DELTA.56-.kappa.B-1/2-,
.DELTA.56-.kappa.B-3-, and .DELTA.56-.kappa.B-M-CAT, reporter
constructs were constructed, where one copy of the
gadd45.beta.-.kappa.B sites or of a mutated site, respectively,
were cloned into .DELTA.56-CAT to drive expression of the CAT gene
(FIG. 1). Each .DELTA.56-CAT construct was then transfected alone
or in combination with increasing amounts of RelA expression
plasmids into Ntera2 cells and CAT activity measured as before. As
shown in FIG. 11, the presence of either .kappa.B-1 plus
.kappa.B-2, or .kappa.B-3 dramatically enhanced the responsiveness
of .DELTA.56-CAT to RelA. As it might have been expected from the
fact that it harbored two, rather than one, .kappa.B sites,
.DELTA.56-.kappa.B-1/2-CAT was induced more efficiently than
.kappa.B3, particularly with the highest amount of pMT2T-RelA. Low,
albeit significant, RelA-dependent CAT activity was also noted with
.DELTA.56-.kappa.B-M-CAT, as well as empty .DELTA.56-CAT vectors,
suggesting that .DELTA.56-CAT contains cryptic .kappa.B sites (FIG.
11). Hence, either the .kappa.B-1 plus .kappa.B-2, or .kappa.B-3
cis-acting elements are sufficient to confer promoter
responsiveness to NF-.kappa.B.
Example 11
The .kappa.B-1, .kappa.B-2, and .kappa.B-3 Elements Bind to
NF-.kappa.B In Vitro
[0183] To assess the ability of .kappa.B elements in the
gadd45.beta. promoter to interact with NF.kappa.B complexes, EMSAs
were performed. Oligonucleotides containing the sequence of
.kappa.B-1, .kappa.B-2, or .kappa.B-3 were radiolabeled and
independently incubated with extracts of NTera-2 cells transfected
before hand with pMT2T-p50, pMT2T-p50 plus pMT2T-RelA, or empty
pMT2T plasmids, and DNA-binding complexes separated by
polyacrylamide gel electrophoresis (FIG. 12A). The incubation of
each .kappa.B probe with various amounts of extract from cells
expressing only p50 generated a single DNA-binding complex
comigrating with p50 homodimers (FIG. 12A, lanes 1-3, 5-7, and
9-11). Conversely, extracts from cells expressing both p50 and RelA
gave rise to two specific bands: one exhibiting the same mobility
of p50/p50 dimers and the other comigrating with p50/RelA
heterodimers (lanes 4, 8, and 12). Extracts from mock-transfected
NTera2 cells did not generate any specific signal in EMSAs (FIG.
12B). Specificity of each complex was confirmed by competition
assays where, in addition to the radiolabeled probe, extracts were
incubated with a 50-fold excess of wild-type or mutated cold
.kappa.B probes. Thus, each of the functionally relevant .kappa.B
elements in the gadd45.beta. promoter can bind to NF-.kappa.B
complexes in vitro.
[0184] To confirm the composition of the DNA binding complexes,
supershift assays were performed by incubating the cell extracts
with polyclonal antibodies raised against human p50 or RelA.
Anti-pSO antibodies completely supershifted the specific complex
seen with extracts of cells expressing p50 (FIG. 12B, lanes 5, 14,
and 23), as well as the two complexes detected with extracts of
cells expressing both p50 and RelA (lanes 8, 17, and 26).
Conversely, the antibody directed against RelA only retarded
migration of the slower complex seen upon concomitant expression of
p50 and RelA (lanes 9, 18, 27) and did not affect mobility of the
faster DNA-binding complex (lanes 6, 9, 15, 18, 24, and 27).
[0185] The gadd45.beta.-.kappa.B sites exhibited apparently
distinct in vitro binding affinities for NF-.kappa.B complexes.
Indeed, with p50/RelA heterodimers, .kappa.B-2 and .kappa.B-3
yielded significantly stronger signals as compared with .kappa.B-1
(FIG. 12B). Conversely, .kappa.B-2 gave rise to the strongest
signal with p50 homodimers, whereas .kappa.B-3 appeared to
associate with this complex most poorly in vitro (FIG. 12B).
Judging from the amounts of p50/p50 and p50/RelA complexes
visualized on the gel, the presence of the antibodies (especially
the anti-RelA antibody) may have stabilized NF-.kappa.B-DNA
interactions (FIG. 12B). Neither antibody gave rise to any band
when incubated with the radiolabeled probe in the absence of cell
extract. The specificity of the supershifted bands was further
demonstrated by competitive binding reactions with unlabeled
competitor oligonucleotides. Hence, consistent with migration
patterns (FIG. 14A), the faster complex is predominantly composed
of p50 homodimers, whereas the lower one is predominantly composed
of p50/RelA heterodimers. These data are consistent with those
obtained with CAT assays and demonstrate that each of the relevant
.kappa.B elements of the gadd45.beta. promoter can specifically
bind to p50/p50 and p50/RelA, NF.kappa.B complexes, in vitro,
thereby providing the basis for the dependence of gadd45.beta.
expression on NF-.kappa.B. Hence, gadd45.beta. is a novel direct
target of NF.kappa.B.
Example 12
JNKK2 (Also Known as MKK7)-Gadd45.beta. Interacting Domains
[0186] JNK1/2/3 are the downstream components of one of the major
mitogen-activated protein kinase (MAPK) cascades, also comprising
the extracellular signal-regulated kinase (ERK1/2) and
p38(.alpha./.beta./.gamma./.delta.) cascades. MAPKs are activated
by MAPK kinases (MAPKKs), which in turn are activated by MAPKK
kinases (MAPKKKs). To understand the basis for the Gadd45.beta.
control of JNK signaling was determined whether Gadd45.beta. could
physically interact with kinases in these cascades. HA-tagged
kinases were transiently expressed in 293 cells, alone or together
with FLAG-Gadd45.beta., and associations were assessed by combined
immunoprecipitation and Western blot assays. Gadd45.beta. bound to
ASK1, but not to other MAPKKKs capable of interacting with TRAF2
(FIG. 26a, left), a factor required for JNK activation by
TNF.alpha.. It also associated with MEKK4/MTK1--a MAPKKK that
instead is not induced by TNF.alpha.. Notably, Gadd45.beta.
interacted strongly with MKK7/JNKK2, but not with the other JNK
kinase, MKK4/JNKK1, the p38-specific activators MKK3b and MKK6, or
the ERK kinase, MEK-1, as well as with MAPKs (FIG. 26a, middle and
right, and FIG. 26b). Gadd45.beta. interactions were confirmed in
vitro. Glutathione S-transferase (GST)-Gadd45.beta., but not GST,
precipitated a large fraction of the MKK7 input (FIG. 26c), whereas
it absorbed only a small fraction of ASK1 or MEKK4. Hence,
Gadd45.beta. interacts with JNK-inducing kinases and most avidly
with MKK7.
[0187] Another question was whether Gadd45.beta. association with
these kinases had functional consequences, in vivo. Remarkably,
whereas in I.kappa.B.alpha.M-Hygro 3DO control clones, TNF.alpha.
activated MKK7 strongly, in clones expressing Gadd45.beta. this
activation was abolished (FIG. 27a). Inhibition was specific since
Gadd45.beta. had no effect on induction of other MAPKKs (i.e. MKK4,
MKK3/6, and MEK1/2) by either TNF.alpha. or PMA plus ionomycin
(P/I; FIG. 27b and FIG. 27c, respectively). ASK1 and MEKK1 were
activated weakly by TNF.alpha., and this activation too was
unaffected by Gadd45.beta. (FIG. 27b). Thus, Gadd45.beta.
selectively blocked induction of MKK7 phosphorylation/activity by
TNF.alpha..
[0188] Gadd45.beta. mediates the suppression of JNK signaling by
NF-.kappa.B. Indeed, MKK7 was inhibited by NF-.kappa.B (FIG. 27d).
Whereas in control 3DO clones (Neo), MKK7 activation by TNF.alpha.
returned to basal levels by 40 minutes--thereby mirroring the JNK
response--in NF-.kappa.B-null clones (I.kappa.B.alpha.M), this
activation remained sustained. MKK7 down-regulation correlated with
Gadd45.beta. induction by NF-.kappa.B. Furthermore, NF-.kappa.B did
not affect MKK4, MKK3/6, or MEK1/2 (FIG. 27d and FIG. 27e), thereby
recapitulating the effects of Gadd45.beta. on MAPK cascades.
[0189] Interaction of endogenous Gadd45.beta. and MKK7 was detected
readily (FIG. 28a). Anti-Gadd45.beta. monoclonal antibodies
co-immunoprecipitated MKK7 from P/I-treated 3DO cells, exhibiting
strong Gadd45.beta. expression (bottom right), but not from
untreated cells, lacking detectable Gadd45.beta.. MKK7 was present
at comparable levels in stimulated and unstimulated cells (bottom,
left) and was not co-precipitated by an isotype-matched control
antibody. The interaction was confirmed by using anti-MKK7
antibodies for immunoprecipitation and the anti-Gadd45.beta.
monoclonal antibody for Western blots (FIG. 28a, top right).
Anti-MEKK1 antibodies failed to co-precipitate Gadd45.beta.,
further demonstrating the specificity of the MKK7-Gadd45.beta.
association. To determine whether Gadd45.beta. binds to MKK7
directly, we used purified proteins (FIG. 28b). Purified GST-MKK7
or GST were incubated, in vitro, with increasing amounts of
purified His.sub.6-Gadd45.beta. or control His.sub.6-JIP1
(6.times.His tag disclosed as SEQ ID NO: 46), and the fraction of
His.sub.6-tagged polypeptides (6.times.His tag disclosed as SEQ ID
NO: 46) that bound to GST proteins was visualized by Western
blotting. His.sub.6-Gadd45.beta. (6.times.His tag disclosed as SEQ
ID NO: 46) specifically associated with GST-MKK7 (FIG. 28c), and
this association was tighter than that of the physiologic MKK7
regulator, JIP1, with the half maximum binding (HMB) values being
.about.390 nM for Gadd45.beta. and above 650 nM for JIP1 (left;
JIP1 was used under non-saturating conditions). Endogenous
Gadd45.beta. and MKK7 likely associate via direct, high-affinity
contact.
[0190] A question was whether Gadd45.beta. inhibited active MKK7,
in vitro. FLAG-MKK7 was immunoprecipitated from TNF.alpha.-treated
or untreated 293 cells, and kinase assays were performed in the
presence of purified His.sub.6-Gadd45.beta. (6.times.His tag
disclosed as SEQ ID NO: 46), GST-Gadd45.beta., or control proteins
(FIG. 28d; see also FIG. 28g). Both Gadd45.beta. polypeptides, but
neither GST nor His.sub.6-EF3 (6.times.His tag disclosed as SEQ ID
NO: 46), blocked GST-JNK1 phosphorylation by MKK7, in a
dose-dependent manner (FIG. 28d). Consistent with the in vivo
findings (FIG. 27), the inhibitory activity of Gadd45.beta. was
specific. In fact, even at high concentrations, this factor did not
hamper MKK4, MKK3b, or--despite its ability to bind to it in
over-expression (FIG. 26a)--ASK1 (FIG. 28e; see also FIG. 28f,
total levels). Hence, Gadd45.beta. is a potent and specific
inhibitor of MKK7. Indeed, the effects of Gadd45.beta. on MKK7
phosphorylation by TNF.alpha. may be due inhibition of the MKK7
ability to auto-phoshorylate and/or to serve as substrate for
upstream kinases. Altogether, the findings identify MKK7 as a
target of Gadd45.beta., and of NF-.kappa.B, in the JNK cascade. Of
interest, MKK7 is a selective activator of JNK, and its ablation
abolishes JNK activation by TNF.alpha.. Thus, blockade of MKK7 is
sufficient on its own to explain the effects of Gadd45.beta. on JNK
signaling--i.e. its specific and near-complete suppression of this
signaling.
[0191] The amino acid sequence of Gadd45.beta. is not similar to
sequences of phosphatases and is not known to have enzymatic
activity. Thus, to understand mechanisms of kinase inactivation,
the Gadd45.beta.-binding region(s) of MKK7 were mapped using sets
of N- and C-terminally truncated MKK7 polypeptides (FIG. 29a and
FIG. 29c, respectively). Full length nucleotide and amino acid
sequences of human and murine MKK7 or JNKK2 are shown in FIG. 31.
As used herein, the amino acid positions refer to a human MKK7 or
JNKK2 amino acid sequence. MKK7/63-401, MKK7/91-401, and
MKK7/132-401 bound to GST-Gadd45.beta. specifically and with
affinity comparable to that of full-length MKK7, whereas mutations
occurring between amino acids 157 and 213 interacted weakly with
GST-Gadd45.beta. (FIG. 29b). Ablation of a region extending to or
beyond residue 232 abolished binding. Analysis of C-terminal
truncations confirmed the presence of a Gadd45.beta.-interaction
domain between residues 141 and 161 (FIG. 29d; compare MKK7/1-140
and MKK7/1-161), but failed to reveal the C-terminal binding region
identified above, suggesting that Gadd45.beta. interacts with this
latter region more weakly. Hence, MKK7 contacts Gadd45.beta.
through two distinct regions located within residues 132-161 and
213-231 (hereafter referred to as region A and B,
respectively).
[0192] To define interaction regions and determine whether they are
sufficient for binding, Gadd45.beta. association with overlapping
peptides spanning these regions (FIG. 29e) was determined. As shown
in FIG. 29f, both regions A and B bound to GST-Gadd45.beta.--even
when isolated from the context of MKK7--and peptides 132-156 and
220-234 (i.e. peptides 1 and 7, respectively) were sufficient to
recapitulate this binding. Both peptides lie within the MKK7 kinase
domain, and peptide 1 spans the ATP-binding site, K149, required
for catalytic function--suggesting that Gadd45.beta. inactivates
MKK7 by masking critical residues. This is reminiscent of the
mechanism by which p27.sup.K1P1 inhibits cyclin-dependent kinase
(CDK)2. A question explored was whether MKK7, Gadd45.beta.-binding
peptides interfered with the Gadd45.beta. ability to suppress
kinase activity. Indeed, peptide 1 prevented MKK7 inhibition by
Gadd45.beta., whereas peptide 7 or control peptides did not (FIG.
30a). Hence, kinase inactivation by Gadd45.beta. requires contact
with region A, but not with region B.
[0193] These data predict that preventing MKK7 inactivation by
Gadd45.beta., in vivo, should sensitize cells to TNF.alpha.-induced
apoptosis. To test this hypothesis, MKK7-mimicking peptides were
fused to a cell-permeable, HIV-TAT peptide and transduced into
cells. Remarkably, peptide 1 markedly increased susceptibility of
I.kappa.B.alpha.M-Gadd45.beta. cells to TNF.alpha.-induced killing,
whereas DMSO-treated cells were resistant to this killing, as
expected (FIG. 30b). Importantly, peptide 1 exhibited marginal
basal toxicity, indicating that its effects were specific for
TNF.alpha. stimulation, and other peptides, including peptide 7,
had no effect on the apoptotic response to TNF.alpha.. Consistent
with the notion that MKK7 is a target of NF-.kappa.B, peptide 1
promoted TNF.alpha.-induced killing in NF-.kappa.B-proficient cells
(Neo; FIG. 30c)--which are normally refractory to this killing. As
seen with Gadd45.beta.-expressing clones, this peptide exhibited
minimal toxicity in untreated cells. Together, the findings support
that Gadd45.beta. halts the JNK cascade by inhibiting MKK7 and
causally link the Gadd45.beta. protective activity to this
inhibition. Furthermore, blockade of MKK7 is a factor in the
suppression of apoptosis by NF-.kappa.B, and this blockade is
mediated, at least in part, by induction of Gadd45.beta..
[0194] A mechanism for the control of JNK signaling by Gadd45.beta.
was identified. Gadd45.beta. associates tightly with MKK7, inhibits
its enzymatic activity by contacting critical residues in the
catalytic domain, and this inhibition is a factor in its
suppression of TNF.alpha.-induced apoptosis. Interactions with
other kinases do not appear relevant to the Gadd45.beta. control of
JNK activation and PCD by TNF.alpha., because MEKK4 is not involved
in TNF-R signaling, and ASK1 is apparently unaffected by
Gadd45.beta.. Indeed, peptides that interfere with Gadd45.beta.
binding to MKK7 blunt the Gadd45.beta. protective activity against
TNF.alpha. (FIG. 30a and FIG. 30b). The targeting of MKK7 is a
factor in the suppression of apoptosis by NF-.kappa.B.
