U.S. patent application number 13/679439 was filed with the patent office on 2013-06-06 for compositions and methods of inhibiting apoptosis.
This patent application is currently assigned to CASE WESTERN RESERVE UNIVERSITY. The applicant listed for this patent is Case Western Reserve University. Invention is credited to Shigemi Matsuyama.
Application Number | 20130143823 13/679439 |
Document ID | / |
Family ID | 43535273 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130143823 |
Kind Code |
A1 |
Matsuyama; Shigemi |
June 6, 2013 |
Compositions and methods of inhibiting apoptosis
Abstract
A method of inhibiting apoptosis in a cell includes
administering to a cell an effective amount of a cell penetrating
peptide (CPP), wherein the CPP consists of about 5 to about 41
amino acids and is substantially homologous to a portion of the
C-terminal region of IFN.gamma.R2.
Inventors: |
Matsuyama; Shigemi;
(Beachwood, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Case Western Reserve University; |
Cleveland |
OH |
US |
|
|
Assignee: |
CASE WESTERN RESERVE
UNIVERSITY
Cleveland
OH
|
Family ID: |
43535273 |
Appl. No.: |
13/679439 |
Filed: |
November 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12853122 |
Aug 9, 2010 |
8324170 |
|
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13679439 |
|
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61232050 |
Aug 7, 2009 |
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Current U.S.
Class: |
514/18.9 ;
530/300; 530/330 |
Current CPC
Class: |
A61P 9/10 20180101; C07K
2319/01 20130101; C07K 7/06 20130101; A61K 38/08 20130101; A61K
38/00 20130101; C07K 14/7156 20130101; A61P 31/00 20180101 |
Class at
Publication: |
514/18.9 ;
530/300; 530/330 |
International
Class: |
C07K 7/06 20060101
C07K007/06; A61K 38/08 20060101 A61K038/08 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
Nos. PCOCA1037 and R01AG031903 awarded by The National Institutes
of Health. The United States government has certain rights in the
invention.
Claims
1-26. (canceled)
27: A synthetic peptide for inhibiting apoptosis in a cell, the
peptide consisting of about 5 to about 41 amino acids and being at
least 90% homologous to a portion of SEQ ID NO: 1.
28: The peptide of claim 27, upon administration to a cell binding
Bax in the cell and inhibiting Bax mediated apoptosis in the
cell.
29: The peptide of claim 28, the apoptosis inhibiting activity
being independent of the Jak/Stat signal transduction pathway.
30: The peptide of claim 27 comprising PILEA (SEQ ID NO: 2).
31: The peptide of claim 27, consisting of about 5 to about 10
amino acids and including SEQ ID NO:2.
32: The peptide of claim 27, consisting essentially of SEQ ID
NO:2.
33: A cell penetrating peptide (CPP) for inhibiting apoptosis in a
cell consisting of about 5 to about 10 amino acids and including
SEQ ID NO:2.
34: The CPP of claim 33 consisting essentially of SEQ ID NO:2.
35: The CPP of claim 33, consisting of SEQ ID NO: 2.
36: A pharmaceutical composition comprising: a synthetic peptide,
the peptide consisting of about 5 to about 41 amino acids and being
at least 90% homologous to a portion of SEQ ID NO: 1; and a
pharmaceutically acceptable carrier.
37: The pharmaceutical composition of claim 36, the peptide
consisting of about 5 to about 41 amino acids and being homologous
to a portion of SEQ ID NO: 1.
38: The pharmaceutical composition of claim 36, the peptide upon
administration to a cell binding Bax in the cell and inhibiting Bax
mediated apoptosis in the cell, independent of the Jak/Stat signal
transduction pathway.
39: The pharmaceutical composition of claim 36, the peptide
comprising PILEA (SEQ ID NO: 2).
40: The pharmaceutical composition of claim 36, the peptide
consisting of about 5 to about 10 amino acids and including SEQ ID
NO:2.
41: The pharmaceutical composition of claim 36, the peptide
consisting essentially of SEQ ID NO: 2.
42: The pharmaceutical composition of claim 36, further comprising
a Ku70-derived Bax-inhibiting peptide having the formula
X.sup.1PX.sup.2LX.sup.3X.sup.4 (SEQ ID NO:4), wherein X.sup.1 is
selected for amino acids with non-polar side chain, X.sup.2 is
selected for amino acids with non-polar side chain, X.sup.3 is
selected for amino acids with charged polar side chain, X.sup.4 is
selected for amino acids with charged polar side chain, and either
X.sup.1 or X.sup.4 may be absent.
43: The pharmaceutical composition of claim 42; wherein the
synthetic peptide comprises PILEA (SEQ ID NO:2); and the
Ku70-derived Bax-inhibiting peptide is selected from the group
consisting of VPMLKE (SEQ ID NO: 5), VPMLK (SEQ ID NO: 6), PMLKE
(SEQ ID NO: 7), VPTLK (SEQ ID NO: 9), and VPALR (SEQ ID NO: 10).
Description
RELATED APPLICATION
[0001] This application is a Continuation of U.S. application Ser.
No. 12/853,122, filed Aug. 9, 2010, which claims priority from U.S.
Provisional Application No. 61/232,050, filed Aug. 7, 2009, the
subject matter of which are incorporated herein by reference in
their entirety.
BACKGROUND
[0003] The process of programmed cell death or apoptosis has been
shown to be centrally involved in the pathogenesis of the
significant majority of human illnesses and injury states. The
cellular attrition observed in most degenerative conditions is
apoptotic in nature; conversely, a failure of apoptosis has been
proposed to underlie many forms of cancer. The central role of
apoptosis in human disease clearly brings with it clinical promise;
for example, the strong possibility exists that attenuation of
apoptotic death will significantly modulate the severity of
degenerative disorders.
[0004] Abnormal regulation of apoptosis is a cause of several
diseases, including cancer and neurodegenerative disorders among
others. Bax is a 21-kDa member of the conserved Bcl-2 family of
proteins involved in regulating programmed cell death. Bax plays a
key role in the intrinsic pathway of apoptosis. Bcl-2 family
proteins are characterized by the presence of four Bcl-2 homology
(BH) domains. Antiapoptotic members (e.g., Bcl-2, Bcl-XL and Mc1-1)
have all four BH domains (BH1-4). The proapoptotic members are
further divided into multi-domain proteins (e.g., Bax, Bak and Bok)
containing three BH domains (BH 1-3) or BH3-only proteins (e.g.,
Bim, Bid and PUMA, etc.,) containing just the BH-3 domain. The
molecular mechanisms, by which these proteins function and interact
is not fully understood, but their role in apoptosis is
indisputable. Although it has been extensively studied how Bcl-2
family proteins influence each other, it is not well known how
these proteins are regulated by non-Bcl-2 family proteins.
SUMMARY
[0005] The present invention relates to a method of inhibiting
apoptosis in a cell. The method includes administering to the cell
a therapeutically effective amount of cell penetrating peptide
(CPP). The CPP consists of about 5 to about 41 amino acids and is
substantially homologous to a portion of the C-terminal region of
interferon gamma receptor 2 (IFN.gamma.R2). In some embodiments,
the CPP comprises SEQ ID NO:1. In other embodiments the CPP
comprises SEQ ID NO:2. In other embodiments the CPP consists
essentially of SEQ ID NO:2. In still other embodiments the CPP
comprises about 5 to about 10 amino acids and includes SEQ ID
NO:2.
[0006] In one specific embodiment, a Ku70-derived Bax-inhibiting
peptide selected from the group consisting of the VPMLKE (SEQ ID
NO:5), VPMLK (SEQ ID NO:6), PMLKE (SEQ ID NO:7), PMLK (SEQ ID
NO:8), VPTLK (SEQ ID NO:9), and VPALR (SEQ ID NO:10) can be
administered.
[0007] In another embodiment, the Ku70-derived Bax-inhibiting
peptide is of the following formula: X.sup.1PX.sup.2LX.sup.3X.sup.4
(SEQ ID NO: 4), wherein X1 is selected from amino acids with a
non-polar side chain; X.sup.2 is selected from amino acids with a
non-polar side chain; X.sup.3 is selected from amino acids with a
charged polar side chain; X.sup.4 is selected from amino acids with
a charged polar side chain; and either X.sup.1 or X.sup.4 may be
absent, although both may not be absent.
[0008] In some embodiments, the CPP binds to Bax in the cell and
inhibits Bax mediated apoptosis in the cell. In some embodiments,
the CPP inhibits Bax activation. In some embodiments, the CPP is
capable of binding to inactive Bax and N-terminus exposed Bax
molecules. The CPP apoptosis inhibiting activity can be independent
of the Jak/Stat signal transduction pathway. The CPP can also
suppress Bim dependent activation but not apoptosis induced by Bak
expression in the cell.
[0009] The therapeutically effective amount can be an amount of CPP
effective to inhibit apoptosis induced by Bax overexpression in the
cell. In some embodiments, the Bax overexpression in the cell is
induced by cytotoxic stresses elicited from chemo- and
radiotherapy.
[0010] The present invention also relates to a cell penetrating
peptide (CPP) for inhibiting apoptosis in a cell. The CPP consists
of about 5 to about 10 amino acids and includes the amino acids of
SEQ ID NO:2. In some aspects, the CPP includes SEQ ID NO:2. In
other aspects, the CPP consists essentially of SEQ ID NO:2.
[0011] The present invention also relates to a pharmaceutical
composition. The pharmaceutical composition includes a CPP. The CPP
consists of about 5 to about 41 amino acids and is substantially
homologous to a portion of the C-terminal region of IFN.gamma.R2.
The composition also includes a Ku70-derived Bax-inhibiting peptide
of the following formula: X.sup.1PX.sup.2LX.sup.3X.sup.4 (SEQ ID
NO: 4), wherein X.sup.1 is selected from amino acids with a
non-polar side chain; X.sup.2 is selected from amino acids with a
non-polar side chain; X.sup.3 is selected from amino acids with
charged a polar side chain; X.sup.4 is selected from amino acids
with a charged polar side chain; and either X.sup.1 or X.sup.4 may
be absent, although both may not be absent. The pharmaceutical
composition further includes a pharmaceutical carrier.
[0012] In an aspect of the invention, the pharmaceutical
composition includes a CPP that comprises SEQ ID NO:2. The
pharmaceutical composition also includes a Ku70-derived
Bax-inhibiting peptide selected from the group consisting of VPMLKE
(SEQ ID NO:5), VPMLK (SEQ ID NO:6), PMLKE (SEQ ID NO:7), PMLK (SEQ
ID NO:8), VPTLK (SEQ ID NO:9), and VPALR (SEQ ID NO:10) and a
pharmaceutical carrier.
[0013] The present invention also includes a method of treating an
apoptotic disease in a subject. The method includes administering
to the subject a therapeutically effective amount of CPP, wherein
the CPP consists of about 5 to about 41 amino acids and is
substantially homologous to a portion of the C-terminal region of
IFN.gamma.R2. In some embodiments, the CPP comprises about 5 to
about 10 amino acids and includes SEQ ID NO:2. In some embodiments,
the CPP includes SEQ ID NO:2. In other embodiments, the CPP
consists essentially of SEQ ID NO:2. In some embodiments, the
method further includes administering a Ku70-derived Bax-inhibiting
peptide selected from the group consisting of VPMLKE (SEQ ID NO:5),
VPMLK (SEQ ID NO:6), PMLKE (SEQ ID NO:7), PMLK (SEQ ID NO:8), VPTLK
(SEQ ID NO:9), and VPALR (SEQ ID NO:10).
[0014] The therapeutically effective amount of CPP can include an
amount effective to inhibit apoptosis induced by Bax overexpression
in one or more cells in the subject. In some embodiments, the
apoptotic disease includes at least one of ischemic disease,
stroke, myocardial infarction, degenerative disease, and an
infectious agent. In some embodiments, the apoptotic disease is
induced by the administration of one or more anticancer drug(s) or
UV/X-ray irradiation to the subject.
[0015] The present invention also relates to a method of treating a
proliferative disorder in a subject. The method includes
administering to the subject a therapeutically effective amount of
an anti-proliferative agent and a therapeutically effective amount
of a CPP, wherein the CPP consists of about 5 to about 41 amino
acids and is substantially homologous to a portion of the
C-terminal region of IFN.gamma.R2. In some embodiments, the CPP
comprises about 5 to about 10 amino acids and includes SEQ ID NO:2.
In some embodiments, the CPP includes SEQ ID NO:2. In other
embodiments the CPP consists essentially of SEQ ID NO:2.
[0016] The proliferative disorder can include cancer and the
antiproliferative agent can be administered at an amount effective
to treat the cancer. In some embodiments, the therapeutically
effective amount of CPP is an amount effective to mitigate
chemotherapy induced apoptosis of megakaryoctyes of the subject
without substantially inhibiting platelet formation of the
megakaryocytes.
[0017] In certain embodiments the method further includes
administering a Ku70-derived Bax-inhibiting peptide selected from
the group consisting of VPMLKE (SEQ ID NO:5), VPMLK (SEQ ID NO:6),
PMLKE (SEQ ID NO:7), PMLK (SEQ ID NO:8), VPTLK (SEQ ID NO:9), and
VPALR (SEQ ID NO:10).
[0018] The present invention further relates to a method of
preserving cells and organs for transfusions or transplantation.
The method includes storing the cells or organs in a
therapeutically effective amount of CPP, wherein the CPP consists
of about 5 to about 41 amino acids and is substantially homologous
to a portion of the C-terminal region of IFN.gamma.R2. In some
embodiments the CPP comprises about 5 to about 10 amino acids and
includes SEQ ID NO:2. In some embodiments, the CPP includes SEQ ID
NO:2. In other embodiments, the CPP consists essentially of SEQ ID
NO:2.
[0019] In certain embodiments, the method further includes storing
the cells in a Ku70-derived Bax-inhibiting peptide selected from
the group consisting of VPMLKE (SEQ ID NO:5), VPMLK (SEQ ID NO:6),
PMLKE (SEQ ID NO:7), PMLK (SEQ ID NO:8), VPTLK (SEQ ID NO:9), and
VPALR (SEQ ID NO:10).
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 A-B illustrates that IFN.gamma.R2 cytoplasmic domain
inhibits Bax-induced cell death. (A) The C-terminus (amino acids
263-337) of interferon gamma receptor beta chain (IFN.gamma.R2) was
cloned as a Bax suppressor in a yeast-based functional screen for
Bax inhibitors. The image shows rescue of yeast growth from Bax
expression by IFN.gamma.R2 (amino acids 263-337) and Ku70 (served
as a positive control). (B) Schematic representation of
IFN.gamma.R2 wild-type (w.t.) protein and its truncated forms used
in this study.
[0021] FIG. 2 A-E illustrates that IFN.gamma.R2 protects cells from
Bax-induced cell death independent of the Jak2-signaling pathway.
(A) The IFN.gamma.R2C-terminal region (296-337) inhibited apoptosis
induced by Bax overexpression. HEK293 cells were transiently
co-transfected with pcDNA3-h Bax and pEGFP C2-IFN.gamma.R2wild
type, -IFN.gamma.R2.sub.1-295 or -IFN.gamma.R2.sub.296-337, and
apoptosis was determined 24 h after the transfection, as described
in the Materials and Methods section. Each bar represents the mean
of triplicate samples and standard errors, and statistical
significance was determined by an unpaired student t test:
***p<0.001. (B) Plasmid-mediated expression of IFN.gamma.R2
inhibited etoposide-induced apoptosis in IFN.gamma.R2 null cells.
IFN.gamma.R2 null cells were transiently transfected (see Materials
and Methods) with pEGFP-C2-IFN.gamma.R2wild type,
-IFN.gamma.R2.sub.1-295 or -IFN.gamma.R2.sub.296-337. One day after
the transfection, cells were treated with 10 .mu.M etoposide for 24
h, and apoptosis was detected by using Hoechst nuclear staining.
Each bar represents the mean of triplicate samples and standard
errors, and statistical significance was determined by an unpaired
student t test: *p<0.05, and ***p<0.001. (C) Plasmid-mediated
expression of IFN.gamma.R2 inhibited etoposide-induced Caspase 3
activation in IFN.gamma.R2 null cells. IFN.gamma.R2 null cells were
transiently transfected (see Materials and Methods) with
pEGFP-C2-IFN.gamma.R2wild type, -IFN.gamma.R2.sub.1-295 or
-IFN.gamma.R2.sub.296-337. One day after transfection, the cells
were treated with 10 .mu.M etoposide for 24 h. Caspase 3 activity
was measured as described in the Materials and Methods section.