NF-.kappa.B-deficient cells fail to down-modulate MKK7 induction by
TNF.alpha., and MKK7-mimicking peptides can hinder the ability of
NF-.kappa.B to block cytokine-induced killing (FIG. 30c). These
results appear consistent with a model whereby NF-.kappa.B
activation induces transcription of Gadd45.beta., which in turn
inhibits MKK7, leading to the suppression of JNK signaling, and
ultimately, apoptosis triggered by TNF.alpha..
[0195] Chronic inflammatory conditions such as rheumatoid arthritis
and inflammatory bowel disease are driven by a positive feedback
loop created by mutual activation of TNF.alpha. and NF-.kappa.B.
Furthermore, several malignancies depend on NF-.kappa.B for their
survival--a process that might involve suppression of JNK
signaling. These results suggest that blockade of the NF-.kappa.B
ability to shut down MKK7 may promote apoptosis of
self-reactive/pro-inflammatory cells and, perhaps, cancer cells,
thereby identifying the MKK7-Gadd45.beta. interaction as a
potential therapeutic target. Interestingly, pharmacological
compounds that disrupt Gadd45.beta. binding to MKK7 might uncouple
anti-apoptotic and pro-inflammatory functions of NF-.kappa.B, and
so, circumvent the potent immunosuppressive side-effects seen with
global NF-.kappa.B blockers--currently used to treat these
illnesses. The pro-apoptotic activity of MKK7 peptides in
NF-.kappa.B-proficient cells implies that, even if NF-.kappa.B were
to induce additional MKK7 inhibitors, these inhibitors would target
MKK7 through its Gadd45.beta.-binding surface, thereby proving in
principle the validity of this therapeutic approach.
Example 13
MKK7 Inactivation by Gadd45.beta. In Vivo, Sensitizes Cells to
TNF.alpha.-Induced Apoptosis
[0196] NF-.kappa.B/Rel transcription factors regulate apoptosis or
programmed cell death (PCD), and this regulation plays a role in
oncogenesis, cancer chemo-resistance, and to antagonize tumor
necrosis factor (TNF).alpha.-induced killing. Upon TNF.alpha.
induction, the anti-apoptotic activity of NF-.kappa.B involves
suppressing the c-Jun-N-terminal kinase (JNK) cascade.
Gadd45.beta./Myd118, a member of the Gadd45 family of inducible
factors plays an important role in this suppressive activity of
NF-.kappa.B. However, the mechanisms by which Gadd45.beta. blunts
JNK signaling are not understood. MKK7/JNKK2 is identified as a
specific and an essential activator of JNK signaling and as a
target of Gadd45.beta. and also NF-.kappa.B itself. Gadd45.beta.
binds to MKK7 directly and blocks its catalytic activity, thereby
providing a molecular link between the NF-.kappa.B and JNK
pathways. Gadd45.beta. is required to antagonize TNF.alpha.-induced
cytotoxicity, and peptides disrupting the Gadd45.beta./MKK7
interaction hinder the ability of Gadd45.beta., as well as of
NF-.kappa.B, to suppress this cytotoxicity. These results establish
a basis for the NF-.kappa.B control of JNK activation and identify
MKK7 as a potential target for anti-inflammatory and anti-cancer
therapy.
[0197] These data predict that preventing. MKK7 inactivation by
Gadd45.beta., in vivo, sensitizes cells to TNF.alpha.-induced
apoptosis. MKK7-mimicking peptides were fused to a cell-permeable,
HIV-TAT peptide and transduced into cells. As shown by flow
cytometry (FCM) and confocal microscopy, peptides entered cells
with equivalent efficiency (FIG. 34 a-d). Peptide 1 markedly
increased susceptibility of I.kappa.B.alpha.M-Gadd45.beta. cells to
TNF.alpha.-induced killing, whereas DMSO-treated cells were
resistant to this killing, as expected (FIG. 33 a, left;). Peptide
1 exhibited marginal basal toxicity indicating that its effects
were specific for TNF.alpha. stimulation, and other peptides,
including peptide 7, had no effect on the apoptotic response to
TNF.alpha.. Further linking the in vivo effects of peptide 1 to
Gadd45.beta., pro-apoptotic activity of Ala mutant peptides
correlated with their apparent binding affinity for Gadd45.beta.,
in vitro (FIGS. 32 d and 33 a, right). Consistent with the notion
that MKK7 is a target of NF-.kappa.B, peptide 1 promoted
TNF.alpha.-induced killing in NF-.kappa.B-proficient cells (Neo;
FIG. 33 b)--which are normally refractory to this killing. As seen
with Gadd45.beta.-expressing clones, this peptide exhibited minimal
toxicity in untreated cells, and mutation of residues required for
interaction with Gadd45.beta. abolished its effects on TNF.alpha.
cytotoxicity (FIG. 33 b, right). Together, the findings demonstrate
that Gadd45.beta. halts the JNK cascade by inhibiting MKK7 and
causally link the Gadd45.beta. protective activity to this
inhibition. Furthermore, blockade of MKK7 is crucial to the
suppression of apoptosis by NF-.kappa.B, and this blockade is
mediated, at least in part, by induction of Gadd45.beta..
[0198] Chronic inflammatory conditions such as rheumatoid arthritis
and inflammatory bowel disease are driven by a positive feedback
loop created by mutual activation of TNF.alpha. and NF-.kappa.B.
Furthermore, several malignancies depend on NF-.kappa.B for their
survival--a process that might involve the suppression of JNK
signaling. The results suggest that blockade of the NF-.kappa.B
ability to shut down MKK7 may promote apoptosis of
self-reactive/pro-inflammatory cells and, perhaps, of cancer cells,
thereby identifying the MKK7-Gadd45.beta. interaction as a
potential therapeutic target. Pharmacological compounds that
disrupt Gadd45.beta. binding to MKK7 might uncouple anti-apoptotic
and pro-inflammatory functions of NF-.kappa.B, and so, circumvent
the potent immunosuppressive side-effects seen with global
NF-.kappa.B blockers-currently used to treat these illnesses. The
pro-apoptotic activity of MKK7 peptides in NF-.kappa.B-proficient
cells indicates that critical NF-.kappa.B-inducible inhibitors
target MKK7 through or in vicinity of its Gadd45.beta.-binding
surface, thereby proving in principle the validity of this
therapeutic approach.
Example 14
Cell-Specific Modulation of JNKK2 Activity
[0199] In mouse embryonic fibroblasts (MEFs), Gadd45.beta. ablation
was reported not to affect TNF.alpha.-induced PCD. The effects of
MKK7-derived peptides were tested in these cells. The peptide 2 (aa
142-166 of MKK7/JNKK2) has an amino acid sequence
NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1) and the TAT
fusion version has an amino acid sequence NH2-GRKKRRQRRRPP
TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 45).
[0200] FIGS. 33A-B shows that the Gadd45.beta.-mediated suppression
of MKK7 is required to block TNF.alpha.-induced apoptosis. This is
shown by the finding that MKK7-mimicking peptide 1, which prevents
the Gadd45.beta.-mediated inhibition of MKK7, sensitizes
I.kappa.B.alpha.M-Gadd45.beta. (FIG. 33A) and Neo (FIG. 33B) 3DO
clones, respectively, to TNF.alpha.-induced apoptosis.
MKK7-mimicking peptides were fused to a cell-permeable, HIV-TAT
peptide and transduced into cells. As shown in FIG. 34, peptides
entered cells with equivalent efficiency. Remarkably, peptide 1
markedly increased susceptibility of I.kappa.B.alpha.M-Gadd45.beta.
cells to TNF.alpha.-induced killing, whereas DMSO-treated cells
were resistant to this killing (FIG. 33A, left; see also FIG. 35A),
as expected (De Smaele et al., 2001). Other peptides, including
peptide 7, had no effect on the apoptotic response to TNF.alpha..
Peptide 1 exhibited marginal basal toxicity (FIG. 35A, left)
indicating that its effect was specific for cytokine stimulation.
Further linking the in vivo effect of peptide 1 to Gadd45.beta.,
pro-apoptotic activity of Ala mutant peptides correlated with their
apparent binding affinity for Gadd45.beta., in vitro (FIG. 32).
[0201] FIG. 33B shows that, consistent with the notion that MKK7 is
a target of NF-.kappa.B, peptide 1 promoted TNF.alpha.-induced
killing in NF-.kappa.B-proficient cells (Neo; FIG. 33B; see also
FIG. 35B)--which are expected to be refractory to this killing (De
Smaele et al., 2001). As seen with Gadd45.beta.-expressing clones,
this peptide exhibited minimal toxicity in untreated cells (FIG.
35B, left), and mutation of residues required for interaction with
Gadd45.beta. abolished its effects on TNF.alpha. cytotoxicity (FIG.
33B, right). Together, the findings demonstrate that Gadd45.beta.
halts the JNK cascade by inhibiting MKK7 and causally links the
Gadd45.beta. protective activity to this inhibition. Furthermore,
blockade of MKK7 is crucial to the suppression of apoptosis by
NF-.kappa.B, and this blockade is mediated, at least in part, by
induction of Gadd45.beta..
[0202] FIG. 33C-D depicts apoptosis assays showing that both
peptide 1 and peptide 2 facilitate TNF.alpha.-induced killing in
wild-type MEFs, and that only peptide 2 promotes this killing in
Gadd45.beta. null MEFs, respectively. MEFs were from twin embryos
and were used at passage (p).sub.4. This figure shows that
Gadd45.beta. is required to block MKK7 activation and apoptosis
induction by TNF.alpha.. It also shows that in some cell types
(e.g. fibroblasts), at least another factor, distinct from
Gadd45.beta., is essential to execute these functions. A recent
report suggested that, in mouse embryonic fibroblasts (MEFs),
Gadd45.beta. ablation does not affect TNF.alpha.-induced PCD
(Amanullah et al., 2003). The effects of MKK7-derived peptides were
tested in these cells. Surprisingly, in wild-type fibroblasts
cytokine-induced toxicity was enhanced by both peptide 1 and
peptide 2, whereas other peptides had no effect on this toxicity
(FIG. 33C, see also FIG. 35C). This contrasts with what was seen in
3DO lymphoid cells, where only peptide 1 promoted killing by
TNF.alpha. (FIG. 33B). Because peptide 2 does not bind to
Gadd45.beta. (FIG. 29), its pro-apoptotic activity is most likely
due to displacement of another inhibitory factor(s) from MKK7.
[0203] Consistent with this notion, activity of peptide 2 was
retained (and, in fact, enhanced) in gadd45.beta..sup.-/- MEFs
(FIG. 33D; see also FIG. 35D). Remarkably, however, Gadd45.beta.
ablation rendered these cells completely insensitive to the
cytotoxic effects of peptide 1 (FIGS. 33D and 35D), indicating that
in wild-type fibroblasts, these effects were due to Gadd45.beta.
inactivation. Together, these findings demonstrate that the MKK7
inhibitory mechanism activated in response to TNF.alpha. is
tissue-specific (shown by the distinct effects of MKK7 peptides in
3DO cells and fibroblasts; FIGS. 33B-D), and that, at least in
MEFs, this mechanism is functionally redundant. They also provide
compelling evidence that Gadd45.beta. is required to antagonize
TNF.alpha.-induced killing (FIG. 35C). Indeed, the apparent lack of
apoptotic phenotype previously reported in gadd45.beta..sup.-/-
fibroblasts (Amanullah et al., 2003) appears due to activation of
compensatory mechanisms in these cells--mechanisms that are not
mounted during acute Gadd45.beta. inactivation by peptide 1.
[0204] A mechanism for the control of JNK signaling by Gadd45.beta.
is identified. Gadd45.beta. associates tightly with MKK7, inhibits
its enzymatic activity by contacting critical residues in the
catalytic domain, and this inhibition is crucial to the suppression
of TNF.alpha.-induced apoptosis. Interactions with other kinases do
not appear relevant to the Gadd45.beta. control of JNK activation
and PCD by TNF.alpha., as MEKK4 is not involved in TNF-R signaling,
and ASK1 is seemingly unaffected by Gadd45.beta. (FIGS. 21-22).
Indeed, peptides that interfere with Gadd45.beta. binding to MKK7
blunt the Gadd45.beta. protective activity against TNF.alpha.
(FIGS. 33A, 33C, 33D, 35A, 35C, 35D). The targeting of MKK7 effects
suppression of apoptosis by NF-.kappa.B itself.
NF-.kappa.B-deficient cells fail to down-modulate MKK7 induction by
TNF.alpha., and MKK7-mimicking peptides disrupting the
Gadd45.beta./MKK7 interaction hinder the ability of NF-.kappa.B to
block TNF.alpha.-induced cytotoxicity (FIGS. 33B-C). A model is
that NF-.kappa.B activation induces expression of Gadd45.beta.,
which in turn inhibits MKK7, leading to the suppression of JNK
signaling, and ultimately, apoptosis triggered by TNF.alpha.. These
findings identify a molecular link between the NF-.kappa.B and JNK
pathways, and establish a basis for the NF-.kappa.B control of JNK
activation. Indeed, the relevance of this link is underscored by
knockout studies showing that Gadd45.beta. is essential to
antagonize TNF.alpha.-induced apoptosis (FIGS. 33B-C). Yet, in some
tissues, other NF-.kappa.B-inducible factors might contribute to
suppress MKK7 induction by TNF.alpha. (FIGS. 33B-C).
[0205] Chronic inflammatory conditions such as rheumatoid arthritis
and inflammatory bowel disease are driven by a positive feedback
loop created by mutual activation of TNF.alpha. and NF-.kappa.B.
Furthermore, several malignancies depend on NF-.kappa.B for their
survival--a process that might involve suppression of JNK
signaling. Blockade of the NF-.kappa.B ability to shut down MKK7
may promote apoptosis of self-reactive/pro-inflammatory cells and,
perhaps, of cancer cells, thereby identifying the MKK7-Gadd45.beta.
interaction as a potential therapeutic target. Pharmacological
compounds that disrupt Gadd45.beta. binding to MKK7 might uncouple
anti-apoptotic and pro-inflammatory functions of NF-.kappa.B, and
so, circumvent the potent immunosuppressive side-effects seen with
global NF-.kappa.B blockers--currently used to treat these
illnesses. The pro-apoptotic activity of MKK7 peptides in
NF-.kappa.B-proficient cells implies that NF-.kappa.B-inducible
factors target MKK7 through or in proximity of its
Gadd45.beta.-binding surface, thereby proving in principle the
validity of this therapeutic approach.
Example 15
Regions of Gadd45.beta. that Bind to and Inhibit MKK7
[0206] FIG. 36 shows that the 69-86 amino acid region of
Gadd45.beta. is sufficient to bind to MKK7 in vitro. GST pull-down
assays were performed using GST- or GST-MKK7-coated beads and in
vitro-translated, Gadd45.beta. products corresponding to the
polypeptidic fragments indicated in FIG. 36A.
[0207] FIG. 37 shows that the Gadd45.beta.-mediated inhibition of
MKK7 requires a polypeptidic region of Gadd45.beta. including the
region between amino acid 60 and 86. Active MKK7 was
immunoprecipitated from TNF.alpha.-activated 293 cells and MKK7
kinase assays were performed using GST-JNK1 substrates and pure
recombinant Gadd45.beta. polypeptides (FIG. 37B; a schematic
diagram representing the Gadd45.beta. polypeptides used is shown in
FIG. 37A). FIGS. 37D-E show that the amino acid regions contained
in the overlapping, Gadd45.beta.-derived peptides 2 and 8 are
sufficient to recapitulate most of the inhibitory activity of
Gadd45.beta. on MKK7. MKK7 kinase assays were performed as in FIG.
37B, except that pure synthetic Gadd45.beta. peptides (whose
sequences are shown in FIG. 37C) were used instead of pure
recombinant Gadd45.beta. proteins. The amino acid region between
amino acids 58 and 77 of Gadd45.beta. is used for the
Gadd45.beta.-mediated inhibition of MKK7. Thus, it is expected that
cell-permeable forms of these peptides can be used in cells to
block apoptosis induced by TNF.alpha. or other pro-apoptotic
agents. These peptides could also used in the whole animal to block
apoptosis in inflammatory diseases, neurodegenerative disorders,
stroke, and myocardial infarction.
Materials and Methods
[0208] 1. Library Preparation and Enrichment
[0209] cDNA was prepared from TNF.alpha.-treated NIH-3T3 cells and
directionally inserted into the pLTP vector (Vito et al., 1996).
For the enrichment, RelA-/- cells were seeded into
1.5.times.10.sup.6/plate in 100 mm plates and 24 hours later used
for transfection by of the spheroplasts fusion method. A total of
4.5.times.10.sup.6 library clones were transfected for the first
cycle. After a 21-hours treatment with TNF.alpha. (100 units/ml)
and CHX (0.25 .mu.g/ml), adherent cells were harvested for the
extraction of episomal DNA and lysed in 10 mM EDTA, 0.6% SDS for
the extraction of episomal DNA after amplification, the library was
used for the next cycle of selection. A total of 4 cycles were
completed.