Each bar represents the mean of triplicate samples and standard
errors, and statistical significance was determined by an unpaired
student t test: ***p<0.001. (D and E) Bax inhibition by
IFN.gamma.R2 is independent of Jak2. (D) Jak2 null cells were
transiently transfected with pEGFP-C2-IFN.gamma.R2wild type,
-IFN.gamma.R2.sub.1-295 or -IFN.gamma.R2.sub.296-337. One day after
the transfection, the cells were treated with 10 .mu.M etoposide
for 24 h. Apoptosis was detected by using Hoechst nuclear staining
as described in the Materials and Methods section. Each bar
represents the mean of triplicate samples and standard errors, and
statistical significance was determined by an unpaired student t
test: ***p<0.001. (E) Jak2 null cells were transiently
transfected with pEGFP-C2-IFN.gamma.R2wild type,
-IFN.gamma.R2.sub.1-295 or -IFN.gamma.R2.sub.296-337. One day after
transfection the cells were treated with etoposide 10 .mu.M for 24
h. Caspase 3 activity was measured as described in the Materials
and Methods section. Each bar represents the mean of triplicate
samples and standard errors, and statistical significance was
determined by an unpaired student t test: ***p<0.001.
[0022] FIG. 3 A-B illustrates that IFN.gamma.R2 knock-down
sensitizes HeLa cells to apoptosis. (A) IFN.gamma.R2 shRNA was
expressed in HeLa cells from a lentivirus vector. For control
shRNA, non-IFN.gamma.R2-targeted shRNA (targeting GFP), and empty
vector were used. IFN.gamma.R2 expression levels were analyzed by
western blotting using IFN.gamma.R2 C-20 antibody (Santa Cruz).
Actin was used for a loading control. (B) HeLa cells expressing
shRNA against IFN.gamma.R2 were treated with 10 .mu.M etoposide for
24 h. Apoptosis was detected by Hoechst dye nuclear staining as
described in the Materials and Methods section. Each bar represents
the mean of triplicate samples and standard errors, and statistical
significance was determined by an unpaired student t test:
***p<0.001.
[0023] FIG. 4 A-B illustrates that IFN.gamma.R2 C-terminus inhibits
Bim-induced apoptosis but not Bak-induced apoptosis. (A)
IFN.gamma.R2 inhibited Bim-induced apoptosis in HEK293T cells.
HEK293T cells were transiently co-transfected with pcDNA3-h Bim EL
and pEGFP-C2-IFN.gamma.R2wild type, -IFN.gamma.R2.sub.1-295 or
IFN.gamma.R2.sub.296-337 as described in Materials and Methods.
Apoptosis induction was analyzed 24 h after the transfection by
using Hoechst dye nuclear staining. Each bar represents the mean of
triplicate samples and standard errors, and statistical
significance was determined by an unpaired student t test:
***p<0.001. (B) IFN.gamma.R2 did not inhibit Bak-induced
apoptosis. HEK293 cells were transiently co-transfected (see
Materials and Methods) with pcDNA3-h Bak and
pEGFP-C2-IFN.gamma.R2wild type, -IFN.gamma.R2.sub.1-295 or
-IFN.gamma.R2.sub.296-337, and apoptosis induction was analyzed 24
h after the transfection. Each bar represents the mean of
triplicate samples and standard errors.
[0024] FIG. 5 A-K illustrates that localization of IFN.gamma.R2.
HeLa and HEK293 cells were transfected with pEGFP-C2
IFN.gamma.R2wild type, pEGFP-C2-IFN.gamma.R2.sub.1-295 or
pEGFP-C2-IFN.gamma.R2.sub.296-337. One day after the transfection,
the subcellular locations of the IFN.gamma.R2-EGFP fusion proteins
were determined by fluorescence microscopy. (A and B) HeLa cells
transfected with pEGFP vector. (C and D) HeLa cells transfected
with pEGFP-C2-IFN.gamma.R2wild type. (E and F) HeLa cells
transfected with pEGFP-C2-IFN.gamma.R2.sub.296-337. (G and H) HeLa
cells transfected with pEGFP-C2-IFN.gamma.R21-295. (I) HEK293 cells
transfected with pEGFP-C2-IFN.gamma.R2wild type. (J) Mito-tracker
staining of HE293 cells transfected with pEGFP-C2-IFN.gamma.R2wild
type as in (I). Images are 40.times. magnification. (K) The
percentages of cells showing a punctate pattern of GFP signals
(mitochondrion-like distribution) in each group are presented in K.
Each bar represents the mean of triplicate samples and standard
errors.
[0025] FIG. 6 A-D illustrates a Western blot analysis of
EGFP-tagged IFN.gamma.R2 proteins (A) Expression of IFN.gamma.R2
EGFP fusion proteins in IFN.gamma.R2 null (mutant HT1080) cells
after the transient transfection. Cells were transfected with
pEGFP-C2-IFN.gamma.R2wild type, pEGFP-C2-IFN.gamma.R2.sub.1-295 or
pEGFP-C2-IFN.gamma.R2.sub.296-337. One day after the transfection,
the cells were collected for western blot analysis with anti-EGFP
polyclonal antibody (Abcam). IFN.gamma.R2-GFP fusion proteins were
detected as bands with the expected molecular weights. However,
smaller forms, probably resulting from protease-dependent cleavage,
were also detected (* indicates a cleaved form of IFN.gamma.R2-GFP
fusion proteins). (B) Western blot analysis of IFN.gamma.R2-GFP
fusion proteins in HEK293 cells. Cells were transfected with
pEGFP-C2-IFN.gamma.R2wild type, pEGFP-C2-IFN.gamma.R2.sub.1-295 or
pEGFP-C2-IFN.gamma.R2.sub.296-337. One day after the transfection,
cells were collected for western blot analysis using EGFP
polyclonal antibody (Abcam). Although intact (non-cleaved)
GFP-IFN.gamma.R2.sub.296-337 was detected, intact IFN.gamma.R2wild
type-GFP and IFN.gamma.R2.sub.1-295-GFP were not detected. Instead,
cleaved forms of these proteins were observed (* indicates these
cleaved forms). (C and D) Confirmation of expression of
IFN.gamma.R2-GFP fusion proteins by enrichment of the fusion
proteins using immunoprecipitation. One day after the transfection
of pEGFP-C2-IFN.gamma.R2 wild type (C),
pEGFP-C2-IFN.gamma.R2.sub.1-295 (C), or
pEGFP-C2-IFN.gamma.R2.sub.296-337 (D), HEK293 cells were collected
and immunoprecipitation was performed by using anti-GFP polyclonal
antibody (Abcam). The samples were subjected to western blot
analysis using anti IFN.gamma.R2 detecting the N-terminal portion
of IFN.gamma.R2 as an epitope (IFN.gamma.R2 antibody from
Fitzgerald) (C) or anti-IFN.gamma.R2 detecting the C-terminal 20
amino acids of IFN.gamma.R2 (IFN.gamma.R2 C20 antibody from Santa
Cruz) (D). Intact fusion proteins were detected as bands with the
expected molecular weights (approximately 67 kDa in (C) and 34 kDa
in (D)). In the "Input" lanes of (C), the strong background
staining prevented the detection of both endogenous IFN.gamma.R2
and INF.gamma.R2-GFP fusion proteins. In the "Input" lanes of (D),
endogenous IFN.gamma.R2 as well as fusion protein were detected by
anti-IFN.gamma.R2 antibody. Seven hundred micrograms of total
protein were used in the IP experiments, and 150 .mu.g of total
protein was used for input level.
[0026] FIG. 7 A-E illustrates that the C-terminal portion of
IFN.gamma.R2 (amino acids 296-337) is sufficient to inhibit Bax
activation. HeLa cells were transfected with pEGFP-C2 vector (A),
pEGFP-C2-IFN.gamma.R2.sub.1-295 (B), -IFN.gamma.R2.sub.296-337 (C)
or -IFN.gamma.R2wild type (D) as described in Materials and
Methods. After 24 h of transfection, the cells were treated with
staurosporine 100 nM for 3 h. Then, the cells were washed with
phosphate buffer pH 7.4 (PBS), fixed by paraformaldehyde 1%,
permeabilized with Triton X-100 (0.02%), blocked with goat serum,
and the activation of Bax was analyzed by immunostaining with
anti-Bax 6A7 monoclonal antibody (which recognizes active Bax).
Arrowheads indicate cells both positive for GFP and active Bax (Bax
6A7+ cells). Images are at 20.times. magnification. (E) Percentages
of cells that were stained by Bax 6A7 Ab among GFP positive cells
are shown. Each bar represents the mean of triplicate samples and
standard errors, and statistical significance was determined by an
unpaired student t test: ***p<0.001.
[0027] Fig. A-E 8 illustrates that IFN.gamma.R2 interacts with Bax.
(A and B) Co-immunoprecipitation of endogenous IFN.gamma.R2 and
Bax. HEK293T cells were lysed using CHAPS or NP40 buffer as
described in Materials and Methods. Immunoprecipitation (IP) was
performed in the same buffer (NP40 or CHAPS) with an anti-Bax
polyclonal (A) or an anti-IFN.gamma.R2 mouse monoclonal antibody
(B). (A) HEK293T cell lysates were prepared in CHAPS or NP40
buffer. IP and western blot (WB) were performed with anti-Bax
polyclonal (BD Pharmingen) and anti-IFN.gamma.R2 monoclonal
(Fitzgerald) antibodies, respectively. (B) HEK293T cells were lysed
with NP40 buffer, IP and WB were performed with anti-IFN.gamma.R2
monoclonal (Fitzgerald) and anti-Bax polyclonal (N20),
respectively. (C) Examination of the interaction of purified
recombinant Bax and IFN.gamma.R2 proteins. Protein G sepharose
beads were pre-incubated with pre-immune mouse IgG (IgG) or
anti-Bax monoclonal antibody B9 (Anti-Bax (B9)). Then, recombinant
proteins of human Bax .DELTA.TM (Bax C-terminal transmembrane
truncated human Bax) and IFN.gamma.R2.sub.263-337 tagged with
thioredoxin (rTrx) were incubated with these beads as described in
Materials and Methods. After extensive washing of the beads with
buffer, beads were boiled in Laemmli buffer. Samples were analyzed
by western blot using anti-IFN.gamma.R2 antibody (C20, Santa Cruz)
and anti-Bax antibody (N20, Santa Cruz). (D) Bcl-2 competed with
IFN.gamma.R2 to bind Bax. HEK 293T cells were lysed using NP40
buffer. Co-immunoprecipitation was performed with anti-Bax
polyclonal antibody (BD Biosciences) in the presence of exogenously
added recombinant Bcl-2 (final concentration is indicated in the
figure). Western blot analysis of immunoprecipiated samples was
performed by using anti-Bcl-2 monoclonal antibody (Santa Cruz) or
anti-IFN.gamma.R2 monoclonal antibody (Fitzgerald), or anti-Bax
monoclonal antibody (B9, Santa Cruz). (E) Bax inhibiting peptide
(BIP) derived from Ku70 (VPTLK) did not compete with IFN.gamma.R2
to bind Bax. HEK 293T cells were lysed in CHAPS buffer, and BIP was
added to the cell lysate to examine its effect on the interaction
of Bax and IFN.gamma.R2 as described in Materials and Methods.
Co-immunoprecipitation was performed with anti-IFN.gamma.R2
monoclonal antibody (Fitzgerald), and western blotting was done
using anti-Bax N20 polyclonal antibody (Santa Cruz).
[0028] FIG. 9 A-C illustrates the detection of the C-terminal
fragment of IFN.gamma.R2 in transformed cells. (A) western blot
analysis of IFN.gamma.R2 in DAMI (human megakaryoblast cell line),
HEK293T (SV40 Large T-transformed cell line), and HUVEC
(non-transformed cells). Total cell lysates (150 .mu.g
protein/lane) were analyzed. INF.gamma.R2 null cells were used as a
negative control sample to distinguish specific bands from
nonspecific bands (A, right lane). (B) Enrichment of the C terminal
fragment by immunoprecipitation (IP). Cells (HUVEC, DAMI and
HEK293T cells) were lysed using RIPA buffer. Both IP and WB were
performed with anti-IFN.gamma.R2 (C20, Santa Cruz) polyclonal
antibody. The C-terminal fragment of IFN.gamma.R2 was detected in
DAMI and HEK293T cells, but not in HUVEC. (C) Western blot analysis
of IFN.gamma.R2 in various human cell types: human normal mammary
epithelial cell line (4A100), non-tumorigenic immortalized breast
cell line (HME1), tumorigenic human breast cancer cell line (MDAMD
468), non-tumorigenic (RWPE1) and tumorigenic (RWPE2) human
prostate cell lines, and tumorigenic prostate cancer cell lines
(LNCap and PC3). INF.gamma.R2 null cells were used as a negative
control to distinguish specific bands from non-specific bands
detected with the IFN.gamma.R2 C20 antibody.
[0029] FIG. 10 A-B illustrates the measurement of cellular
concentrations of IFN.gamma.R2 and Bax. Purified recombinant
proteins of rTrx-IFN.gamma.R2.sub.263-337 and Bax (ATM) were used
as standards. DAMI cells were lysed in hypotonic buffer
supplemented with protease inhibitors. Each subcellular fraction
from equivalent cell numbers, and sequential dilutions of protein
standards were subjected to SDS-PAGE, and transferred to
nitrocellulose membrane. Bax-N20-HRP antibody (Santa Cruz) and
IFN.gamma.R2 C-20 antibody (Santa Cruz) were used to detect Bax (A)
and IFN.gamma.R2 (B), respectively. Signal intensities were
analyzed by using BioRad Gel Doc and Quantity One 4.5.1 software
from BioRad. LDH, F1.alpha. and YY1 were used as cytosolic,
mitochondrial and nuclear markers, respectively.
DETAILED DESCRIPTION
[0030] The present invention generally relates to a method of
inhibiting or mitigating apoptosis in a cell and particularly
relates to method of inhibiting or mitigating apoptosis in cells of
tissue using a Bax inhibitor. It was found that the cytoplasmic
domain (or C-terminal domain) of the interferon gamma receptor beta
chain (IFN.gamma.R2) acts as a novel Bax suppressor in mammalian
cells. Bax is a well-known pro-apoptotic protein that mediates
intrinsic cell-death signaling. The C-terminal domain of
IFN.gamma.R2 can be used to design cell penetrating peptides (CPPs)
that can inhibit the activation of Bax in human cell lines, thereby
inhibiting Bax mediated apoptosis of the cells. CPPs in accordance
with one aspect of the present invention that can inhibit
activation of Bax in human cells can have an amino acid sequence
substantially homologous to a portion of the C-terminal region 5 to
41 amino acid sequence (SEQ ID NO:1) of the C-terminus of
IFN.gamma.R2 (IFN.gamma.R2.sub.296-337).
[0031] CPPs derived from or based on the amino acid sequence of the
C-terminus of IFN.gamma.R2 do not contain the Jak2-binding domain;
and therefore, the anti-apoptotic function of IFN.gamma.R2 is
independent of JAK/STAT signaling. CPPs derived from or based on
the C-terminus of IFN.gamma.R2 can also rescue human cells from
apoptosis induced by overexpression of Bax but not Bak. By way of
example, CPPs derived from or based on the amino acid sequence of
the C-terminus of IFN.gamma.R2 can rescue Bim induced apoptosis
(Bax mediated) (FIG. 4A), but not rescue cells from apoptosis
induced by Bak mediated apoptosis (FIG. 4B).
[0032] CPPs derived from or based on the amino acid sequence of the
C-terminus of IFN.gamma.R2 can bind to both inactive Bax and active
N-terminus exposed Bax molecules. Bax activation involves exposure
of the protein's N-terminus by a conformational change followed by
Bax translocation from the cytosol to mitochondria (Li et al., Cell
Death Differ 2007, Nechushtan et al., EMBO J, 1999; 18:2330-41;
Wolter et al., J Cell Biol 139:1281-92, 1997). The Example below
shows that IFN.gamma.R2 is able to bind to the active exposed
N-terminus Bax molecules and inhibits the conformational change and
mitochondrial translocation of Bax (FIG. 7). The Example below
further illustrates that IFN.gamma.R2 interacts with Bax in a
detergent-free buffer (CHAPS) that maintains the inactive
conformation of Bax (FIG. 8), indicating that the inventive CPPs
bind to and inhibit inactive Bax.
[0033] One aspect of the present invention therefore relates to a
method of inhibiting apoptosis in a cell by administering to the
cell a therapeutically effective amount of cell penetrating peptide
(CPP). The CPP can consist of about 5 to about 41 amino acids and
be substantially homologous to a portion of the amino acid sequence
of the C-terminal region of interferon gamma receptor 2
(IFN.gamma.R2). An "effective amount" or "therapeutically effective
amount" of CPP administered to a cell is the amount of the CPP
effective to mitigate Bax mediated apoptosis in the cell. In some
embodiments, the Bax mediated apoptosis is induced by cytotoxic
stresses elicited from chemo- and radiotherapy delivered to the
cell.