[0210] 2. Constructs
[0211] I.kappa.B.alpha.M was excised from pCMX-I.kappa.B.alpha.M
(Van Antwerp et al., 1996) and ligated into the EcoRI site of
pcDNA3-Neo (Invitrogen). Full length human RelA was PCR-amplified
from BS-RelA (Franzoso et al., 1992) and inserted into the BamHI
site of pEGFP-C1 (Clontech). Gadd45.beta., Gadd45.alpha. and
Gadd45.gamma. cDNAs were amplified by PCR for the pLTP library and
cloned into the XhoI site and pcDNA 3.1-Hygro (Invitrogen) in both
orientations. To generate pEGFP-Gadd45.beta., Gadd45.beta. was
excised from pcDNA Hygro with XhoI-XbaI and ligated with the linker
5'-CTAGAGGAACGCGGAAGTGGTGGAAGTGGTGGA-3' (SEQ ID NO: 13) into the
XbaI-BamHI sites of pEGFP-N1. pcDNA-Gadd45.alpha. was digested with
EcoRI-XhoI and ligated with XhoI-BamHI opened pEGFP-C1 and the
linker 5'-GTACAAGGGAAGTGGTGGAAGTGTGGAATGACTTTGGAGG-3' (SEQ ID NO:
14). pEGFP-N1-Gadd45.gamma. was generated by introducing the
BspEI-XhoI fragment of pcDNA-Hygro-Gadd45.gamma. along with the
adapter 5'-ATTGCGTGGCCAGGATACAGTT-3' (SEQ ID NO: 15) into
pEGFP-C1-Gadd45.alpha., where Gadd45.alpha. was excised by
EcoRI-SalI. All constructs were checked by sequencing. pSR.alpha.3
plasmids expressing DN-JNKK1 (S257A, T261A), DN-JNKK2 (K149M,
S271A, T275A) and MKK3bDN (S128A, T222A) were previously described
(Lin et al., 1995; Huang et al., 1997).
[0212] 3. Anti Sense Constructs of Gadd45.beta.
[0213] Modulators of the JNK pathway, such as Gadd45.beta., can be
modulated by molecules that directly affect RNA transcripts
encoding the respective functional polypeptide. Antisense and
ribozyme molecules are examples of such inhibitors that target a
particular sequence to achieve a reduction, elimination or
inhibition of a particular polypeptide, such as a Gadd45 sequence
or fragments thereof.
[0214] Antisense methodology takes advantage of the fact that
nucleic acids tend to pair with "complementary" sequences.
Antisense constructs specifically form a part of the current
invention, for example, in order to modulate the JNK pathway. In
one embodiment of the invention, antisense constructs comprising a
Gadd45 nucleic acid are envisioned, including antisense constructs
comprising nucleic acid sequence in antisense orientation, as well
as portions of fragments thereof.
[0215] By complementary, it is meant that polynucleotides are those
which are capable of base-pairing according to the standard
Watson-Crick complementarity rules. That is, the larger purines
will base pair with the smaller pyrimidines to form combinations of
guanine paired with cytosine (G:C) and adenine paired with either
thymine (A:T) in the case of DNA, or adenine paired with uracil
(A:U) in the case of RNA. Inclusion of less common bases such as
inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others
in hybridizing sequences doe not interfere with pairing.
[0216] Targeting double-stranded (ds) DNA with polynucleotides
leads to triple-helix formation; targeting RNA will lead to
double-helix formation. Antisense polynucleotides, when introduced
into a target cell, specifically bind to their target
polynucleotide and interfere with transcription, RNA processing,
transport, translation and/or stability. Antisense RNA constructs,
or DNA encoding such antisense RNAs, may be employed to inhibit
gene transcription or translation of both within a host cell,
either in vitro or in vivo, such as within a host animal, including
a human subject.
[0217] Antisense constructs, including synthetic anti-sense
oligonucleotides, may be designed to bind to the promoter and other
control regions, exons, introns or even exon-intron boundaries of a
gene. It is contemplated that the most effective antisense
constructs may include regions complementary to intron/exon splice
junctions. Thus, antisense constructs with complementarily to
regions within 50-200 bases of an intron-exon splice junction may
be used. It has been observed that some exon sequences can be
included in the construct without seriously affecting the target
selectivity thereof. The amount of exonic material included will
vary depending on the particular exon and intron sequences used.
One can readily test whether too much exon DNA is included simply
by testing the constructs in vitro to determine whether normal
cellular function is affected or whether the expression of related
genes having complementary sequences is affected.
[0218] It may be advantageous to combine portions of genomic DNA
with cDNA or synthetic sequences to generate specific constructs.
For example, where an intron is desired in the ultimate construct,
a genomic clone will need to be used. The cDNA or a synthesized
polynucleotide may provide more convenient restriction sites for
the remaining portion of the construct and, therefore, would be
used for the rest of the sequence.
[0219] 4. Cell Lines, Transfections and Treatments
[0220] MEF and 3DO cells were cultured in 10% Fetal bovine
serum-supplemented DMEM and RPMI, respectively. Transient
transfections in RelA-/- MEF were performed by Superfect according
to the manufacturer's instructions (Qiagen). After cytotoxic
treatment with CHX (Sigma) plus or minus TNF.alpha. (Peprotech),
adherent cells were counted and analyzed by FCM (FACSort, Becton
Dickinson) to assess numbers of live GFP.sup.+ cells. To generate
3DO stable lines, transfections were carried out by
electroporatoration (BTX) and clones were grown in appropriate
selection media containing Geneticin (Gibco) and/or Hygromycin
(Invitrogen). For the assessment of apoptosis, 2DO cells were
stained with PI (Sigma) and analyzed by FCM, as previously
described (Nicoletti et al, 1991). Daunorubicin, PMA, lonomycin,
hydrogen peroxide, and sorbitol were from Sigma; Cisplatin
(platinol AQ) was from VHAplus, PD98059 and SB202190 were from
Calbiochem.
[0221] 5. Northern Blots, Western blots, EMSAs, and Kinase
Assays
[0222] Northern blots were performed by standard procedures using 6
.mu.g of total RNA. The EMSAs with the palindromic probes and the
preparation of whole cell L extracts were as previously described
(Franzoso et al., 1992). For western blots, cell extracts were
prepared either in a modified lysis buffer (50 mM Tris, pH 7.4, 100
mM NaCl, 50 mM NaF, 1 mM NaBo.sub.4, 30 mM pyrophosphate, 0.5%
NP-40, and protease inhibitors (FIG. 1B; Boehringer Mannheim), in
Triton X-100 buffer (FIG. 4A; Medema et al, 1997) or in a lysis
buffer containing 1% NP-40 350 mM NaCl, 20 mM HEPES (pH 8.0), 20%
glycerol, 1 mM MgCl.sub.2, 0.1 mM EGTA, 0.5 mM DTT, 1 mM
Na.sub.3VO.sub.4, 50 mM NaF and protease inhibitors. Each time,
equal amounts of proteins (ranging between 15 and 50 .mu.g) were
loaded and Western blots prepared according to standard procedures.
Reactions were visualized by ECL (Amersham). Antibodies were as
follows: I.kappa.B.alpha., Bid, and .beta.-actin from Santa Cruz
Biotechnology; caspase-6, -7 and -9, phospho and total -p38, phosph
and total -ERK, phospho and total -JNK from Cell Signaling
Technology; caspase-8 from Alexis; Caspase-2 and -3 from R&D
systems. The Gadd45.beta.-specific antibody was generated against
an N-Terminal peptide. Kinase assays were performed with
recombinant GST-c-jun and anti-JNK antibodies (Pharmingen), (Lin et
al., 1995).
[0223] 6. Measurement of Caspase Activity and Mitochondrial
Transmembrane Potential
[0224] For caspase in vitro assays, cells were lysed in Triton
X-100 buffer and lysates incubated in 40 .mu.M of the following
amino trifluoromethyl coumarin (ATC)-labeled caspase-specific
peptides (Bachem): xVDVAD (SEQ ID NO: 55) (caspase 2), zDEVD (SEQ
ID NO: 56) (caspases 3/7), xVEID (SEQ ID NO: 57) (caspase 6), xIETD
(SEQ ID NO: 58) (caspase 8), and Ac-LEHD (SEQ ID NO: 59) (caspase
9). Assays were carried out as previously described (Stegh et al.,
2000) and specific activities were determined using a fluorescence
plate reader. Mitochondrial transmembrane potential was measured by
means of the fluorescent dye JC-1 (Molecular Probes, Inc.) as
previously described (Scaffidi et al., 1999). After TNF.alpha.
treatment, cells were incubated with 1.25 .mu.g/ml of the dye for
10 min at 37.degree. C. in the dark, washed once with PBS and
analyzed by FCM.
[0225] 7. Therapeutic Application of the Invention
[0226] The current invention provides methods and compositions for
the modulation of the JNK pathway, and thereby, apoptosis. In one
embodiment of the invention, the modulation can be carried out by
modulation of Gadd45.beta. and other Gadd45 proteins or genes.
Alternatively, therapy may be directed to another component of the
JNK pathway, for example, JNK1, JNK2, JNK3, MAPKKK (Mitogen
Activated Protein Kinase Kinase Kinase): GCK, GCKR, ASK1/MAPKKK5,
ASK2/MAPKKK6, DLK/MUK/ZPK, LZK, MEKK1, MEKK2, MEKK3, MEKK4/MTK1,
MLK1, MLK2/MST, MLK3/SPRK/PTK1, TAK1, Tp1-2/Cot. MAPKK (Mitogen
Activated Protein Kinase Kinase): MKK4/SEK1/SERK1/SKK1/JNKK1,
MKK7/SEK2/SKK4/JNKK2. MAPK (Mitogen Activated Kinase):
JNK1/SAPK.gamma./SAPK1c, JNK2/SAPK.alpha./SAPK1a,
JNK3/SAPK.beta./SAPK1b/p49F12.
[0227] Further, there are numerous phosphatases, scaffold proteins,
including JIP1/IB1, JIP2/IB2, JIP3/JSAP and other activating and
inhibitory cofactors, which are also important in modulating JNK
signaling and may be modulated in accordance with the invention.
Therapeutic uses are suitable for potentially any condition that
can be affected by an increase or decrease in apoptosis. The
invention is significant because many diseases are associated with
an inhibition or increase of apoptosis. Conditions that are
associated with an inhibition of apoptosis include cancer;
autoimmune disorders such as systemic lupus erythemaosus and
immune-mediated glomerulonephritis; and viral infections such as
Herpesviruses, Poxviruses and Adenoviruses. The invention therefore
provides therapies to treat these, and other conditions associated
with the inhibition of apoptosis, which comprise administration of
a JNK pathway modulator that increases apoptosis. As upregulation
of Gadd45 blocks apoptosis, diseases caused by inhibition of
apoptosis will benefit from therapies aimed to increase JNK
activation, for example via inhibition of Gadd45. one example of a
way such inhibition could be achieved is by administration of an
antisense Gadd45 nucleic acid.
[0228] Particular uses for the modulation of apoptosis, and
particularly the increase of apoptosis, are for the treatment of
cancer. In these instances, treatments comprising a combination of
one or more other therapies may be desired. For example, a
modulator of the JNK pathway might be highly beneficial when used
in combination with conventional chemo- or radio-therapies. A wide
variety of cancer therapies, known to one of skill in the art, may
be used individually or in combination with the modulators of the
JNK pathway provided herein. Combination therapy can be used in
order to increase the effectiveness of a therapy using an agent
capable of modulating a gene or protein involved in the JNK
pathway. Such modulators of the JNK pathway may include sense or
antisense nucleic acids.
[0229] One example of a combination therapy is radiation therapy
followed by gene therapy with a nucleic acid sequence of a protein
capable of modulating the JNK pathway, such as a sense or antisense
Gadd45.beta. nucleic acid sequence. Alternatively, one can use the
JNK modulator based anti-cancer therapy in conjunction with surgery
and/or chemotherapy, and/or immunotherapy, and/or other gene
therapy, and/or local heat therapy. Thus, one can use one or
several of the standard cancer therapies existing in the art in
addition with the JNK modulator-based therapies of the present
invention.
[0230] The other cancer therapy may precede or follow a JNK pathway
modulator-based therapy by intervals ranging from minutes to days
to weeks. In embodiments where other cancer therapy and a
Gadd45.beta. inhibitor-based therapy are administered together, one
would generally ensure that a significant period of time did not
expire between the time of each delivery. In such instances, it is
contemplated that one would administer to a patient both modalities
without about 12-24 hours of each other and, more preferably,
within about 6-12 hours of each other, with a delay time of only
about 12 hours being most preferred. In some situations, it may be
desirable to extend the time period for treatment significantly,
however, where several days (2, 3, 4, 5, 6 or 7) to several weeks
(1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective
administrations.
[0231] It also is conceivable that more than one administration of
either another cancer therapy and a Gadd45.beta. inhibitor-based
therapy will be required to achieve complete cancer cure. Various
combinations may be employed, where the other cancer therapy is "A"
and a JNK pathway modulator-based therapy treatment, including
treatment with a Gadd45 inhibitor, is "B", as exemplified
below:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A/ B/AB/A B/A/A/B B/B/B/A A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
[0232] Other combinations also are contemplated. A description of
some common therapeutic agents is provided below.
[0233] 8. Chemotherapeutic Agents
[0234] In the case of cancer treatments, another class of agents
for use in combination therapy are chemotherapeutic agents. These
agents are capable of selectively and deleteriously affecting tumor
cells. Agents that cause DNA damage comprise one type of
chemotherapeutic agents. For example, agents that directly
cross-link DNA, agents that intercalate into DNA, and agents that
lead to chromosomal and mitotic aberrations by affecting nucleic
acid synthesis. Some examples of chemotherapeutic agents include
antibiotic chemotherapeutics such as Doxorubicin, Daunorubucin,
Mitomycin (also known as mutamycin and/or mitomycin-C),
Actinomycine D (Dactinomycine), Bleomycin, Plicomycin. Plant
alkaloids such as Taxol, Vincristine, Vinblastine. Miscellaneous
agents such as Cisplatin, VP16, Tumor Necrosis Factor. Alkylating
Agents such as, Carmustine, Melphalan (also known as alkeran,
L-phenylalanine mustard, phenylalanine mustard, L-PAM, or
L-sarcolysin, is a phenylalanine derivative of nitrogen mustard),
Cyclophosphamide, Chlorambucil, Busulfan (also known as myleran),
Lomustine. And other agents for example, Cisplatin (CDDP),
Carboplatin, Procarbazine, Mechlorethamine, Camptothecin,
Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene,
Estrogen Receptor Binding Agents, Gemcitabien, Mavelbine,
Farnesyl-protein transferase inhibitors, Transplatinum,
5-Fluorouracil, and Methotrexate, Temaxolomide (an aqueous form of
DTIC), or any analog or derivative variant of the foregoing.
[0235] a. Cisplatinum
[0236] Agents that directly cross-link nucleic acids, specifically
DNA, are envisaged to facilitate DNA damage leading to a
synergistic, anti-neoplastic combination with a mutant oncolytic
virus. Cisplatinum agents such as cisplatin, and other DNA
alkylating agents may be used. Cisplatinum has been widely used to
treat cancer, with efficacious doses used in clinical applications
of 20 mg/m.sup.2 for 5 days every three weeks for a total of three
courses. Cisplatin is not absorbed orally and must therefore be
delivered via injection intravenously, subcutaneously,
intratumorally or intraperitoneally.
[0237] b. Daunorubicin
[0238] Daunorubicin hydrochloride, 5,12-Naphthacenedione,
(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)ox-
y]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,
hydrochloride; also termed cerubidine and available from Wyeth.
Daunorubicin intercalates into DNA, blocked DNA-directed RNA
polymerase and inhibits DNA synthesis. It can prevent cell division
in doses that do not interfere with nucleic acid synthesis.
[0239] In combination with other drugs it is included in the
first-choice chemotherapy of acute myelocytic leukemia in adults
(for induction of remission), acute lymphocytic leukemia and the
acute phase of chronic myelocytic leukemia. Oral absorption is
poor, and it must be given intravenously. The half-life of
distribution is 45 minutes and of elimination, about 19 hr. the
half-life of its active metabolite, daunorubicinol, is about 27 hr.
daunorubicin is metabolized mostly in the liver and also secreted
into the bile (ca 40%). Dosage must be reduced in liver or renal
insufficiencies.