[0034] In one embodiment of the invention, the CPP can have an
amino acid sequence substantially homologous to the amino acid
sequence of SEQ ID NO: 1. By "substantially homologous" to the
amino acid sequence of SEQ ID NO: 1 it is meant that the CPP can be
a fragment, analog or derivative of mammalian SEQ ID NO: 1 that
differs from the amino acid sequence of SEQ ID NO: 1 in one or more
amino acids. The amino sequence of such CPPs can feature a
deletion, addition, or substitution of one or more amino acids of
SEQ ID NO: 1. Amino acid insertions are preferably of about 1 to 4
contiguous amino acids, and deletions are preferably of about 1 to
10 contiguous amino acids. CPP fragments substantially homologous
to the amino acid sequence of SEQ ID NO: 1 can substantially
maintain the CPPs functional activity of inhibiting Bax mediated
apoptosis in cells. In one example, a CPP having an amino acid
sequence substantially homologous to SEQ ID NO:1 can have an amino
acid sequence that is at least about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
or about 99% homologous to the amino acid sequence of SEQ ID NO:
1.
[0035] CPP Bax inhibiting fragments of the amino acid sequence of
SEQ ID NO: 1 can be obtained by screening peptides recombinantly
produced or chemically synthesized and tested for Bax inhibiting
activity. For example, a CPP fragment of a peptide having an amino
acid sequence of SEQ ID NO: 1 may be arbitrarily divided into
fragments of desired length with no overlap of the fragments, or
preferably divided into overlapping fragments of a desired length.
The fragments can be produced and tested to identify those peptidyl
fragments, which can function as Bax inhibiting peptides.
[0036] In some embodiments, the CPP can be a peptide of about 5 to
about 10 amino acids in length and include each of the individual
amino acids of SEQ ID NO:2 (PILEA). It is further contemplated that
each of the individual amino acids of SEQ ID NO:2 can be included
in such a peptide at any position relative to each other. In one
particular embodiment of the invention, a five amino acid peptide
corresponding to SEQ ID NO:2, has cell penetration activity and
upon administration to the cell is sufficient to inhibit Bax
mediated apoptosis.
[0037] The five amino acid peptide of SEQ ID NO:2, as well as
variants and modified peptides (e.g., modified for better membrane
permeablization or longer stability) are also cell penetrating
peptides, and may be used to protect cells from apoptosis in
accordance with the present invention. For example, CPPs of the
present invention may further include peptides with slight
modifications (e.g., substitution of similar charged amino acids or
addition of 1, 2 or 3 innocuous amino acids at either end or by the
addition of an innocuous entity or moiety) to the peptide sequences
described herein. By "innocuous", it is meant that the amino
acid(s) or entities do not substantially reduce the Bax-inhibiting
activity of the core peptide sequence PILEA (SEQ ID NO: 2).
Therefore, in some embodiments, a composition comprising a
Bax-inhibiting peptide of the present invention includes a peptide
described herein (e.g., PILEA (SEQ ID NO: 2)), with additions of 1,
2 or 3 innocuous amino acids at either end and/or innocuous amino
acid substitutions. The present invention can also include peptides
in which sequences described above are repeated multiple times.
[0038] However, peptide delivery and therapeutic administration may
be limited by permeability and selectivity problems involving the
cell membrane (Morris et al., Nat. Biotechnology. 19(12):1173-1176,
2001). Therefore, in some embodiments CPPs can further include the
addition of small protein domains called protein transduction
domains (PTD's) to either end of the peptide. PTD's can aid in
either the transport of CPPs to specific target cells or to aid the
stability of the molecule as they have been shown to cross
biological membranes and act independently from transporters or
specific receptors to promote delivery of peptides into cells (see
Hawiger, Curr. Opin. Chem. Biol. 3(1):88-94, 1999).
[0039] A CPP of the present invention may be administered to a cell
in any manner known in the art, which allows for the delivery of
the CPP inside the cell. In some embodiments, there is no need to
use a delivery tool, such as a liposome to administer a CPP to a
cell given the cell penetrating property of the peptide. One
example of a method of administering a CPP to a cell is to add an
effective amount of CPP directly into culture media to protect
cells from cytotoxic stresses.
[0040] In some embodiments of the invention, an additional membrane
permeable peptide, which inhibits Bax-mediated apoptosis, may be
co-administered to a cell with the inventive CPPs described above.
In some particular embodiments, the co-administered peptide
includes Ku-70 derived Bax-inhibiting peptides (BIPs). These BIPs
are described in U.S. Pat. No. 7,314,866 B2, which is incorporated
herein by reference.
[0041] A BIP for use in the present invention can include VPMLKE
(SEQ ID NO: 5), VPMLK (SEQ ID NO: 6), PMLKE (SEQ ID NO: 7), PMLK
(SEQ ID NO: 8), VPTLK (SEQ ID NO: 9), or VPALR (SEQ ID NO: 10).
Advantageously VPMLK (SEQ ID NO:6), PMLKE (SEQ ID NO:7), PMLK (SEQ
ID NO:8), VPTLK (SEQ ID NO:9), and VPALR (SEQ ID NO:10) are also
cell membrane permeable and do not require a cell delivery system,
such as liposomes.
[0042] In another particular embodiment, a BIP for use in the
methods of the present invention includes a peptide of the general
formula X.sup.1PX.sup.2LX.sup.3X.sup.4 (SEQ ID NO:4), wherein:
[0043] X.sup.1=Amino acids with a non-polar side chain, such as
Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleu-65 cine
(1), Methionine (M), Proline (P), Phenylalanine (F), or Tryptophan
(W).
[0044] X.sup.2=Amino acids with a non-polar side chain, such as
Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (1),
Methionine (M), Proline (P), Phenylalanine (F), Tryptophan (W) or
Threonine (T).
[0045] X.sup.3=Amino acids with a charged polar side chain, such as
Lysine (K), Arginine (R), Histidine (H), Aspartic acid (D),
Glutamic acid (E), and
[0046] X.sup.4=Amino acids with a charged polar side chain, such as
Lysine (K), Arginine (R), Histidine (H), Aspartic acid (D),
Glutamic acid (E).
[0047] Either X.sup.1 or X.sup.4 may be absent.
[0048] The present invention also relates to a pharmaceutical
composition comprising a CPP as described above. A pharmaceutical
composition in accordance with invention can further include a BIP
as described above and/or a suitable pharmaceutical carrier. In one
exemplary embodiment, the pharmaceutical composition includes a
five amino acid long peptide of SEQ ID NO:2, a BIP selected from
the group consisting of the VPMLKE (SEQ ID NO:5), VPMLK (SEQ ID
NO:6), PMLKE (SEQ ID NO:7), PMLK (SEQ ID NO:8), VPTLK (SEQ ID
NO:9), and VPALR (SEQ ID NO:10), and a pharmaceutical carrier.
[0049] In another exemplary embodiment, the pharmaceutical
composition includes a five amino acid long peptide of SEQ ID NO:2,
a BIP having the following formula: X.sup.1PX.sup.2LX.sup.3X.sup.4
(SEQ ID NO: 4), wherein X.sup.1 is selected from amino acids with a
non-polar side chain; X.sup.2 is selected from amino acids with a
non-polar side chain; X.sup.3 is selected from amino acids with a
charged polar side chain; X.sup.4 is selected from amino acids with
a charged polar side chain; and either X.sup.1 or X.sup.4 may be
absent, although both may not be absent, and a pharmaceutical
carrier.
[0050] The pharmaceutical compositions of the present invention can
be administered to a subject by any means that achieve their
intended purpose. For example, administration can be by parenteral,
subcutaneous, intravenous, intraarticular, intrathecal,
intramuscular, intraperitoneal, or intradermal injections, or by
transdermal, buccal, oromucosal, ocular routes or via inhalation.
Alternatively or concurrently, administration can be by the oral
route. The dosage administered will be dependent upon the age,
health, and weight of the patient, kind of concurrent treatment, if
any, frequency of treatment, and the nature of the effect
desired.
[0051] The pharmaceutical compositions of the present invention can
include pharmaceutically acceptable carriers comprising excipients
and auxiliaries that facilitate processing of the active agents
into preparations that can be used pharmaceutically. The
pharmaceutical preparations of the present invention are
manufactured in a manner that is, itself, known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes.
[0052] Formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form, for
example, water-soluble salts and alkaline solutions. Especially
preferred salts are maleate, fumarate, succinate, S,S tartrate, or
R,R tartrate. In addition, suspensions of the active compounds as
appropriate oily injection suspensions can be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for example,
ethyl oleate or triglycerides or polyethylene glycol-400 (the
compounds are soluble in PEG-400). Aqueous injection suspensions
can contain substances that increase the viscosity of the
suspension, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension may also contain
stabilizers.
[0053] It is well known that apoptosis, and particularly Bax
mediated apoptosis, is centrally involved in the pathogenesis of
many human illnesses and injury states. The following references
describe the Bax protein playing a key role in various diseases:
Injury-induced neuron death-Deckwerth, et al. Neuron. 17:401-411,
1996; Martin, et al., J. Compo Neurol. 433:299-311, 2001; Kirkland,
et al., J. Neurosci. 22:6480-90, 2002; Alzheimer disease-MacGibbon,
et al., Brain Res. 750:223-234, 1997; Selznick, et al., J.