[0240] Suitable doses are (base equivalent), intravenous adult,
younger than 60 yr. 45 mg/m.sup.2/day (30 mg/m2 for patients older
than 60 yr.) for 1, 2 or 3 days every 3 or 4 wk or 0.8 mg/kg/day
for 3 to 6 days every 3 or 4 wk; no more than 550 mg/m.sup.2 should
be given in a lifetime, except only 450 mg/m2 if there has been
chest irradiation; children, 25 mg/m.sup.2 once a week unless the
age is less than 2 yr. or the body surface less than 0.5 m, in
which case the weight-based adult schedule is used. It is available
in injectable dosage forms (base equivalent) 20 mg (as the base
equivalent to 21.4 mg of the hydrochloride). Exemplary doses may be
10 mg/m.sup.2, 20 mg/m.sup.2, 30 mg/m.sup.2, 50 mg/m.sup.2, 100
mg/m.sup.2, 150 mg/m.sup.2, 175 mg/m.sup.2, 200 mg/m.sup.2, 225
mg/m.sup.2, 250 mg/m.sup.2, 275 mg/m.sup.2, 300 mg/m.sup.2, 350
mg/m.sup.2, 400 mg/m.sup.2, 425 mg/m.sup.2, 450 mg/m.sup.2, 475
mg/m.sup.2, 500 mg/m.sup.2. Of course, all of these dosages are
exemplary, and any dosage in-between these points is also expected
to be of use in the invention.
[0241] 9. Immunotherapy
[0242] In accordance with the invention, immunotherapy could be
used in combination with a modulator of the JNK pathway in
therapeutic applications. Alternatively, immunotherapy could be
used to modulate apoptosis via the JNK pathway. For example,
anti-Gadd45.beta. antibodies or antibodies to another component of
the JNK pathway could be used to disrupt the function of the target
molecule, thereby inhibiting Gadd45 and increasing apoptosis.
Alternatively, antibodies can be used to target delivery of a
modulator of the JNK pathway to a cell in need thereof. For
example, the immune effector may be an antibody specific for some
marker on the surface of a tumor cell. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associate antigen, fetal antigen, tyrosinse (97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0243] In an embodiment of the invention the antibody may be an
anti-Gadd45.beta. antibody. The antibody alone may serve as an
effector of therapy or it may recruit other cells to actually
effect cell killing. The antibody also may be conjugated to a drug
or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera
toxin, pertussis toxin, etc.) and serve merely as a targeting
agent. Alternatively, the effector may be a lymphocyte carrying a
surface molecule that interacts, either directly or indirectly,
with a target in a tumor cell, for example Gadd45.beta.. Various
effector cells include cytotoxic T cells and NK cells. These
effectors cause cell death and apoptosis. The apoptotic cancer
cells are scavenged by reticuloendothelial cells including
dendritic cells and macrophages and presented to the immune system
to generate anti-tumor immunity (Rovere et al., 1999; Steinman et
al., 1999). Immune stimulating molecules may be provided as immune
therapy: for example, cytokines such as IL-2, IL-4, IL-12, GM-CSF,
gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors
such as FLT ligand. Combining immune stimulating molecules, either
as proteins or using gene delivery in combination with Gadd45
inhibitor will enhance anti-tumor effects. This may comprise: (i)
Passive Immunotherapy which includes: injection of antibodies
alone; injection of antibodies coupled to toxins or
chemotherapeutic agents; injection of antibodies coupled to
radioactive isotopes; injection of anti-idiotype antibodies; and
finally, purging of tumor cells in bone marrow; and/or (ii) Active
Immunotherapy wherein an antigenic peptide, polypeptide or protein,
or an autologous or allogenic tumor cell composition or "vaccine"
is administered, generally with a distinct bacterial adjuvant
(Ravindranath & Morton, 1991; Morton & Ravindranath, 1996;
Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993)
and/or (iii) Adoptive Immunotherapy wherein the patient's
circulating lymphocytes, or tumor infiltrated lymphocyltes, are
isolated in vitro, activated by lymphokines such as IL-2 or
transduced with genes for tumor necrosis, and readministered
(Rosenberg et al., 1998; 1989).
[0244] 10. Gene therapy
[0245] Therapy in accordance with the invention may comprise gene
therapy, in which one or more therapeutic polynucleotide is
administered to a patient in need thereof. This can comprise
administration of a nucleic acid that is a modulator of the JNK
pathway, and may also comprise administration of any other
therapeutic nucleotide in combination with a modulator of the JNK
pathway. One embodiment of cancer therapy in accordance with the
invention comprises administering a nucleic acid sequence that is
an inhibitor of Gadd45.beta., such as a nucleic acid encoding a
Gadd45.beta. inhibitor polypeptide or an antisense Gadd45.beta.
sequence. Delivery of a vector encoding a JNK inhibitor polypeptide
or comprising an antisense JNK pathway modulator in conjunction
with other therapies, including gene therapy, will have a combined
anti-hyperproliferative effect on target tissues. A variety of
proteins are envisioned by the inventors as targets for gene
therapy in conjunction with a modulator of the JNK pathway, some of
which are described below.
[0246] 11. Clinical Protocol
[0247] A clinical protocol has been described herein to facilitate
the treatment of cancer using a modulator of the JNK pathway, such
as an inhibitor of a Gadd45 protein, including the activity or
expression thereof by a Gadd45 gene. The protocol could similarly
be used for other conditions associated with a decrease in
apoptosis. Alternatively, the protocol could be used to assess
treatments associated with increased apoptosis by replacing the
inhibitor of Gadd45 with an activator of Gadd45.
[0248] 12. Therapeutic kits
[0249] Therapeutic kits comprising a modulator of the JNK pathway
are also described herein. Such kits will generally contain, in
suitable container means, a pharmaceutically acceptable formulation
of at least one modulator of the JNK pathway. The kits also may
contain other pharmaceutically acceptable formulations, such as
those containing components to target the modulator of the JNK
pathway to distinct regions of a patient or cell type where
treatment is needed, or any one or more of a range of drugs which
may work in concert with the modulator of the JNK pathway, for
example, chemotherapeutic agents.
[0250] The kits may have a single container means that contains the
modulator of the JNK pathway, with or without any additional
components, or they may have distinct container means for each
desired agent. When the components of the kit are provided in one
or more liquid solutions, the liquid solution is an aqueous
solution, with a sterile aqueous solution being particularly
preferred. However, the components of the kit may be provided as
dried powder(s). When reagents or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent also may be
provided in another container means. The container means of the kit
will generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which the
monoterpene/triterpene glycoside, and any other desired agent, may
be placed and, preferably, suitably aliquoted. Where additional
components are included, the kit will also generally contain a
second vial or other container into which these are placed,
enabling the administration of separated designated doses. The kits
also may comprise a second/third container means for containing a
sterile, pharmaceutically acceptable buffer or other diluent.
[0251] The kits also may contain a means by which to administer the
modulators of the JNK pathway to an animal or patient, e.g., one or
more needles or syringes, or even an eye dropper, pipette, or other
such like apparatus, from which the formulation may be injected
into the animal or applied to a diseased area of the body. The kits
of the present invention will also typically include a means for
containing the vials, or such like, and other component, in close
confinement for commercial sale, such as, e.g., injection or
blow-molded plastic containers into which the desired vials and
other apparatus are placed and retained.
[0252] 13. Gadd45 Compositions
[0253] Certain aspects of the current invention involve modulators
of Gadd45. In one embodiment of the invention, the modulators may
Gadd45 or other genes or proteins. In particular embodiments of the
invention, the inhibitor is an antisense construct. An antisense
construct may comprise a full length coding sequence in antisense
orientation and may also comprise one or more anti-sense
oligonucleotides that may or may not comprise a part of the coding
sequence. Potential modulators of the JNK pathway, including
modulators of Gadd45.beta., may include synthetic peptides, which,
for instance, could be fused to peptides derived from the
Drosophila Antennapedia or HIV TAT proteins to allow free migration
through biological membranes; dominant negative acting mutant
proteins, including constructs encoding these proteins; as well as
natural and synthetic chemical compounds and the like. Modulators
in accordance with the invention may also upregulate Gadd45, for
example, by causing the overexpression of a Gadd45 protein.
Similarly, nucleic acids encoding Gadd45 can be delivered to a
target cell to increase Gadd45. The nucleic acid sequences encoding
Gadd45 may be operably linked to a heterologous promoter that may
cause overexpression of the Gadd45.
[0254] Exemplary Gadd45 gene can be obtained from Genbank Accession
No. NM-015675 for the human cDNA, NP 056490.1 for the human
protein, NM-008655 for the mouse cDNA and NP-032681.1 for the mouse
protein. Similarly, for Gadd45.alpha. nucleotide and protein
sequences the Genbank Accession NOS. are: NM-001924 for the human
cDNA; NP-001915 for the human protein; NM-007836 for the mouse cDNA
and NP-031862.1 for the mouse protein. For Gadd45.gamma. nucleotide
and protein sequences the Genbank Accession Nos. are: NM-006705 for
the human cDNA, NP-006696.1 for the human protein, NM-011817 for
the mouse cDNA and NP-035947.1 for the mouse protein. Also forming
part of the invention are contiguous stretches of nucleic acids,
including about 25, about 50, about 75, about 100, about 150, about
200, about 300, about 400, about 55, about 750, about 100, about
1250 and about 1500 or more contiguous nucleic acids of these
sequences. The binding sites of the Gadd45 promoter sequence,
include the core binding sites of kB-1, kB-2 and kB-3, given by any
of these sequences may be used in the methods and compositions
described herein.
[0255] Further specifically contemplated by the inventors are
arrays comprising any of the foregoing sequences bound to a solid
support. Proteins of Gadd45 and other components of the JNK pathway
may also be used to produce arrays, including portions thereof
comprising about 5, 10, 15, 20, 25, 30, 40, 50, 60 or more
contiguous amino acids of these sequences.
[0256] 14. Ribozymes
[0257] The use of ribozymes specific to a component in the JNK
pathway including Gadd45.beta. specific ribozymes, is also a part
of the invention. The following information is provided in order to
complement the earlier section and to assist those of skill in the
art in this endeavor.
[0258] Ribozymes are RNA-protein complexes that cleave nucleic
acids in the site-specific fashion. Ribozymes have specific
catalytic domains that possess endonuclease activity (Kim and Cech,
1987; Gerlack et al., 1987; Forster and Symons, 1987). For example,
a large number of ribozymes accelerate phosphoester transfer
reactions with a high degree of specificity, often cleaving only
one of several phosphoesters in an oligonucleotide substrate (Cech
et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub,
1992). This specificity has been attributed to the requirement that
the substrate bind via specific base-pairing interactions to the
internal guide sequence ("IGS") of the ribozyme prior to chemical
reaction.
[0259] 15. Proteins
[0260] a. Encoded Proteins
[0261] Protein encoded by the respective gene can be expressed in
any number of different recombinant DNA expression systems to
generate large amounts of the polypeptide product, which can then
be purified and used to vaccinate animals to generate antisera with
which further studies may be conducted. In one embodiment of the
invention, a nucleic acid that inhibits a Gadd45 gene product or
the expression thereof can be inserted into an appropriate
expression system. Such a nucleic acid may encode an inhibitor of
Gadd45, including a dominant negative mutant protein, and may also
comprise an antisense Gad45 nucleic acid. The antisense sequence
may comprise a full length coding sequence in antisense orientation
and may also comprise one or more anti-sense oligonucleotides that
may or may not comprise a part of the coding sequence. Potential
modulators of the JNK pathway, including modulators of
Gadd45.beta., may include synthetic peptides, which, for instance,
could be fused to peptides derived from a Drosophila Antennapedia
or HIV TAT proteins to allow free migration through biological
membranes; dominant negative acting mutant proteins, including
constructs encoding these proteins; as well as natural and
synthetic chemical compounds and the like.
[0262] Examples of other expression systems known to the skilled
practitioner in the art include bacteria such as E. coli, yeast
such as Pichia pastoris, baculovirus, and mammalian expression
fragments of the gene encoding portions of polypeptide can be
produced.
[0263] b. Mimetics
[0264] Another method for the preparation of the polypeptides
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules which mimic elements of protein
secondary structure. See, for example, Johnson et al., "Peptide
Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds.,
Chapman and Hall, New York (1993). The underlying rationale behind
the use of peptide mimetics is that the peptide backbone of
proteins exists chiefly to orient amino acid side chains in such a
way as to facilitate molecular interactions, such as those of
antibody and antigen. A peptide mimic is expected to permit
molecular interactions similar to the natural molecule.
[0265] 16. Pharmaceutical Formulations and Delivery
[0266] In an embodiment of the present invention, a method of
treatment for a cancer by the delivery of an expression construct
comprising a Gadd45 inhibitor nucleic acid is contemplated. A
"Gadd45 inhibitor nucleic acid" may comprise a coding sequence of
an inhibitor of Gadd45, including polypeptides, anti-sense
oligonucleotides and dominant negative mutants. Similarly, other
types of inhibitors, including natural or synthetic chemical and
other types of agents may be administered. The pharmaceutical
formulations may be used to treat any disease associated with
aberrant apoptosis levels.
[0267] An effective amount of the pharmaceutical composition,
generally, is defined as that amount of sufficient to detectably
and repeatedly to ameliorate, reduce, minimize or limit the extent
of the disease or its symptoms. More rigorous definitions may
apply, including elimination, eradication or cure of the
disease.
[0268] 17. Methods of Discovering Modulators of the JNK Pathway
[0269] An aspect of the invention comprises methods of screening
for any one or more properties of Gadd45, including the inhibition
of JNK or apoptosis. The modulators may act at either the protein
level, for example, by inhibiting a polypeptide involved in the JNK
pathway, or may act at the nucleic acid level by modulating the
expression of such a polypeptide. Alternatively, such a modulator
could affect the chemical modification of a molecule in the JNK
pathway, such as the phosphorylation of the molecule. The screening
assays may be both for agents that modulate the JNK pathway to
increase apoptosis as well as those that act to decrease apoptosis.
In screening assays for polypeptide activity, the candidate
substance may first be screened for basic biochemical
activity--e.g., binding to a target molecule and then tested for
its ability to regulate expression, at the cellular, tissue or
whole animal level. The assays may be used to detect levels of
Gadd45 protein or mRNA or to detect levels of protein or nucleic
acids of another participant in the JNK pathway.
[0270] Exemplary procedures for such screening are set forth below.
In all of the methods presented below, the agents to be tested
could be either a library of small molecules (i.e., chemical
compounds), peptides (e.g., phage display), or other types of
molecules.
[0271] a. Screening for Agents that Bind Gadd45.beta. In Vitro
[0272] 96 well plates are coated with the agents to be tested
according to standard procedures. Unbound agent is washed away,
prior to incubating the plates with recombinant Gadd45.beta.
proteins. After, additional washings, binding of Gadd45.beta. to
the plate is assessed by detection of the bound Gadd45, for
example, using anti-Gadd45.beta. antibodies and methodologies
routinely used for immunodetection (e.g. ELISA).
[0273] b. Screening for Agents that Inhibit Binding of Gadd45.beta.
to its Molecular Target in the JNK Pathway
[0274] In certain embodiments, methods of screening and identifying
an agent that modulates the JNK pathway, are disclosed for example,
that inhibits or upregulates Gadd45.beta.. Compounds that inhibit
Gadd45 can effectively block the inhibition of apoptosis, thus
making cells more susceptible to apoptosis. This is typically
achieved by obtaining the target polypeptide, such as a Gadd45
protein, and contacting the protein with candidate agents followed
by assays for any change in activity.
[0275] Candidate compounds can include fragments or parts of
naturally-occurring compounds or may be only found as active
combinations of known compounds which are otherwise inactive. In a
preferred embodiment, the candidate compounds are small molecules.
Alternatively, it is proposed that compounds isolated from natural
sources, such as animals, bacteria, fungi, plant sources, including
leaves and bark, and marine samples may be assayed as candidates
for the presence of potentially useful pharmaceutical agents. It
will be understood that the pharmaceutical agents to be screened
could also be derived or synthesized from chemical compositions or
man-made compounds.
[0276] Recombinant Gadd45.beta. protein is coated onto 96 well
plates and unbound protein is removed by extensive washings. The
agents to be tested are then added to the plates along with
recombinant Gadd45.beta.-interacting protein. Alternatively, agents
are added either before or after the addition of the second
protein. After extensive washing, binding of Gadd45.beta. to the
Gadd45.beta.-interacting protein is assessed, for example, by using
an antibody directed against the latter polypeptide and
methodologies routinely used for immunodetection (ELISA, etc.). In
some cases, it might be preferable to coat plates with recombinant
Gadd45.beta.-interacting protein and assess interaction with
Gadd45.beta. by using an anti-Gadd45.beta. antibody. The goal is to
identify agents that disrupt the association between Gadd45.beta.
and its partner polypeptide.
[0277] C. Screening for Agents that Prevent the Ability of
Gadd45.beta. to Block Apoptosis
[0278] NF-.kappa.B-deficient cell lines expressing high levels of
Gadd45.beta. are protected against TNF.alpha.-induced apoptosis.
Cells (e.g., 3DO-I.kappa.B.alpha.M-Gadd45.beta. clones) are grown
in 96 well plates, exposed to the agents tested, and then treated
with TNF.alpha.. Apoptosis is measured using standard
methodologies, for example, calorimetric MTS assays, PI staining,
etc. Controls are treated with the agents in the absence of
TNF.alpha.. In additional controls, TNF.alpha.-sensitive
NF-.kappa.B-null cells (e.g., 3DO-I.kappa.B.alpha.M cells), as well
as TNF.alpha.-resistant NF-.kappa.B-competent cells (e.g., 3DO-Neo)
are exposed to the agents to be tested in the presence or absence
of TNF.alpha.. The goal is to identify agents that induce apoptosis
in TNF.alpha.-treated 3DO-I.kappa.B.alpha.M-Gadd45.beta., with
animal toxicity in untreated cells and no effect on
TNF.alpha.-induced apoptosis in 3DO-I.kappa.B.alpha.M or 3DO-Neo
cells. Agents that fit these criteria are likely to affect
Gadd45.beta. function, either directly or indirectly.
[0279] d. Screening for Agents that Prevent the Ability of
Gadd45.beta. to Block JNK Activation
[0280] Cell lines, treatments, and agents are as in c. However,
rather than the apoptosis, JNK activation by TNF.alpha. is
assessed. A potential complication of this approach is that it
might require much larger numbers of cells and reagents. Thus, this
type of screening might not be most useful as a secondary screen
for agents isolated, for example, with other methods.
[0281] e. In vitro Assays
[0282] The present embodiment of this invention contemplates the
use of a method for screening and identifying an agent that
modulates the JNK pathway. A quick, inexpensive and easy assay to
run is a binding assay. Binding of a molecule to a target may, in
and of itself, by inhibitory, due to steric, allosteric or
charge-charge interactions. This can be performed in solution or on
a solid phase and can be utilized as a first round screen to
rapidly eliminate certain compounds before moving into more
sophisticated screening assays. The target may be either free in
solution, fixed to a support, express in or on the surface of a
cell. Examples of supports include nitrocellulose, a column or a
gel. Either the target or the compound may be labeled, thereby
permitting determining of binding. In another embodiment, the assay
may measure the enhancement of binding of a target to a natural or
artificial substrate or binding partner. Usually, the target will
be the labeled species, decreasing the chance that the labeling
will interfere with the binding moiety's function. One may measure
the amount of free label versus bound label to determine binding or
inhibition of binding.
[0283] A technique for high throughput screening of compounds is
described in WO 84/03564. In high throughput screening, large
numbers of candidate inhibitory test compounds, which may be small
molecules, natural substrates and ligands, or may be fragments or
structural or functional mimetics thereof, are synthesized on a
solid substrate, such as plastic pins or some other surface.
Alternatively, purified target molecules can be coated directly
onto plates or supports for u se in drug screening techniques.
Also, fusion proteins containing a reactive region (preferably a
terminal region) may be used to link an active region of an enzyme
to a solid phase, or support. The test compounds are reacted with
the target molecule, such as Gadd45.beta., and bound test compound
is detected by various methods (see, e.g., Coligan et al., Current
Protocols in Immunology 1(2): Chapter 5, 1991).
[0284] Examples of small molecules that may be screened including
small organic molecules, peptides and peptide-like molecules,
nucleic acids, polypeptides, peptidomimetics, carbohydrates, lipids
or other organic (carbon-containing) or inorganic molecules. Many
pharmaceutical companies have extensive libraries of chemical
and/or biological mixtures, often fungal, bacterial, or algal
extracts, which can be screened with any of the assays of the
invention to identify compounds that modulate the JNK pathway.
Further, in drug discovery, for example, proteins have been fused
with antibody Fc portions for the purpose of high-throughput
screening assays to identify potential modulators of new
polypeptide targets. See, D. Bennett et al., Journal of Molecular
Recognition, 8: 52-58 (1995) and K. Johanson et al., The Journal of
Biological Chemistry, 270, (16): 9459-9471 (1995).
[0285] In certain embodiments of the invention, assays comprise
binding a Gadd45 protein, coding sequence or promoter nucleic acid
sequence to a support, exposing the Gadd45.beta. to a candidate
inhibitory agent capable of binding the Gadd45.beta. nucleic acid.
The binding can be assayed by any standard means in the art, such
as using radioactivity, immunologic detection, fluorescence, gel
electrophoresis or colorimetry means. Still further, assays may be
carried out using whole cells for inhibitors of Gadd45.beta.
through the identification of compounds capable of initiating a
Gadd45.beta.-dependent blockade of apoptosis (see, e.g., Examples
8-11, below).
[0286] f. In Vivo Assays
[0287] Various transgenic animals, such as mice may be generated
with constructs that permit the use of modulators to regulate the
signaling pathway that lead to apoptosis.
[0288] Treatment of these animals with test compounds will involve
the administration of the compound, in an appropriate form, to the
animal. Administration will be by any route that could be utilized
for clinical or non-clinical purposes including oral, nasal,
buccal, or even topical. Alternatively, administration may be by
intratracheal instillation, bronchial instillation, intradermal,
subcutaneous, intramuscular, intraperitoneal or intravenous
injection. Specifically contemplated are systemic intravenous
injection, regional administration via blood or lymph supply.
[0289] g. In Cyto Assays
[0290] The present invention also contemplates the screening of
compounds for their ability to modulate the JNK pathway in cells.
Various cell lines can be utilized for such screening assays,
including cells specifically engineered for this purpose. Depending
on the assay, culture may be required. The cell is examined using
any of a number of different assays for screening for apoptosis or
JNK activation in cells.
[0291] In particular embodiments of the present invention,
screening may generally include the steps of: [0292] (a) obtaining
a candidate modulator of the JNK pathway, wherein the candidate is
potentially any agent capable of modulating a component of the JNK
pathway, including peptides, mutant proteins, cDNAs, anti-sense
oligonucleotides or constructs, synthetic or natural chemical
compounds, etc.; [0293] (b) admixing the candidate agent with a
cancer cell; [0294] (c) determining the ability of the candidate
substance to modulate the JNK pathway, including either
upregulation or downregulation of the JNK pathway and assaying the
levels up or down regulation.
[0295] The levels up or down regulation will determine the extent
to which apoptosis is occurring in cells and the extent to which
the cells are, for example, receptive to cancer therapy. In order
to detect the levels of modulation, immunodetection assays such as
ELISA may be considered.
[0296] 18. Methods of Assessing Modulators of Apoptotic Pathways
Involving Gadd45.beta. In Vitro and In Vivo
[0297] After suitable modulators of Gadd45.beta. are identified,
these agents may be used in accordance with the invention to
increase or decrease Gadd45.beta. activity either in vitro and/or
in vivo.
[0298] Upon identification of the molecular target(s) of
Gadd45.beta. in the JNK pathway, agents are tested for the
capability of disrupting physical interaction between Gadd45.beta.
and the Gadd45.beta.-interacting protein(s). This can be assessed
by employing methodologies commonly used in the art to detect
protein-protein interactions, including immunoprecipitation, GST
pull-down, yeast or mammalian two-hybrid system, and the like. For
these studies, proteins can be produced with various systems,
including in vitro transcription translation, bacterial or
eukaryotic expression systems, and similar systems.
[0299] Candidate agents are also assessed for their ability to
affect the Gadd45.beta.-dependent inhibition of JNK or apoptosis.
This can be tested by using either cell lines that stably express
Gadd45.beta. (e.g. 3DC-I.kappa.B.alpha.M-Gadd45.beta.) or cell
lines transiently transfected with Gadd45.beta. expression
constructs, such as HeLa, 293, and others. Cells are treated with
the agents and the ability of Gadd45.beta. to inhibit apoptosis or
JNK activation induced by various triggers (e.g., TNF.alpha.)
tested by using standard methodologies. In parallel, control
experiments are performed using cell lines that do not express
Gadd45.beta..
[0300] Transgenic mice expressing Gadd45.beta. or mice injected
with cell lines (e.g., cancer cells) expressing high levels of
Gadd45.beta. are used, either because they naturally express high
levels of Gadd45.beta. or because they have been engineered to do
so (e.g., transfected cells). Animals are then treated with the
agents to be tested and apoptosis and/or JNK activation induced by
various triggers is analyzed using standard methodologies. These
studies will also allow an assessment of the potential toxicity of
these agents.
[0301] 19. Methods of Treating Cancer with Modulators of Apoptotic
Pathways Involving Gadd45.beta.
[0302] This method provides a means for obtaining potentially any
agent capable of inhibiting Gadd45.beta. either by way of
interference with the function of Gadd45.beta. protein, or with the
expression of the protein in cells. Inhibitors may include:
naturally-occurring or synthetic chemical compounds, particularly
those isolated as described herein, anti-sense constructs or
oligonucleotides, Gadd45.beta. mutant proteins (i.e., dominant
negative mutants), mutant or wild type forms of proteins that
interfere with Gadd45.beta. expression or function,
anti-Gadd45.beta. antibodies, cDNAs that encode any of the above
mentioned proteins, ribozymes, synthetic peptides and the like.
[0303] a. In Vitro Methods
[0304] i) Cancer cells expressing high levels of Gadd45.beta., such
as various breast cancer cell lines, are treated with candidate
agent and apoptosis is measured by conventional methods (e.g., MTS
assays, PI staining, caspase activation, etc.). The goal is to
determine whether the inhibition of constitutive Gadd45.beta.
expression or function by these agents is able to induce apoptosis
in cancer cells. ii) In separate studies, concomitantly with the
agents to be tested, cells are treated with TNF.alpha. or the
ligands of other "death receptors" (DR) (e.g., Fas ligand binding
to Fas, or TRAIL binding to both TRAIL-R1 and -R2). The goal of
these studies is to assess whether the inhibition of Gadd45.beta.
renders cancer cells more susceptible to DR-induced apoptosis. iii)
In other studies, cancer cells are treated with agents that inhibit
Gadd45.beta. expression or function in combination with
conventional chemotherapy agents or radiation. DNA damaging agents
are important candidates for these studies. However, any
chemotherapeutic agent could be used. The goal is to determine
whether the inhibition of Gadd45.beta. renders cancer cells more
susceptible to apoptosis induced by chemotherapy or radiation.
[0305] b. In Vivo Methods
[0306] The methods described above are used in animal models. The
agents to be tested are used, for instance, in transgenic mice
expressing Gadd45.beta. or mice injected with tumor cells
expressing high levels of Gadd45.beta., either because they
naturally express high levels of Gadd45.beta. or because they have
been engineered to do so (e.g., transfected cells). Of particular
interest for these studies, are cell lines that can form tumors in
mice. The effects of Gadd45.beta. inhibitors are assessed, either
alone or in conjunction with ligands of DRs (e.g. TNF.alpha. and
TRAIL), chemotherapy agents, or radiation on tumor viability. These
assays also allow determination of potential toxicity of a
particular means of Gadd45.beta. inhibition or combinatorial
therapy in the animal.
[0307] 20. Regulation of the Gadd45.beta. Promoter by
NF-.kappa.B
[0308] .kappa.B binding sites were identified in the gadd45.beta.
promoter. The presence of functional .kappa.B sites in the
gadd45.beta. promoter indicates a direct participation of
NF-.kappa.B complexes in the regulation of Gadd45.beta., thereby
providing an important protective mechanism by NF-.kappa.B.
[0309] 21. Isolation and Analysis of the Gadd45.beta. Promoter
[0310] A BAC clone containing the murine gadd45.beta. gene was
isolated from a 129 SB mouse genomic library (mouse ES I library;
Research Genetics), digested with Xho I, and ligated into the XhoI
site of pBluescript II SK-(pBS; Stratagene). A pBS plasmid
harboring the 7384 bp Xho I fragment of gadd45.beta. (pBS-014D) was
subsequently isolated and completely sequenced by automated
sequencing at the University of Chicago sequencing facility. The
TRANSFAC database (Heinemeyer et al., 1999) was used to identify
putative transcription factor-binding DNA elements, whereas the
BLAST engine (Tatusova et al, 1999) was used for the comparative
analysis with the human promoter.
[0311] 22. Plasmids
[0312] The pMT2T, pMT2T-p50, and pMT2T-RelA expression plasmids
were described previously (Franzoso et al., 1992). To generate the
gadd45.beta.-CAT reporter constructs, portions of the gadd45.beta.
promoter were amplified from pBS-014D by polymerase chain reaction
(PCR) using the following primers:
5'-GGATAACGCGTCACCGTCCTCAAACTTACCAAACGTTTA-3'(SEQ ID NO: 16) and
5'-GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3' (SEQ ID NO: 17)
(-592/+23-gadd45.beta., MluI and EcoRV sites incorporated into
sense and anti-sense primers, respectively, are underlined);
5'-GGATAACGCGTTAGAGCTCTCTGGCTTTTCTAGCTGTC-3' (SEQ ID NO: 18) and
5'-GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3' (SEQ ID NO: 19)
(-265/+23-gadd45.beta.); 5'-GGATAACGCGTAAAGCGCATGCCTCCAGTGGCCACG-3'
(SEQ ID NO: 20) and 5'-GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3'
(SEQ ID NO: 21) (-103/+23-gadd45.beta.);
5'-GGATAACGCGTCACCGTCCTCAAACTTACCAAACGTTTA-3' (SEQ ID NO: 22) and
5'-GGATGGATATCCAAGAGGCAAAAAAACCTTCCCGTGCGA-3' (SEQ ID NO: 23)
(-592/+139-gadd45.beta.);
5'-GGATAACGCGTTAGAGCTCTCTGGCTTTTCTAGCTGTC-3' (SEQ ID NO: 24) and
5'-GGATGGATATCCAAGAGGCAAAAAAACCTTCCCGTGCGA-3' (SEQ ID NO: 25)
(-265/+139-gadd45.beta.). PCR products were digested with MluI and
EcoRV and ligated into the MluI and SmaI sites of the promoterless
pCAT3-Basic vector (Promega) to drive ligated into the MluI and
SmaI sites of the promoterless pCAT2-Basic vector (Promega) to
drive expression of the chloramphenicol acetyl-transferase (CAT)
gene. All inserts were confirmed by sequencing. To generate
-5407/+23-gadd45.beta.-CAT and -3465/+23-gadd45.beta.-CAT, pBS-014D
was digested with XhoI or EcoNI, respectively, subjected to Klenow
filling, and further digested with BssHII. The resulting 5039 bp
XhoI-BssHII and 3097 bp EcoNI-BssH II fragments were then
independently inserted between a filled-in MluI site and the BssHII
site of -592/+23-gadd45.beta.-CAT. The two latter constructs
contained the gadd45.beta. promoter fragment spanning from either
-5407 or -3465 to -368 directly joined to the -38/+23 fragment.
Both reporter plasmids contained intact .kappa.B-1, .kappa.B-2, and
.kappa.B-3 sites (see FIG. 10).
[0313] .kappa.B-1M-gadd45.beta.-CAT, .kappa.B-2M-gadd45.beta.-CAT,
and .kappa.B-3M-gadd45.beta.-CAT were obtained by site-directed
mutagenesis of the -592+23-gadd45.beta.-CAT plasmid using the
QuikChangem.TM. kit (Stratagene) according to the manufacturer's
instructions. The following base substitution were introduced:
5'-TAGGGACTCTCC-3' (SEQ ID NO: 26) to 5'-AATATTCTCTCC-3' (SEQ ID
NO: 27) (.kappa.B-1M-gadd45.beta.-CAT; .kappa.B sites and their
mutated counterparts are underlined; mutated nucleotides are in
bold); 5'-GGGGATTCCA-3' (SEQ ID NO: 28) to 5'-ATCGATTCCA-3' (SEQ ID
NO: 29) (.kappa.B-2M-gadd45.beta.-CAT); and 5'-GGAAACCCCG-3' (SEQ
ID NO: 30) to 5'-GGAAATATTG-3' (SEQ ID NO: 31)
(.kappa.B-3M-gadd45.beta.-CAT). .kappa.B-1/2-gadd45.beta.-CAT,
containing mutated .kappa.B-1 and .kappa.B-2 sites, was derived
from .kappa.B-2M-gadd45.beta.-CAT by site-directed mutagenesis of
.kappa.B-1, as described above. With all constructs, the -592/+23
promoter fragment, including mutated .kappa.B elements, and the
pCAT-3-Basic region spanning from the SmaI cloning site to the end
of the CAT poly-adenylation signal were confirmed by
sequencing.