Neuropathol. Exp. Neurol. 59:271-279, 2000; Cao, et al., J. Cereb.
Blood Flow Metab. 21:321-333, 2001; Zhang, et al., J. Cell Biol.
156:519-529, 2002; Ischemia induced cell damage-Kaneda, et al.,
Brain Res. 815: 11-20, 1999; Gibson, et al., Mol. Med. 7:644-655,
2001; HIV (AIDS) and Bax: Castedo, et al., J. Exp. Med. 45
194:1097-1110, 2001; Drug-induced neuron death-Dargusch, et al., J.
Neurochem. 76:295-301, 2001; Parkinson's disease-Ploix and Spier,
Trends Neurosci. 24:255, 2001; Huntington's disease-Antonawich, et
al., Brain Res. Bull. 57:647-649, 2002.
[0054] Therefore, in another embodiment, a pharmaceutical
composition of the present invention can be administered to a
subject for the treatment of an apoptotic disease. The method
includes administering a therapeutically effective amount of a
pharmaceutical composition comprising CPP to the subject. The term
"therapeutically effective amount" refers to the amount of an
inventive pharmaceutical composition required to reduce the
severity and/or frequency of symptoms, elimination of symptoms
and/or underlying cause, prevention of the occurrence of symptoms
and/or their underlying cause, and improvement or remediation of
disease. For example, a therapeutically effective amount of a
pharmaceutical composition of the present invention encompasses the
reduction of Bax mediated cell or tissue death in a subject.
[0055] Apoptotic diseases and related disorders as contemplated by
the present invention, can include stroke, heart attack, ischemia,
degenerative diseases (neuron and muscle, e.g., Alzheimer disease,
Parkinson's disease, cardiomyocyte degeneration, etc), macular
degeneration, hypoxia induced apoptosis, ischemia reperfusion
injury, atrophy, infection by parasitic organisms (virus, bacteria,
yeast, or protozoa, etc), side effects of other drugs (e.g.,
anti-cancer drugs), UV/X-ray irradiation, and several other
pathological conditions triggering cell death signals.
[0056] As described above, the compositions described herein can be
used to inhibit Bax mediated cell death wherein Bax overexpression
in the cell is induced by chemo- and radiotherapy. In one exemplary
embodiment, a pharmaceutical composition described above in
accordance with the present invention can protect megakaryocytes
from chemotherapy induced apoptosis without substantially affecting
the ability of megakaryocytes to produce and release platelets.
[0057] It is further contemplated that the pharmaceutical
compositions in accordance with the invention can be used in a
combination therapy or adjunctive therapy with antiproliferative
agents or chemotherapeutic agents for the treatment of
proliferative disorders, such as neoplastic disorders or cancer.
The phrase "combination therapy" embraces the administration of the
pharmaceutical compositions including CPPs and a therapeutic agent
as part of a specific treatment regimen intended to provide a
beneficial effect from the co-action of these therapeutic
agents.
[0058] Administration of these therapeutic agents in combination
typically is carried out over a defined time period (usually
minutes, hours, days or weeks depending upon the combination
selected). "Combination therapy" is intended to embrace
administration of these therapeutic agents in a sequential manner,
that is, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents, in a
substantially simultaneous manner. Substantially simultaneous
administration can be accomplished, for example, by administering
to the subject a single capsule having a fixed ratio of each
therapeutic agent or in multiple, single capsules for each of the
therapeutic agents. Sequential or substantially simultaneous
administration of each therapeutic agent can be effected by any
appropriate route including, but not limited to, oral routes,
intravenous routes, intramuscular routes, and direct absorption
through mucous membrane tissues. The therapeutic agents can be
administered by the same route or by different routes. For example,
a first therapeutic agent of the combination selected may be
administered by intravenous injection while the other therapeutic
agents of the combination may be administered orally.
Alternatively, for example, all therapeutic agents may be
administered orally or all therapeutic agents may be administered
by intravenous injection. The sequence in which the therapeutic
agents are administered is not narrowly critical. "Combination
therapy" also can embrace the administration of the therapeutic
agents as described above in further combination with other
biologically active ingredients (such as, but not limited to, a
second and different therapeutic agent) and non-drug therapies
(such as, but not limited to, surgery or radiation treatment).
Where the combination therapy further comprises radiation
treatment, the radiation treatment may be conducted at any suitable
time so long as a beneficial effect from the co-action of the
combination of the therapeutic agents and radiation treatment is
achieved. For example, in appropriate cases, the beneficial effect
is still achieved when the radiation treatment is temporally
removed from the administration of the therapeutic agents, perhaps
by days or even weeks.
[0059] The phrase "adjunctive therapy" encompasses treatment of a
subject with agents that reduce or avoid side effects associated
with the combination therapy of the present invention, including,
but not limited to, those agents, for example, that reduce the
toxic effect of anticancer drugs, e.g., bone resorption inhibitors,
cardioprotective agents; prevent or reduce the incidence of nausea
and vomiting associated with chemotherapy, radiotherapy or
operation; or reduce the incidence of infection associated with the
administration of myelosuppressive anticancer drugs.
[0060] The apoptotic disease treated by the combination therapy can
include proliferative diseases, such as neoplastic disorders (e.g.,
leukemia) and cancer. Besides being useful for human treatment, the
combination therapy is also useful for veterinary treatment of
companion animals, exotic and farm animals, including rodents,
horses, dogs, and cats.
[0061] In another embodiment of the invention, the therapeutic
agents administered in combination therapy with the inventive CPP
pharmaceutical compositions can comprise at least one
anti-proliferative agent selected from the group consisting of a
chemotherapeutic agent, an antimetabolite, an antitumorgenic agent,
an antimitotic agent, an antiviral agent, an antineoplastic agent,
an immunotherapeutic agent, and a radiotherapeutic agent.
[0062] The phrase "anti-proliferative agent" can include agents
that exert antineoplastic, chmotherapeutic, antiviral, antimitotic,
antitumorgenic, and/or immunotherapeutic effects, e.g., prevent the
development, maturation, or spread of neoplastic cells, directly on
the tumor cell, e.g., by cytostatic or cytocidal effects, and not
indirectly through mechanisms such as biological response
modification. There are large numbers of anti-proliferative agents
available in commercial use, in clinical evaluation and in
pre-clinical development, which could be included in the present
invention by combination drug chemotherapy. For convenience of
discussion, anti-proliferative agents are classified into the
following classes, subtypes and species: ACE inhibitors, alkylating
agents, angiogenesis inhibitors, angiostatin, anthracyclines/DNA
intercalators, anti-cancer antibiotics or antibiotic-type agents,
antimetabolites, antimetastatic compounds, asparaginases,
bisphosphonates, cGMP phosphodiesterase inhibitors, calcium
carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA
topoisomerase, endostatin, epipodophylotoxins, genistein, hormonal
anticancer agents, hydrophilic bile acids (URSO), immunomodulators
or immunological agents, integrin antagonists, interferon
antagonists or agents, MMP inhibitors, miscellaneous antineoplastic
agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine
decarboxylase inhibitors, pBATTs, radio/chemo
sensitizers/protectors, retinoids, selective inhibitors of
proliferation and migration of endotheliai cells, selenium,
stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids.
[0063] The major categories that some anti-proliferative agents
fall into include antimetabolite agents, alkylating agents,
antibiotic-type agents, hormonal anticancer agents, immunological
agents, interferon-type agents, and a category of miscellaneous
antineoplastic agents. Some anti-proliferative agents operate
through multiple or unknown mechanisms and can thus be classified
into more than one category.
[0064] The CPPs in accordance with the present invention allow the
combination therapeutic agents and therapies of the present
invention to be administered at a higher dose, that is, at a dose
higher than has been conventionally used in clinical situations
because of the risk of thrombocytopenia.
[0065] The present invention also provides a method of preserving
tissues and organs for transfusions or transplantation. According
to the present invention, the cells, tissue, or organ can be stored
in and/or contacted with a composition including an effective
amount of CPP. The effective amount of CPP is an amount effective
to mitigate Bax mediated apoptosis of the cells, tissue, or organ
of interest. In some embodiments, a composition for storing cells
or organs can include an effective amount of CPP and an organ
preservation solution. In some embodiments, the composition can
further comprise a Ku70-derived BIP peptide.