[0314] .DELTA.56-.kappa.B-1/2-CAT, .DELTA.56-.kappa.B-3-CAT, and
A56-.kappa.B-M-CAT reporter plasmids were constructed by inserting
wild-type or mutated oligonucleotides derived from the mouse
gadd45.beta. promoter into .DELTA.56-CAT between the BglII and XhoI
sites, located immediately upstream of a minimal mouse c-fos
promoter. The oligonucleotides used were:
5'-GATCTCTAGGGACTCTCCGGGGACAGCGAGGGGATTCCAGACC-3' (SEQ ID NO: 32)
(.kappa.B-1/2-CAT; .kappa.B-1 and .kappa.B-2 sites are underlined,
respectively); 5'-GATCTGAATTCGCTGGAAACCCCGCAC-3' (SEQ ID NO: 33)
(.kappa.B-3-CAT; .kappa.B-3 is underlined); and
5'-GATCTGAATTCTACTTACTCTCAAGAC-3' (SEQ ID NO: 34)
(.kappa.B-M-CAT).
[0315] 23. Transfections, CAT Assays, and Electrophoretic Mobility
Shift Assays (EMSAs)
[0316] Calcium phosphate-mediate transient transfection of NTera-2
cells and CAT assays, involving scintillation vial counting, were
performed as reported previously (Franzoso et al., 1992, 1993).
EMSA, supershifting analysis, and antibodies directed against
N-terminal peptides of human p50 and RelA were as described
previously (Franzoso et al., 1992). Whole cell extracts from
transfected NTera-2 cells were prepared by repeated freeze-thawing
in buffer C (20 mM HEPES [pH 7.9], 0.2 mM EDTA; 0.5 mM MgCl.sub.2,
0.5 M NaCl, 25% glycerol, and a cocktail of protease inhibitors
[Boehringer Mannheim]), followed by ultracentrifugation, as
previously described.
[0317] 24. Generation and Treatments of BJAB Clones and Oropidium
Iodide Staining Assays
[0318] To generate stable clones, BJAB cells were transfected with
pcDNA-HA-Gadd45.beta. or empty pcDNA-HA plamids (Invitrogen), and
24 hours later, subjected to selection in G418 (Cellgro; 4 mg/ml).
Resistant clones where expanded and HA-Gadd45.beta. expression was
assessed by Western blotting using anti-HA antibodies or, to
control for loading, anti-.beta.-actin antibodies.
[0319] Clones expressing high levels of HA-Gadd45.beta. and control
HA clones (also referred to as Neo clones) were then seeded in
12-well plates and left untreated or treated with the agonistic
anti-Fas antibody APO-1 (1 .mu.g/ml; Alexis) or recombinant TRAIL
(100 ng/ml; Alexis). At the times indicated, cells were harvested,
washed twice in PBS and incubated overnight at 4.degree. C. in a
solution containing 0.1% Na citrate (pH 7.4), 50 .mu.g/ml propidium
iodide (PI; Sigma), and 0.1% Triton X-100. Cells were then examined
by flow cytometry (FCM) in both the FL-2 and FL-3 channels, and
cells with DNA content lesser than 2N (sub-G1 fraction) were scored
as apoptotic.
[0320] For the protective treatment with the JNK blocker SP600125
(Calbiochem), BJAB cells were left untreated or pretreated for 30
minutes with various concentrations of the blocker, as indicated,
and then incubated for an additional 16 hours with the agonistic
anti-Fas antibody APO-1 (1 .mu.g/ml). Apoptosis was scored in PI
assays as described herein.
[0321] 25. Treatments, Viral Tranduction, and JNK Kinase Assays
with JNK Null Fibroblasts
[0322] JNK null fibroblast--containing the simultaneous deletion of
the jnk1 and jnk2 genes--along with appropriate control
fibroblasts, were obtained from Dr. Roger Davis (University of
Massachusetts). For cytotoxicity experiments, knockout and
wild-type cells were seeded at a density of 10,000 cells/well in
48-well plates, and 24 hours later, treated with TNF.alpha. alone
(1,000 U/ml) or together with increasing concentrations of
cycloheximide (CHX). Apoptosis was monitored after a 8-hour
treatment by using the cell death detection ELISA kit
(Boehringer-Roche) according to the manufacturer's instructions.
Briefly, after lysing the cells directly in the wells, free
nucleosomes in cell lysates were quantified by ELISA using a
biotinylated anti-histone antibody. Experiments were carried out in
triplicate.
[0323] The MIGR1 retroviral vector was obtained from Dr. Harinder
Singh (University of Chicago). MIGR1--JNKK2-JNK1, expressing
constitutively active JNK1, was generated by excising the
HindIII-BglII fragment of JNKK2-JNK1 from pSR.alpha.-JNKK2-JNK1
(obtained from Dr. Anning Lin, University of Chicago), and after
filling-in this fragment by Klenow's reaction, inserting it into
the filled-in XhoI site of MIGR1. High-titer retroviral
preparations were obtained from Phoenix cells that had been
transfected with MIGR1 or MIGR1-JNKK2-JNK1. For viral transduction,
mutant fibroblasts were seeded at 100,000/well in 6-well plates and
incubated overnight with 4 ml viral preparation and 1 ml complete
DMEM medium in 5 .mu.g/ml polybrene. Cells were then washed with
complete medium, and 48 hours later, used for cytotoxic assays.
[0324] For JNK kinase assays, cells were left untreated or treated
with TNF.alpha. (1,000 U/ml) for 10 minutes, and lysates were
prepared in a buffer containing 20 mM HEPES (pH 8.0), 350 mM NaCl,
20% glycerol, 1% NP-40, 1 mM MgCl.sub.2, 0.2 mM EGTA, 1 mM DTT, 1
mM Na.sub.3VO.sub.4, 50 mM NaF, and protease inhibitors. JNK was
immunoprecipitated from cell lysates by using a commercial anti-JNK
antibody (BD Pharmingen) and kinase assays were performed as
described for FIGS. 6 and 7 using GST-c-Jun substrates.
[0325] 26. Treatment of WEHI-231 Cells and Electrophoretic Mobility
Shift Assays
[0326] WEHI-231 cells were cultured in 10% FBS-supplemented RPMI
medium according to the recommendations of the American Type
Culture Collection (ATCC). For electrophoretic mobility shift
assays (EMSAs), cells were treated with 40 .mu.g/ml
lypopolysaccharide (LPS; Escherichia coli serotype 0111:B4), and
harvested at the times indicated. Cell lysates were prepared by
repeated freeze-thawing in buffer C (20 mM HEPES [pH 7.9], 0.2 mM
EDTA, 0.5 mM DTT, 1.5 mM MgCl.sub.2, 0.42 M NaCl, 25% glycerol, and
protease inhibitors) followed by ultracentrifugation. For in vitro
DNA binding assays, 2 .mu.l cell extracts were incubated for 20
minutes with radiolabeled probes derived from each of the three
.kappa.B sites found in the murine gadd45.beta. promoter.
Incubations were carried out in buffer D (20 mM HEPES [pH 7.9], 20%
glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF)
containing 1 .mu.g/ml polydI-dC and 0.1 .mu.g/ml BSA, and
DNA-binding complexes were resolved by polyacrilamide gel
electrophoresis. For supershifts, extracts were pre-incubated for
10 minutes with 1 .mu.l of antibodies reacting with individual
NF-.kappa.B subunits.
[0327] 27. Treatments of BT-20 and MDA-MD-231 Cells
[0328] Breast cancer cell lines were cultured in complete DMEM
medium supplemented with 10% FCS and seeded at 100,000/well in
12-well plates. After 24 hours, cultures were left untreated or
pre-treated for 1 hour with the indicated concentrations of the
SP600125 inhibitor (Calbiochem), after which the NF-.kappa.B
inhibitors prostaglandin A1, CAPE, or parthenolide (Biomol) were
added as shown in FIG. 20. At the indicated times, cell death was
scored morphologically by light microscopy.
[0329] 28. Co-Immunoprecipitations with 293 Cell Lysates
[0330] 293 cells were transfected by the calcium phosphate method
with 15 .mu.g pcDNA-HA plasmids expressing either full-length (FL)
human MEKK1, MEKK3, GCK, GCKR, ASK1, MKK7/JNKK2, and JNK3, or
murine MEKK4 and MKK4/JNKK1 along with 15 .mu.g
pcDNA-FLAG-Gadd45.beta.--expressing FL murine Gadd45.beta.--or
empty pcDNA-FLAG vectors. pcDNA vectors (Invitrogen). 24 hours
after transfection, cells were harvested, and cell lysates were
prepared by resuspending cell pellets in CO-IP buffer (40 mM TRIS
[pH 7.4], 150 mM NaCl, 1% NP-40, 5 mM EGTA, 20 mM NaF, 1 mM
Na.sub.3VO.sub.4, and protease inhibitors) and subjecting them to
ultracentrifugation.
[0331] For co-immunoprecipitations (co-IP), 200 .mu.g cell lysate
were incubated with anti-FLAG(M2)-coated beads (Sigma) in CO-IP
buffer for 4 hours at 4.degree. C. After incubation, beads were
washed 4 times and loaded onto SDS-polyacrylamide gels, and Western
bots were performed by using anti-HA antibodies (Santa Cruz).
[0332] 29. GST Fusion Proteins Constructions and GST Pull-Down
Assays
[0333] Murine Gadd45.beta. and human JNKK2 were cloned into the
EcoRi and BamHI sites of the pGEX-3X and pGEX-2T bacterial
expression vectors (both from Amersham), respectively. These
constructs and the pGEX-3X vector an without insert were introduced
into E. coli BL21 cells in order to express GST-Gadd45.beta.,
GST-JNKK2, and GST proteins. Following induction with 1 mM IPTG,
cells were lysed by sonication in PBS and then precipitated with
glutathione-sepharose beads (Sigma) in the presence of 1% Triton
X-100, and washed 4 times in the same buffer.
[0334] In vitro transcription and translation reactions were
carried out by using the TNT coupled reticulocyte lysate system
(Promega) according to the manufacturer's instructions in the
presence of [.sup.35S]methionine. To prime in vitro reactions,
cDNAs were cloned into the pBluescript (pBS) SK-plasmid
(Stratagene). FL murine MEKK4 was cloned into the SpeI and EcoRI
sites of pBS and was transcribed with the T3 polymerase; FL human
JNKK2, FL murine JNKK1, and FL human ASK1, were cloned into the
XbaI-EcoRI, NotI-EcoRI, and XbaI-ApaI sites of pBS, respectively,
and were transcribed by using the T7 polymerase.
pBS-C-ASK1-encoding amino acids 648-1375 of human ASK1--was derived
from pBS-FL-ASK1 by excision of the Earl and XbaI fragment of ASK1
and insertion of the following oligonucleotide linker:
5'-CGCCACCATGGAGATGGTGAACACCAT-3' (SEQ ID NO: 47). N-ASK1-encoding
the 1-756 amino acid fragment of ASK1--was obtained by priming the
in vitro transcription/translation reaction with pBS-FL-ASK1
digested with PpuMI.
[0335] pBS plasmids expressing N-terminal deletions of human JNKK2
were generated by digestion of pBS-FL-JNKK2 with BamHI and
appropriate restriction enzymes cleaving within the coding sequence
of JNKK2 and replacement of the excised fragments with an
oligonucleotide containing (5' to 3'): a BamHI site, a Kozak
sequence, an initiator ATG, and a nucleotide sequence encoding
between 7 and 13 residues of JNKK2. resulting pBS plasmids encoded
the carboxy-terminal amino acidic portion of JNKK2 that is
indicated in FIG. 28. To generate JNKK2 C-terminal deletions,
pBS-FL-JNKK2 was linearized with SacII, PpuMI, NotI, XcmI, BsgI,
BspEI, BspHI, or PflMI, prior to be used to prime in vitro
transcription/translation reactions. The resulting polypeptide
products contain the amino-terminal amino acidic sequence of JNKK2
that is indicated in FIG. 28.
[0336] To generate Gadd45.beta. polypeptides, in vitro reactions
were primed with pBS-GFP-Gadd45.beta. plasmids, encoding green
fluorescent protein (GFP) directly fused to FL or truncated
Gadd45.beta.. To obtain these plasmids, pBS-Gadd45.beta. (FL),
pBS-Gadd45.beta. (41-160), pBS-Gadd45.beta. (60-160),
pBS-Gadd45.beta. (69-160), pBS-Gadd45.beta. (87-160), and
pBS-Gadd45.beta. (113-160)--encoding the corresponding amino acid
residues of murine Gadd45.beta. were generated--by cloning
appropriate gadd45.beta. cDNA fragments into the XhoI and HindIII
sites of pBS SK--. These plasmids, encoding either FL or truncated
Gadd45.beta., were then opened with KpnI and XhoI, and the excised
DNA fragments were replaced with the KpnI-BsrGI fragment of
pEGFP-N1 (Clontech; containing the GFP-coding sequence) directly
joined to the following oligonucleotide linker:
5'-GTACAAGGGTATGGCTATGTCAATGGGAGGTAG-3' (SEQ ID NO: 48). These
constructs were designated as pBS-GFP-Gadd45.beta.. Gadd45.beta.
C-terminal deletions were obtained as described for the JNKK2
deletions by using pBS-GFP-Gadd45.beta. (FL) that had been digested
with the NgoMI, SphI, or EcoRV restriction enzymes to direct
protein synthesis in vitro. These plasmids encoded the 1-134, 1-95,
and 1-68 amino acid fragments of Gadd45.beta., respectively. All
pBS-Gadd45.beta. constructs were transcribed using the T7
polymerase.
[0337] For GST pull-down experiments, 5 .mu.l of in
vitro-translated and radio-labeled proteins were mixed with
glutathione beads carrying GST, GST-JNKK2 (only with Gadd45.beta.
translation products), or GST-Gadd45.beta. (only with ASK1, MEKK4,
JNKK1, and JNKK2 translation products) and incubated for 1 hour at
room temperature in a buffer containing 20 mM TRIS, 150 mM NaC, and
0.2% Triton X-100. The beads were then precipitated and washed 4
times with the same buffer, and the material was separated by SDS
polyacrylamide gel electrophoresis. Alongside of each pair of GST
and GST-JNKK2 or GST-Gadd45.beta. beads were loaded 2 .mu.l of
crude in vitro transcription/translation reaction (input).
[0338] 30. Kinase Assays
[0339] To test the inhibitory effects of recombinant Gadd45.beta.
proteins on kinase activity, HEK-293 cells were transfected by
using the calcium phosphate method with 1 to 10 .mu.g of
pcDNA-FLAG-JNKK2, pcDNA-FLAG-JNKK1, pcDNA-FLAG-MKK3b or
pcDNA-FLAG-ASK1, and empty pcDNA-FLAG to 30 .mu.g total DNA. 24
hours later, cells were treated for 20 minutes with human
TNF.alpha. (1,000 U/ml) or left untreated, harvested, and then
lysed in a buffer containing 20 mM HEPES (pH 8.0), 350 mM NaCl, 20%
glycerol, 1% NP-40, 1 mM MgCl.sub.2, 0.2 mM EGTA, 1 mM DTT, 1 mM
Na.sub.3VO.sub.4, 50 mM NaF, and protease inhibitors, and subjected
to ultracentrifugation. Immunoprecipitations were performed using
anti-FLAG(M2)-coated beads (Sigma) and 200 .mu.g cell lysates.
After immunoprecipitation, beads were washed twice in lysis buffer
and twice more in kinase buffer. To assay for kinase activity of
immunoprecipitates, beads were pre-incubated for 10 minutes with
increasing amounts of recombinant His.sub.6-Gadd45.beta.,
GST-Gadd45.beta., or control proteins in 30 .mu.l kinase buffer
containing 10 M ATP and 10 .mu.Ci [.sup.32P]{tilde over
(.gamma.)}ATP, and then incubated for 1 additional hour at
30.degree. C. with 1 .mu.g of the appropriate kinase substrate, as
indicated. the following kinase buffers were used: 20 mM HEPES, 20
mM MgCl.sub.2, 20 mM .beta.-glycero-phosphate, 1 mM DTT, and 50
.mu.M Na.sub.3VO.sub.4 for JNKK2; 20 mM HEPES, 10 mM MgCl.sub.2, 20
mM .beta.-glycero-phosphate, and 0.5 mM DTT for JNKK1; 25 mM HEPES,
25 mM MgCl.sub.2, 25 mM .beta.-glycero-phosphate, 0.5 mM DTT, and
50 .mu.M Na.sub.3VO.sub.4 for MKK3; 20 mM Tris HCl, 20 mM
MgCl.sub.2, 20 mM .beta.-glycero-phosphate, 1 mM DTT, and 50 .mu.M
Na.sub.3VO.sub.4 for ASK1.
[0340] To assay activity of endogenous kinases,
immunoprecipitations were performed by using appropriate commercial
antibodies (Santa Cruz) specific for each enzyme and cell lysates
obtained from 3DO-I.kappa.B.alpha.M-Gadd45.beta. and
3DO-I.kappa.B.alpha.M-Hygro clones prior and after stimulation with
TNF.alpha. (1,000 U/ml), as indicated. Kinase assays were performed
as described above, but without pre-incubating immunoprecipitates
with recombinant Gadd45.beta. proteins.
[0341] 31. Cytoprotection Assays in RelA Knockout Cells and
pEGFP-Gadd45.beta. Constructs
[0342] Plasmids expressing N- and C-terminal truncations of murine
Gadd45.beta. were obtained by cloning appropriate gadd45.beta. cDNA
fragments into the XhoI and BamHI sites of pEGFP-N1 (Clontech).
These constructs expressed the indicated amino acids of
Gadd45.beta. directly fused to the N-terminus of GFP. For
cytoprotection assays, GFP-Gadd45.beta.-coding plasmids or empty
pEGFP were transfected into RelA-/- cells by using Superfect
(Qiagen) according to the manufacturer's instructions, and 24 hours
later, cultures were treated with CHX alone (0.1 .mu.g/ml) or CHX
plus TNF.alpha. (1,000 U/ml). After a 12-hour treatment, live cells
adhering to tissue culture plates were counted and examined by FCM
to assess GFP positivity. Percent survival values were calculated
by extrapolating the total number of live GFP.sup.+ cells present
in the cultures that had been treated with CHX plus TNF.alpha.
relative to those treated with CHX alone.
[0343] 32. Plasmids in Example 12.
[0344] pcDNA-HA-GCKR, pCEP-HA-MEKK1, pcDNA-HA-ASK1, pCMV5-HA-MEKK3,
pCMV5-HA-MEKK4, pcDNA-HA-MEK1, pMT3-HA-MKK4, pSR.alpha.-HA-JNK1,
pMT2T-HA-JNK3, pcDNA-HA-ERK1, pSR.alpha.-HA-ERK2,
pcDNA-FLAG-p38.alpha., pcDNA-FLAG-p38.beta., pcDNA-FLAG-p38.gamma.,
and pcDNA-FLAG-p38.delta. were provided by A. Leonardi, H. Ichijo,
J. Landry, R. Vaillancourt, P. Vito, T. H. Wang, J. Wimalasena, and
H. Gram. pcDNA-HA-Gadd45.beta., pGEX-JNK1, pET28-His.sub.6/T7-JIP1
(expressing the MKK7-binding domain of JIP1b), and
pProEx-1.His.sub..quadrature.-EF3 (expressing edema factor 3). All
other FLAG- or HA-coding constructs were generated using pcDNA
(Invitrogen). For bacterial expression, sub-clonings were in the
following vectors: His.sub.6/T7-Gadd45.beta. in pET-28 (Novagen);
His.sub.6-Gadd45.beta. in pProEx-1.H.sub.6.sup.20; GST-p38.alpha.,
GST-MKK7, and GST-Gadd45.beta. in pGEX (Amersham). To prime in
vitro transcription/translations, pBluescript(BS)-MEKK4, pBS-ASK1,
and pBS-MKK7 were generated (FIG. 26); pBS-based plasmids
expressing N-terminal truncations and polypeptidic fragments of
human MKK7. To enhance radio-labeling, the latter peptides were
expressed fused to enhanced green fluorescent protein (eGFP,
Clontech). ASK1.sup.1-757 (encoding amino acids 1-757 of ASK1) and
C-terminal MKK7 truncations were obtained by linearizing pBS-ASK1
and pBS-MKK7, respectively, with appropriate restriction
enzymes.
[0345] 33. Treatments and Apoptosis Assays.
[0346] Treatments were as follows: murine TNF.alpha. (Peprotech),
1,000 U/ml (FIG. 27) or 10 U/ml (FIG. 30); human TNF.alpha.
(Peprotech), 2,000 U/ml; PMA plus ionomycin (Sigma), 100 ng/ml and
1 .mu.M, respectively. In FIG. 30, pre-treatment with HIV-TAT
peptides (5 .mu.M) or DMSO was for 30 minutes and incubation with
TNF.alpha. was for an additional 7 and 3.5 hours, respectively.
Apoptosis was measured by using the Cell Death Detection
ELISA.sup.PLUS kit (Roche).
[0347] 34. Binding Assays, Protein Purification, and Kinase
Assays.
[0348] GST precipitations with in vitro-translated proteins or
purified proteins (FIG. 26-30), and kinase assays were performed.
His.sub.6/T7-Gadd45.beta., His.sub.6/T7-JIP1,
His.sub.6-Gadd45.beta., His.sub.6-EF3 (6.times.His tag disclosed as
SEQ ID NO: 46), and GST proteins were purified from bacterial
lysates as detailed elsewhere, and dialyzed against buffer A.sup.19
(FIG. 28) or 5 mM Na.sup.+ phosphate buffer (pH 7.6; FIG. 28, 30).
Kinase pre-incubation with recombinant proteins was for 10 minutes
(FIG. 28, 30), and GST-Gadd45.beta. pre-incubation with peptides or
DMSO (-) was for an additional 20 minutes (FIG. 30). MKK7
phosphorylation was monitored by performing immunoprecipitations
with anti-P-MKK7 antibodies (developed at Cell Signaling) followed
by Western blots with anti-total MKK7 antibodies. For
co-immunoprecipitations, extracts were prepared in IP buffer.
[0349] 35. Antibodies.
[0350] The anti-MKK7 antibodies were: FIG. 27, kinase assays (goat;
Santa Cruz); FIG. 27, Western blots, and FIG. 3a, top right,
immunoprecipitations (rabbit; Santa Cruz); FIG. 28, top left,
Western blot (mouse monoclonal; BD Pharmingen). Other antibodies
were: anti-FLAG from Sigma; anti-P-MKK4, anti-P-MKK3/6,
anti-P-MEK1/2, anti-total MKK3, and anti-total MEK1/2 from Cell
Signaling; anti-T7 from Novagen; anti-HA, anti-total MKK4,
anti-total ASK1 (kinase assays and Western blots), and anti-total
MEKK1 (kinase assays, Western blots, and co-immunoprecipitations)
from Santa Cruz. There was an anti-Gadd45.beta. monoclonal antibody
(5D2.2).
[0351] 36. Peptide Intracellular Incorporation Assays, Treatments,
and Apoptosis Assays.
[0352] Treatments were as follows: murine TNF.alpha. (Peprotech),
1,000 U/ml, 10 U/ml, or 1,000 U/ml plus 0.3 .mu.g/ml cycloheximide
(CHX; FIG. 33); human TNF.alpha..alpha.(Peprotech), 2,000 U/ml; PMA
plus ionomycin (Sigma), 100 ng/ml and 1 .mu.M, respectively.
Treatments with H.sub.2O.sub.2 and sorbitol were as described
previously. In FIG. 33, pre-treatment with HIV-TAT peptides (5
.mu.M) or DMSO was for 30 minutes and incubation with TNF.alpha.
was for an additional 4 and 3.5 hours, respectively. In FIG. 33,
peptides were used at 10 .mu.M and incubation with
TNF.alpha..alpha. was for 4 hours. Apoptosis was measured by using
the Cell Death Detection ELISA.sup.PLUS kit (Roche). To assess
intracellular incorporation, peptides were labeled with FITC either
at the N-terminus during synthesis or after HPLC purification by
using the FluoReporter FITC protein labeling kit (Molecular
Probes). Cells were then incubated with 5 .mu.M peptides for 20
minutes, subjected to trypsinization, washed three times with PBS,
and examined by FCM or confocal microscopy.
[0353] 37. Generation of Gadd45.beta..sup.-/- Fibroblasts.
[0354] Gadd45.beta. null mice were generated with the help of the
Transgenic and Knockout facility at the University of Chicago by
using standard homologous recombination-based technology in ES
cells. MEFs were isolated from mouse embryos at day 14
post-coitum.
[0355] 38. Methods to Identify Peptide 2-Interacting Factors
[0356] Methods to identify peptide 2-interacting factors include
techniques such as two-hybrid system, phage display, affinity
purification, and GST-pull downs.
[0357] Phage display describes a selection technique in which a
peptide or protein is expressed as a fusion with a coat protein of
a bacteriophage, resulting in display of the fused protein on the
exterior surface of the phage virion, while the DNA encoding the
fusion resides within the virion. Phage display has been used to
create a physical linkage between a vast library of random peptide
sequences to the DNA encoding each sequence, allowing rapid
identification of peptide ligands for a variety of target molecules
(antibodies, enzymes, cell-surface receptors, signal transducers
and the like) by an in vitro selection process called "panning".
Commercially available systems such as Ph.D..TM. Phage Display
Peptide Library Kits (New England Biolabs, MA) can be used.
[0358] Affnity column-based purification systems can also be used
to identify interacting proteins. Commercially available affinity
purification systems such as the Strep-tag.TM. purification system
based on the highly selective binding of engineered streptavidin,
called Strep-Tactin, to Strep-tag II fusion proteins are useful
(IBA GmbH, Germany). This technology allows one-step purification
of recombinant protein under physiological conditions, thus
preserving its bioactivity. The Strep-tag system can be used to
purify functional Strep-tag II proteins from any expression system
including baculovirus, mammalian cells, yeast, and bacteria. Unique
Strep-Tactin affinity columns have been developed for this purpose
and the corresponding operating protocols are described below.
Because of its small size, Strep-tag generally does not interfere
with the bioactivity of the fusion partner.
[0359] The yeast two-hybrid system is a widespread method used to
study protein-protein interactions. In this system, one protein,
the "bait" molecule, is fused to a DNA-binding domain (e.g.,
Escherichia coli LexA protein), and the other partner, the "prey"
molecule, is fused to an activation domain (e.g., yeast GAL4
protein). When these two hybrid proteins interact, a bipartite
transcription factor is reconstituted and can transactivate
reporter genes, such as lacZ (encoding beta-galactosidase) or his3
(encoding imidazole acetol phosphate transaminase enzyme), which
are downstream of DNA-binding sites for the bait protein's
DNA-binding domain. The system is also of great use for detecting
and characterizing new binding partners for a specific protein that
is fused to the DNA-binding domain. This is achieved by screening a
library of cDNAs fused to the sequence of the activation domain. In
a typical screening protocol, the plasmid DNA from each yeast clone
must be isolated in order to identify the cDNA. Commercially
available systems such as Checkmate.TM. Mammalian Two-Hybrid System
(Promega, Madison, Wis.) can be used to identify interacting
factors.
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Sequence CWU 1
1
61125PRTHomo sapiens 1Thr Gly His Val Ile Ala Val Lys Gln Met Arg
Arg Ser Gly Asn Lys1 5 10 15Glu Glu Asn Lys Arg Ile Leu Met Asp 20
25227PRTUnknownDescription of Unknown Organism Gadd45-beta amino
acid sequence 2Ala Ile Asp Glu Glu Glu Glu Asp Asp Ile Ala Leu Gln
Ile His Phe1 5 10 15Thr Leu Ile Gln Ser Phe Cys Cys Asp Asn Asp 20
25318PRTUnknownDescription of Unknown Organism Gadd45-beta amino
acid sequence 3Ile Ala Leu Gln Ile His Phe Thr Leu Ile Gln Ser Phe
Cys Cys Asp1 5 10 15Asn Asp 425PRTHomo sapiens 4Gly Pro Val Trp Lys
Met Arg Phe Arg Lys Thr Gly His Val Ile Ala1 5 10 15Val Lys Gln Met
Arg Arg Ser Gly Asn 20 25515PRTHomo sapiens 5Gly Lys Met Thr Val
Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys1 5 10 15625PRTHomo sapiens
6Gly Ala Ala Ala Ala Met Arg Phe Arg Lys Thr Gly His Val Ile Ala1 5
10 15Val Lys Gln Met Arg Arg Ser Gly Asn 20 25725PRTHomo sapiens
7Gly Pro Val Trp Lys Ala Ala Ala Arg Lys Thr Gly His Val Ile Ala1 5
10 15Val Lys Gln Met Arg Arg Ser Gly Asn 20 25825PRTHomo sapiens
8Gly Pro Val Trp Lys Met Arg Phe Arg Ala Ala Ala Ala Val Ile Ala1 5
10 15Val Lys Gln Met Arg Arg Ser Gly Asn 20 25925PRTHomo sapiens
9Gly Pro Val Trp Lys Met Arg Phe Arg Lys Thr Gly His Ala Ala Ala1 5
10 15Ala Lys Gln Met Arg Arg Ser Gly Asn 20 251025PRTHomo sapiens
10Gly Pro Val Trp Lys Met Arg Phe Arg Lys Thr Gly His Val Ile Ala1
5 10 15Val Ala Ala Ala Ala Arg Ser Gly Asn 20 251125PRTHomo sapiens
11Gly Pro Val Trp Lys Met Arg Phe Arg Lys Thr Gly His Val Ile Ala1
5 10 15Val Lys Gln Met Arg Ala Ala Ala Ala 20 251225PRTHomo sapiens
12Gly Pro Val Trp Lys Met Arg Phe Arg Lys Thr Gly His Val Ala Ile1
5 10 15Val Lys Gln Met Arg Arg Ser Gly Asn 20 251333DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13ctagaggaac gcggaagtgg tggaagtggt gga
331440DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 14gtacaaggga agtggtggaa gtgtggaatg
actttggagg 401522DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 15attgcgtggc caggatacag tt
221639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16ggataacgcg tcaccgtcct caaacttacc aaacgttta
391741DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17ggatggatat ccgaaattaa tccaagaaga cagagatgaa c
411838DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18ggataacgcg ttagagctct ctggcttttc tagctgtc
381941DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19ggatggatat ccgaaattaa tccaagaaga cagagatgaa c
412036DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20ggataacgcg taaagcgcat gcctccagtg gccacg
362141DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 21ggatggatat ccgaaattaa tccaagaaga cagagatgaa c
412239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22ggataacgcg tcaccgtcct caaacttacc aaacgttta
392339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 23ggatggatat ccaagaggca aaaaaacctt cccgtgcga
392438DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24ggataacgcg ttagagctct ctggcttttc tagctgtc
382539DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25ggatggatat ccaagaggca aaaaaacctt cccgtgcga
392612DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26tagggactct cc 122712DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27aatattctct cc 122810DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28ggggattcca 102910DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29atcgattcca 103010DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30ggaaaccccg 103110DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31ggaaatattg 103243DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32gatctctagg gactctccgg ggacagcgag gggattccag acc
433327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 33gatctgaatt cgctggaaac cccgcac
273427DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34gatctgaatt ctacttactc tcaagac
27352695DNAMus musculus 35ggcctctggg attttggttg tgttttaatc
attccttttg actttctatg tgcattggtg 60ttttgcctgt atgcatgtct gtgtgagggt
gtctggtccc ctgaaattgg agttacggat 120ggttgtgagc tgccatattg
aaccctgttc ctctggaaga gcagctagtg ctcttaatct 180ctgagccatt
tctctgcccc tgctgtttgt tttgctttgt cttgttttgg tttcgtttcg
240ttttggtttt tcgagacagg gtttctctgt gtagccctgg ctgtcctgga
actcactctg 300tagcccaggc tggcctcgaa ctcagaaatt cgcctgcctc