[0066] Typically, the tissue or organ has been separated from its
usual nutrient sources, e.g., the blood circulation of a living
animal or person. Organ preservation solutions depend on
contacting, storing and/or perfusing the organ with a supportive
preservation solution designed to provide pH buffering, osmotic
balance and/or some minimal nutritional support, e.g., in the form
of glucose and a limited set of other basic nutrients. This
approach is typically combined with reduction in organ temperature
to just above the freezing point of water. This is intended to
reduce the metabolic rate of organ tissues, thus slowing the
consumption of nutrients and the production of waste products.
Thus, in some embodiments, the CPP containing compositions of the
present invention can be employed at the hypothermic ranges
commonly used in the art, which can range from below 20.degree. C.
to about 4.degree. C. These art-known preservative solutions
include, for example, isotonic saline solutions, that may contain,
in various proportions, salts, sugars, osmotic agents, local
anesthetic, buffers, and other such agents, as described, simply by
way of example, by Berdyaev et al., U.S. Pat. No. 5,432,053; Belzer
et al., described by U.S. Pat. Nos. 4,798,824, 4,879,283; and
4,873,230; Taylor, U.S. Pat. No. 5,405,742; Dohi et al., U.S. Pat.
No. 5,565,317; Stern et al., U.S. Pat. Nos. 5,370,989 and
5,552,267.
[0067] The term, "organ" as used herein encompasses both solid
organs, e.g., kidney, heart, liver, lung, pancreas, as well as
functional parts of organs, e.g., segments of skin, sections of
artery, transplantable lobes of a liver, kidney, lung, and other
organs. The term, "tissue" refers herein to viable cellular
materials in an aggregate form, e.g., small portions of an organ,
as well as dispersed cells, e.g., cells dispersed, isolated and/or
grown from heart muscle, liver or kidney, including bone marrow
cells and progeny cells, blood born stem cells and progeny, and the
various other art-known blood elements, unless otherwise
specified.
[0068] The invention also contemplates using a CPP containing
composition for localized or systemic circulatory or perfusion
support for organs or tissues acutely deprived of normal blood
circulation caused by trauma, e.g., infusions or temporary
circulation of the inventive compositions to support a partially
severed limb, or analogous conditions, until surgical repair of
damaged vasculature is achieved.
[0069] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples,
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. 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
[0070] We show in the following example that interferon gamma
receptor beta chain (IFN.gamma.R2) is a Bax inhibitor not belonging
to the Bcl-2 family of proteins.
[0071] IFN.gamma.R2 is part of the interferon .gamma. (IFN .gamma.)
receptor complex composed of IFN.gamma.R alpha chain (IFN.gamma.R1)
and IFN.gamma.R2. IFN.gamma.R2 interacts with Jak2 prior to
IFN.gamma. binding. Upon IFN.gamma. binding, a conformational
change in the receptor complex occurs, followed by
auto-phosphorylation of Jak kinase, phosphorylalion of
IFN.gamma.R1, and recruitment of STAT1, leading to STAT1
activation. IFN.gamma.R2 is expressed in the plasma membrane,
endoplasmic reticulum (ER) and mitochondria. At present, the
biological significance of the mitochondrial localization of
IFN.gamma.R2 is not known. IFN.gamma.R2 knock-out mice show no
sensitivity to IFN.gamma. and are unable to prevent infection by
Lsteria monocytogenes. Previous studies showed that IFN.gamma.R2
plays a role in apoptosis regulation as a signal-transduction
molecule of IFN.gamma. (reviewed in ref. 19), but to our knowledge,
there is no report describing the apoptosis regulating activity of
IFN.gamma.R2 itself.
[0072] Here we report that the C-terminus of IFN.gamma.R2 has a
Bax-inhibiting activity that is independent of the Jak/STAT signal
transduction pathway. We also found that certain cancer cell lines
(DAMI cells, MDA-MD468 cells and PC3) express a truncated form of
IFN.gamma.R2 containing the C-terminal Bax-inhibitory domain. The
presence of this C-terminal fragment of IFN.gamma.R2 in the cytosol
may help such cancer cells increase their resistance to cytotoxic
stresses, including those elicited by chemo- and radiotherapy.
Yeast-Based Functional Screening for Bax Inhibitors
[0073] The yeast strain EGY48 was used for the yeast functional
screening for Bax inhibitors. Mouse Bax was expressed under a
galactose-inducible promoter using pGilda vector as reported. Yeast
expression libraries of cDNAs of mouse brain and a human cell line
(HeLa) were prepared in pYES2 and pJG4-5 vectors, respectively.
Cell Culture and Transfection Cell Culture
[0074] HT1080 (wild type, IFN.gamma.R2 null mutant and JAK2 null
mutant) cells were cultured in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS). HeLa, Human
Embryonic Kidney (HEK) 293 and HEK293T cells were purchased from
ATCC, and cultured in DMEM supplemented with 10% FBS. LNCap and PC3
cells were purchased from ATCC and cultured in DMEM F12 medium
supplemented with 10% FBS. RWPE1 and RWPE2 were purchased from ATCC
and cultured in keratinocytes-SFM plus supplements medium (Gibco).
Primary mammary epithelial (4A100) cultures were derived from
organoids isolated from discarded mammary tissue acquired from
patients undergoing reduction mammoplasty surgery. Anonymized
specimens were acquired from patients who had given written
consent, through the Tissue Procurement and Histology Core Facility
of the Case Comprehensive Cancer Center (Case CCC), under a Case
CCC IRB approved protocol. Primary epithelial cultures were grown
in M87A+X medium. Human HME1 cells (Clontech) were grown in medium
171 with mammary epithelial growth supplement (Cascade Biologics)
and penicillin-streptomycin.42 Human breast cancer cell line
MDA-MD468 was cultured in RPMI, 5% FBS, supplemented with
L-glutamine, penicillin-streptomycin and fungizone (Gibco). Human
megakaryocytic cell line DAMI cells was cultured in Iscove's
Modified Dulbecco's Medium (IMDM) supplemented with 10% horse
serum. Human umbilical vein endothelial cells (HUVEC) were cultured
in endothelial cell growth medium with supplements
(EGM.RTM.-2-Endothelial Cell Medium-2-Lonza).
Transfection
[0075] Cells were cultured overnight in DMEM supplemented with 10%
FBS. The transfections were performed using SUPERFECT.RTM. (Qiagen,
Valencia, Calif.) in accordance with the manufacturer's
instructions. Transfection efficiency was analyzed by the
expression of the EGFP-tagged proteins.
Lentivirus
[0076] Five E. coli clones expressing pLKO1-shRNA IFN.gamma.R2
plasmids were purchased from Open Biosystems (cat
#RHS4533-NM.sub.--005534). Lentiviruses were produced in HEK293T
cells by transfection using each of pLKO1-shRNA IFN.gamma.R2, pCMV
DR 8.76 and pMD2G. Viruses were produced and used to infect HeLa
cells with a 1:3 dilution of stock lentivirus for 16 h. Cells were
then cultured for 24 h in complete medium, and then stable clones
expressing the shRNA against IFN.gamma.R2 and shRNA against GFP
(control shRNA) were selected using puromycin. To select the best
shRNA targeting IFN.gamma.R2 mRNA, cell lysates were analyzed by
western blotting, and the best cloneshowing the lowest IFN.gamma.R2
protein expression was used to determine the effects of
IFN.gamma.R2 knock-down in HeLa cells.
Apoptosis Detection
[0077] Apoptosis was induced by transfecting the cells with
pcDNA3-human Bax, or pcDNA3-human Bak, or pcDNA3-human Bim EL, or
by treatment with etoposide (10 .mu.M) or staurosporine (100 nM).
To determine the induction of apoptosis by different apoptotic
stresses, cells were stained with Hoechst 33258 dye, and the
numbers of cells with apoptotic nuclei were counted using
fluorescence microscopy. Three hundred cells were analyzed in
triplicate samples. The data presented in the figures showed the
percentage of apoptosis.+-.SEM of three independent experiments.
Caspase activity were measured by using a fluorogenic caspase 3
substrate II (Calbiochem), IFN.gamma.R2 constructs were cloned in
pEGFP-C2 (IFN.gamma.R2wild type, IFN.gamma.R2.sub.1-295 and
IFN.gamma.R2.sub.296-337) vector.
Apoptosis Induction by Overexpression of Bax, Bak or Bim
[0078] For Bax, Bak or Bim EL overexpression, cells were
transfected with either 1 .mu.g pcDNA3-human Bax, 1 .mu.g
pcDNA3-human Bak, or 1 .mu.g pcDNA3 human Bim EL, and 4 .mu.g of
pEGFP plasmid encoding IFN.gamma.R2, and the apoptosis or caspase
activity was determined 24 h after the transfection.