tgcctcccaa gtgctgggat 360tgaaggcgtg tgccaccact gcctggcaac
aaccagtgtt ctttaaggct gagacatctc 420tctagcccca cccccaggtt
taaaacaggg tctcatttag cccaggctag tctcaaactc 480actacatagc
cctggatgat cctgacctac tgactgatct tccggtctct tccttcctag
540ggctgggatg acaaatgtgt accaccatag ggttcgtgtg gtacaggggt
ggaaaacagc 600gcctcacaca tgctcagtac gtgctctgcc attgaaccat
tgctacagtc cagcagccaa 660tttagactat taaaatacac atctagtaaa
gtttacttat ttgtgtgtga ggacacagta 720cactttggag taggtacgga
gatcagaaga caattcgcag gagtcagctc gaaccctcca 780tcctgtggag
gatgtcttgc ccttcatgtt tgatatttaa aatactgtat gtatagatta
840ttccaggttg ggctatagcg gtatgtagat attggtgatg agcttgctag
gcatcacgaa 900gtcctggatt catcaccagc atcgaaaaaa aaattaataa
aaaaaaaatc gctgggcagt 960ggtggcccac gcctttaatc ccagcaagca
ctagggaggc agaggcaggc ggatctcttg 1020agttcgaggc cagcctggtc
tacagagtga gttccaggac agtcagggct atacagagaa 1080atctgtctca
aaaaaaaaaa aaaaaaaaaa atcattccaa gtgttctctc cccctccctt
1140tccggaagct gcgtgagcag agacctcatg aggccaccag gtgtcgccgc
cgcgcctctc 1200acgccaggga catttcgcat gctgggtggg tggcgcggag
gaagcaggat gcgtcaccag 1260acccgggatc gggggatccg gggatccggg
gaaccgagcc gcgcggccga ggccaggacc 1320caggctggcg gaggaggcga
ctcagggtga ttcaccggga gcccccgtgc accgtgggag 1380aatcccacgc
gggtctatct gcctcgctcg tgtccttgct gtcgactacc agccctcaag
1440ctgtggcttg gaacgccctt ggaagcctca gtttccattt tgcataatgc
agatatcaat 1500tcctttgcct gacaaatctt ggaaagataa atgacacgcg
tggaagaagg ggcttgtgct 1560tcatgctacg cactacaaaa atgccaggga
cataagagcg gctgcctttc agtcacctct 1620ccccgggtca gtacccttcg
ggttttgcca cttggcttcc ccctcagggg ttaagtgtgg 1680cgaatcgatc
tgaggataga cggtgaggca gccggcaggg ggcagggtca ctccgcagag
1740cgtctggagg gctcttcacc tgcgcctccc gtgcacacgt gaaattctcg
gggtgccggg 1800aggagggaga aagggttccg gatctctccc cctgcgatcc
cttagtgctc tgcagccagg 1860acccctgggg caccgccaag ccacctacca
cgaccactag gaagcttcct gtgtgcctct 1920cctcccgcga ccctggcctt
agagggctga gcgttctcaa agcaccttcg tgctggcgat 1980gctagggtgc
cttggtagtt ctcactttgg ggagaggatc ccaccgtcct caaacttacc
2040aaacgtttac tgtataccct agacgttatt taaacactct ccaactctac
aaggccggca 2100gaacacttag taagcctcct ggcgcatgca catcccttct
ttcagagctt gggaaaggct 2160agggactctc cggggacagc gaggggattc
cagacagccc tccccgaaag ttcaggccag 2220cctctcgcgc tggaaacccc
gcgcgcggcc tgcgtagcgc ggctgccggg aaatcaggag 2280agaaacttct
gtggtttttt tttttttttt tttttttttt ttttctctct agagctctct
2340ctctagagct ctctggcttt tctagctgtc gccgctgctg gcgttcacgc
tcctcccagc 2400cctgaccccc acgtggggcc gccggagctc cgagctccgc
cctttccatc tccagccaat 2460ctcagcgcgg gatactcggc cctttgtgca
tctaccaatg ggtggaaagc gcatgcctcc 2520agtggccacg cctccacccg
ggaagtcata taaaccgctc gcagcgcccg cgcgctcact 2580ccgcagcaac
cctgggtctg cgttcatctc tgtcttcttg gattaatttc gagggggatt
2640ttgcaatctt ctttttaccc ctactttttt cttgggaagg gaagtcccac cgcct
26953672PRTMus musculus 36Lys Leu Met Asn Val Asp Pro Asp Ser Val
Val Leu Cys Leu Leu Ala1 5 10 15Ile Asp Glu Glu Glu Glu Asp Asp Ile
Ala Leu Gln Ile His Phe Thr 20 25 30Leu Ile Gln Ser Phe Cys Cys Asp
Asn Asp Ile Asp Ile Val Arg Val 35 40 45Ser Gly Met Gln Arg Leu Ala
Gln Leu Leu Gly Glu Pro Ala Glu Thr 50 55 60Leu Gly Thr Thr Glu Ala
Arg Asp65 703720PRTMus musculus 37Lys Leu Met Asn Val Asp Pro Asp
Ser Val Val Leu Cys Leu Leu Ala1 5 10 15Ile Asp Glu Glu
203820PRTMus musculus 38Leu Leu Ala Ile Asp Glu Glu Glu Glu Asp Asp
Ile Ala Leu Gln Ile1 5 10 15His Phe Thr Leu 203920PRTMus musculus
39Leu Gln Ile His Phe Thr Leu Ile Gln Ser Phe Cys Cys Asp Asn Asp1
5 10 15Ile Asp Ile Val 204020PRTMus musculus 40Asp Asn Asp Ile Asp
Ile Val Arg Val Ser Gly Met Gln Arg Leu Ala1 5 10 15Gln Leu Leu Gly
204120PRTMus musculus 41Arg Leu Ala Gln Leu Leu Gly Glu Pro Ala Glu
Thr Leu Gly Thr Thr1 5 10 15Glu Ala Arg Asp 204220PRTMus musculus
42Asp Asp Ile Ala Leu Gln Ile His Phe Thr Leu Ile Gln Ser Phe Cys1
5 10 15Cys Asp Asn Asp 204320PRTMus musculus 43Ile Asp Glu Glu Glu
Glu Asp Asp Ile Ala Leu Gln Ile His Phe Thr1 5 10 15Leu Ile Gln Ser
204420PRTMus musculus 44Val Leu Cys Leu Leu Ala Ile Asp Glu Glu Glu
Glu Asp Asp Ile Ala1 5 10 15Leu Gln Ile His 204537PRTHomo sapiens
45Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Thr Gly His Val1
5 10 15Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu Glu Asn
Lys 20 25 30Arg Ile Leu Met Asp 35466PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
46His His His His His His1 54727DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 47cgccaccatg
gagatggtga acaccat 274833DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 48gtacaagggt
atggctatgt caatgggagg tag 33491392DNAHomo sapiens 49aattcggcac
gaggtgtttg tctgccggac tgacgggcgg ccgggcggtg cgcggcggcg 60gtggcggcgg
ggaagatggc ggcgtcctcc ctggaacaga agctgtcccg cctggaagca
120aagctgaagc aggagaaccg ggaggcccgg cggaggatcg acctcaacct
ggatatcagc 180ccccagcggc ccaggcccac cctgcagctc ccgctggcca
acgatggggg cagccgctcg 240ccatcctcag agagctcccc gcagcacccc
acgccccccg cccggccccg ccacatgctg 300gggctcccgt caaccctgtt
cacaccccgc agcatggaga gcattgagat tgaccacaag 360ctgcaggaga
tcatgaagca gacgggctac ctgaccatcg ggggccagcg ctaccaggca
420gaaatcaacg acctggagaa cttgggcgag atgggcagcg gcacctgcgg
accggtgtgg 480aagatgcgct tccggaagac cggccacgtc attgccgtta
agcaaatgcg gcgctccggg 540aacaaggagg agaacaagcg catcctcatg
gacctggatg tggtgctgaa gagccacgac 600tgcccctaca tcgtgcagtg
ctttgggacg ttcatcacca acacggacgt cttcatcgcc 660atggagctca
tgggcacctg cgctgagaag ctcaagaagc ggatgcaggg ccccatcccc
720gagcgcattc tgggcaagat gacagtggcg attgtgaagg cgctgtacta
cctgaaggag 780aagcacggtg tcatccaccg cgacgtcaag ccctccaaca
tcctgctgga cgagcggggc 840cagatcaagc tctgcgactt cggcatcagc
ggccgcctgg tggactccaa agccaagacg 900cggagcgccg gctgtgccgc
ctacatggca cccgagcgca ttgacccccc agaccccacc 960aagccggact
atgacatccg ggccgacgta tggagcctgg gcatctcgtt ggtggagctg
1020gcaacaggac agtttcccta caagaactgc aagacggact ttgaggtcct
caccaaagtc 1080ctacaggaag agcccccgct tctgcccgga cacatgggct
tctcggggga cttccagtcc 1140ttcgtcaaag actgccttac taaagatcac
aggaagagac caaagtataa taagctactt 1200gaacacagct tcatcaagcg
ctacgagacg ctggaggtgg acgtggcgtc ctggttcaag 1260gatgtcatgg
cgaagacctg agtcaccgcg gactaacggc gttccttgag ccagccccac
1320cttggcccct tcttcaggtt agcttgcttt ggccggcggc caacccctct
ggggggccag 1380ggcattggcc cc 139250401PRTHomo sapiens 50Met Ala Ala
Ser Ser Leu Glu Gln Lys Leu Ser Arg Leu Glu Ala Lys1 5 10 15Leu Lys
Gln Glu Asn Arg Glu Ala Arg Arg Arg Ile Asp Leu Asn Leu 20 25 30Asp
Ile Ser Pro Gln Arg Pro Arg Pro Thr Leu Gln Leu Pro Leu Ala 35 40
45Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro Gln His
50 55 60Pro Thr Pro Pro Ala Arg Pro Arg His Met Leu Gly Leu Pro Ser
Thr65 70 75 80Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp
His Lys Leu 85 90 95Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile
Gly Gly Gln Arg 100 105 110Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn
Leu Gly Glu Met Gly Ser 115 120 125Gly Thr Cys Gly Pro Val Trp Lys
Met Arg Phe Arg Lys Thr Gly His 130 135 140Val Ile Ala Val Lys Gln
Met Arg Arg Ser Gly Asn Lys Glu Glu Asn145 150 155 160Lys Arg Ile
Leu Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys 165 170 175Pro
Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr Asp Val 180 185
190Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu Lys Lys
195 200 205Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met
Thr Val 210 215 220Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys
His Gly Val Ile225 230 235 240His Arg Asp Val Lys Pro Ser Asn Ile
Leu Leu Asp Glu Arg Gly Gln 245 250 255Ile Lys Leu Cys Asp Phe Gly
Ile Ser Gly Arg Leu Val Asp Ser Lys 260 265 270Ala Lys Thr Arg Ser
Ala Gly Cys Ala Ala Tyr Met Ala Pro Glu Arg 275 280 285Ile Asp Pro
Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg Ala Asp 290 295 300Val
Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly Gln Phe305 310
315 320Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys Val
Leu 325 330 335Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe
Ser Gly Asp 340 345 350Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys
Asp His Arg Lys Arg 355 360 365Pro Lys Tyr Asn Lys Leu Leu Glu His
Ser Phe Ile Lys Arg Tyr Glu 370 375 380Thr Leu Glu Val Asp Val Ala
Ser Trp Phe Lys Asp Val Met Ala Lys385 390 395 400Thr512313DNAMus
musculus 51ggttgtcaga ctcaacgcag tgagtctgta aaaggctcta acatgcagga
gcctttgacc 60tcgtgccgaa ttcggcacga gggaggatcg acctcaactt ggatatcagc
ccacagcggc 120ccaggcccac cctgcaactc ccactggcca acgatggggg
cagccgctca ccatcctcag 180agagctcccc acagcaccct acacccccca
cccggccccg ccacatgctg gggctcccat 240caaccttgtt cacaccgcgc
agtatggaga gcatcgagat tgaccagaag ctgcaggaga 300tcatgaagca
gacagggtac ctgactatcg ggggccagcg ttatcaggca gaaatcaatg
360acttggagaa cttgggtgag atgggcagtg gtacctgtgg tcaggtgtgg
aagatgcggt 420tccggaagac aggccacatc attgctgtta agcaaatgcg
gcgctctggg aacaaggaag 480agaataagcg cattttgatg gacctggatg
tagtactcaa gagccatgac tgcccttaca 540tcgttcagtg ctttggcacc
ttcatcacca acacagacgt ctttattgcc atggagctca 600tgggcatatg
tgcagagaag ctgaagaaac gaatgcaggg ccccattcca gagcgaatcc
660tgggcaagat gactgtggcg attgtgaaag cactgtacta tctgaaggag
aagcatggcg 720tcatccatcg cgatgtcaaa ccctccaaca tcctgctaga
tgagcggggc cagatcaagc 780tctgtgactt tggcatcagt ggccgccttg
ttgactccaa agccaaaaca cggagtgctg 840gctgtgctgc ctatatggct
cccgagcgca tcgaccctcc agatcccacc aagcctgact 900atgacatccg
agctgatgtg tggagcctgg gcatctcact ggtggagctg gcaacaggac
960agttccccta taagaactgc aagacggact ttgaggtcct
caccaaagtc ctacaggaag 1020agcccccact cctgcctggt cacatgggct
tctcagggga cttccagtca tttgtcaaag 1080actgccttac taaagatcac
aggaagagac caaagtataa taagctactt gaacacagct 1140tcatcaagca
ctatgagata ctcgaggtgg atgtcgcgtc ctggtttaag gatgtcatgg
1200cgaagaccga ttccccaagg actagtggag tcctgagtca gcaccatctg
cccttcttca 1260ggtagcctca tggcagcggc cagccccgca ggggccccgg
gccacggcca ccgacccccc 1320ccccaacctg gccaacccag ctgcccatca
ggggacctgg ggacctggac gactgccaag 1380gactgaggac agaaagtagg
gggttcccat ccagctctga ctccctgcct accagctgtg 1440gacaaaaggg
catgctggtt cctaatccct cccactctgg ggtcagccag cagtgtgagc
1500cccatcccac cccgacagac actgtgaacg gaagacagca ggccatgagc
agactcgcta 1560tttattcaat cataacctct gggctggggt aacccccagg
ggcagagaga cggcacgagc 1620tcaaaccaac tctgagtatg gaactctcag
gctctctgaa ctctgacctt atctcctgga 1680ctcactcacc aacagtgacc
acttggatct ttaacagacc tcagcacttc cagcacactg 1740ctgttgggag
ccttgcactc actatagtct caaacacaac aacaacaaca acaataataa
1800caacaacaac aacaacaaca acaagctgcc tctggttagc ttactgcatg
cttccctcag 1860ctcttgagta tcgctttctg ggagggttcc tcgaggtccc
tggacggatg acttcccagc 1920atcgttcact gcacttacta tgcactgaca
taatatgcac cacattttgt gattgcaaga 1980tacacatttg tcttaaaatt
tgccacagct gaaacaaagg gtatattaaa ggtataacgt 2040caaagcttgt
accaagcttt ctcactggtc tgtgggggct tcagccggtg cttggaatac
2100tatcaactgg aggaaactgt tcaagtgttc tgtttagacc acactggaca
gaaaacagat 2160acctatgggg tgaggttcct attctcaggg tttgtttgtt
tgtttgtttg tttgtttgtt 2220tttcagtgca aattagagac agttcatgtt
ttcttgcagt tgtttttttc tggggggata 2280attctggctt tgtttatctc
tcgtgccgaa ttc 231352346PRTMus musculus 52Met Leu Gly Leu Pro Ser
Thr Leu Phe Thr Pro Arg Ser Met Glu Ser1 5 10 15Ile Glu Ile Asp Gln
Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr 20 25 30Leu Thr Ile Gly
Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu 35 40 45Asn Leu Gly
Glu Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met 50 55 60Arg Phe
Arg Lys Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg65 70 75
80Ser Gly Asn Lys Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val
85 90 95Val Leu Lys Ser His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly
Thr 100 105 110Phe Ile Thr Asn Thr Asp Val Phe Ile Ala Met Glu Leu
Met Gly Ile 115 120 125Cys Ala Glu Lys Leu Lys Lys Arg Met Gln Gly
Pro Ile Pro Glu Arg 130 135 140Ile Leu Gly Lys Met Thr Val Ala Ile
Val Lys Ala Leu Tyr Tyr Leu145 150 155 160Lys Glu Lys His Gly Val
Ile His Arg Asp Val Lys Pro Ser Asn Ile 165 170 175Leu Leu Asp Glu
Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser 180 185 190Gly Arg
Leu Val Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala 195 200
205Ala Tyr Met Ala Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro
210 215 220Asp Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Ser
Leu Val225 230 235 240Glu Leu Ala Thr Gly Gln Phe Pro Tyr Lys Asn
Cys Lys Thr Asp Phe 245 250 255Glu Val Leu Thr Lys Val Leu Gln Glu
Glu Pro Pro Leu Leu Pro Gly 260 265 270His Met Gly Phe Ser Gly Asp
Phe Gln Ser Phe Val Lys Asp Cys Leu 275 280 285Thr Lys Asp His Arg
Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His 290 295 300Ser Phe Ile
Lys His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp305 310 315
320Phe Lys Asp Val Met Ala Lys Thr Asp Ser Pro Arg Thr Ser Gly Val
325 330 335Leu Ser Gln His His Leu Pro Phe Phe Arg 340
3455310PRTHuman immunodeficiency virus type 1 53Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg1 5 105413PRTArtificial SequenceDescription of
Artificial Sequence Synthetic fusion peptide 54Gly Gly Tyr Ala Arg
Ala Ala Ala Arg Gln Ala Arg Ala1 5 10555PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Val
Asp Val Ala Asp1 5564PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 56Asp Glu Val
Asp1574PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Val Glu Ile Asp1584PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Ile
Glu Thr Asp1594PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Leu Glu His Asp16025PRTHomo sapiens
60Gly Pro Val Trp Lys Ala Ala Ala Ala Lys Thr Gly His Val Ile Ala1
5 10 15Val Lys Gln Met Arg Arg Ser Gly Asn 20 256125PRTHomo sapiens
61Gly Pro Val Trp Lys Met Arg Phe Arg Lys Thr Gly His Val Ile Ala1
5 10 15Val Lys Ala Ala Ala Ala Ser Gly Asn 20 25
* * * * *