Immunoprecipitations
IFN.gamma.R2-Bax Co-Immunoprecipitation (Co-IP)
[0079] HEK293T cells were lysed in 300 .mu.l NP40 buffer (150 mM
NaCl, 10 mM HEPES at pH 7.4 and 1% NP40) or CHAPS buffer (150 mM
NaCl, 10 mM HEPES at pH 7.4 and 1% CHAPS) supplemented with
protease inhibitors (1:100 dilution of protease inhibitor Cocktail;
Sigma) and PMSF. Samples were pre-cleared by incubating 300-.mu.l
(1,000 mg total protein) cell lysates with 20 .mu.l
protein-G-sepharose (Amersham Biosciences) at 4.degree. C. for 1 h.
Then, the samples were incubated with 20 .mu.l protein-G-sepharose
pre-absorbed with 2 .mu.g of Bax monoclonal antibody (B9, Santa
Cruz) or IFN.gamma.R2 monoclonal antibody (Fitzgerald) at 4.degree.
C. for 2 h. After the incubation, sepharose beads were washed with
lysis buffer. Beads were then boiled in 30 .mu.l Laemmli buffer,
and 15 .mu.l of the sample was analyzed by western blotting.
Western blotting analysis of pre-immunoprecipitation (Input) (100
.mu.g total protein) and immunoprecipitated samples (IP) were
performed with a Bax monoclonal antibody (B9 antibody, Santa Cruz),
Bax polyclonal antibody (N20 antibody, Santa Cruz), or IFN.gamma.R2
polyclonal antibody (Fitzgerald).
Binding of Recombinant Proteins
[0080] Recombinant human Bax .DELTA.TM (Bax C terminal
transmembrane truncated human Bax) was produced by using pHMTc
vector downstream of maltose binding protein (MBP), separated by
the TEV protease site. Overexpressed MBP-Bax was purified through a
maltose-binding column (NEB) and subsequently cleaved by TEV
protease (Invitrogen), followed by Ni-affinity purification to
remove the protease and the His-tagged MBP. IFN.gamma.R2 cytoplamic
domain (amino acids 263 337) was fused with thioredoxin (rTrx) to
increase the recovery rate from bacterial lysates. The production
and the purification of this fusion protein were performed by
Protein X Laboratory (San Diego, Calif.). Recombinant Bax (25 ng)
was loaded onto Sepharose G beads pre-equilibrated with anti-Bax
antibody (Bax B9, Santa Cruz) or pre-immune IgG (control IgG) at
4.degree. C. for 2 h. The excess Bax molecules were washed 3 times
with buffer (50 mM phosphate buffer, pH 7.4). Recombinant
IFN.gamma.R2 (263-337)-rTrx (25 ng) was added to the beads
preloaded with Bax and anti-Bax or control IgG. Beads and
IFN.gamma.R2.sub.263-337-rTrx were incubated at 4.degree. C. for 2
h. After the incubation, beads were extensively washed with the
loading buffer (50 mM phosphate buffer, pH 7.4). Beads were boiled
in Laemmli buffer, and the supernatant was collected as a sample.
Samples were analyzed by western blot using anti IFN.gamma.R2
antibody (C20, Santa Cruz) and Bax (N20, Santa Cruz).
Determination of the Effects of Bcl-2 and BIP on IFN.gamma.R2-Bax
Interaction
[0081] HEK293T cells were lysed by using either NP40 buffer (150 mM
NaCl, 10 mM HEPES at pH 7.4 and 1% NP40) or CHAPS buffer (150 mM
NaCl, 10 mM HEPES at pH 7.4 and 1% CHAPS) supplemented with
protease inhibitors (1:100 dilution of protease inhibitor Cocktail;
Sigma) and PMSF, as previously reported.14 To determine if
recombinant Bcl-2 (Prospect cat #PRO-630) protein competes with
endogenous IFN.gamma.R2 for binding to endogenous Bax, HEK293T cell
lysate prepared in NP40 buffer was used. Three-hundred microliters
(1,000 .mu.g total protein) of the sample was pre-cleared by
incubating in 20 .mu.l protein G-sepharose (Amersham Biosciences)
at 4.degree. C. for 1 hour.
[0082] Cleared samples (300 .mu.l) were incubated (4.degree. C. for
2 h) with or without recombinant Bcl-2 (75 or 150 nM final
concentration) in the presence of protein G sepharose (20 .mu.l)
preabsorbed with 2 .mu.g of Bax polyclonal antibody (BD
Biosciences). Beads were washed and then boiled in 30 .mu.l Laemmli
buffer, and 15 .mu.l of the eluted protein solution was analyzed by
western blotting. Western blotting of pre-immunoprecipitation
(pre-treated) (100 .mu.g total protein) and immunoprecipitated
samples (IP) were performed with IFN.gamma.R2 monoclonal antibody
(Fitzgerald-WB), Bcl-2 monoclonal antibody (Santa Cruz), and Bax
polyclonal antibody (HRP-conjugated N20 antibody, Santa Cruz). To
determine the effects of BIP15 on the interaction of endogenous
IFN.gamma.R2 and Bax, HEK293T cell lysate prepared in CHAPS buffer
was used. Three hundred microliters of the sample was pre-cleared
by incubating with 20 .mu.l protein G-sepharose (Amersham
Biosciences) at 4.degree. C. for 1 h. Cleared samples (300 .mu.A
1,000 .mu.g total protein) were incubated (37.degree. C. for 2 h)
with or without BIP (40 or 200 .mu.M final concentration) in the
presence of protein G sepharose (20 .mu.A preabsorbed with 2 .mu.g
of FN.gamma.R2 monoclonal antibody (Fitzgerald). Beads were washed
and then boiled in 30 .mu.l Laemmli buffer, and 15 .mu.l of the
eluted protein solution was analyzed by western blotting. Western
blot analysis of Bax was performed by using Bax polyclonal antibody
(N20, Santa Cruz).
Inhibition of Bax Activation by IFN.gamma.R2
[0083] HeLa cells were transiently transfected with
pEGFP-C2-IFN.gamma.R2 (wild type 1-295, or 296-337) using
SUPERFECT.RTM. (Qiagen, Valencia, Calif.) in accordance with the
manufacturer's instructions. Four micograms of the plasmid were
used to transfect cells cultured in 6-cm diameter dishes. After 24
h of transfection, the cells were treated with staurosporine (100
nM) for 3 h. Then, the cells were washed with phosphate buffer pH
7.4 (PBS), fixed using paraformaldehyde (1%), permeabilized with
Triton X-100 (0.02%), blocked with goat serum, and the activation
of Bax was analyzed by immunocytochemistry using monoclonal Bax 6A7
antibody (BDPharmingen) and ALEXA FLUOR.RTM. 568-labeled anti-mouse
IgG secondary antibody (Invitrogen).
Determination of Concentrations of Endogenous IFN.gamma.R2 and Bax
in Cultured Cells
[0084] Recombinant Bax.DELTA.TM and IFN.gamma.R2263-337-rTrx were
used as standards. Cells were harvested and lysed in NP40 buffer
(10 mM HEPES, 150 mM NaCl and 1% NP40 pH 7.4), or with hypotonic
buffer for subcellular fractionation (20 mM HEPES, 10 mM KCl, 1.5
mM MgCl2, 1 mM EDTA and 250 mM sucrose); both buffers were
supplemented with protease inhibitors cocktail (Sigma) and PMSF
(Sigma). LDH, F1.alpha. and YY1 proteins were used as makers of the
cytosolic, mitochondrial and nuclear fractions, respectively. Cell
lysates from equivalent cell numbers, and sequential dilutions of
protein standards were subjected to SDS-PAGE (BioRad). Bax antibody
conjugated with horseradish peroxidase (HRP) (anti-Bax N20-HRP,
Santa Cruz) was used to detect Bax, and IFN.gamma.R2 antibody
(C-20, Santa Cruz) was used to detect IFN.gamma.R2. HRP-conjugated
anti-rabbit goat IgG was used as a secondary antibody. Signal
intensities were analyzed by using BioRad Gel Doc and Quantity One
4.5.1 software from BioRad.
Protein Identification by Mass Spectrometry
[0085] To enrich for the immunoreactive (ir) IFN.gamma.R2 fragment
expressed in transformed cells, DAMI and HEK293T cell lysates were
incubated with anti-IFN.gamma.R2 antibody (C-20, Santa Cruz)
overnight at 4.degree. C., and the antibody-protein complexes were
recovered by incubation of the mixture with protein G sepharose.
The sepharose gels were then boiled in 30 .mu.l Laemmli buffer, and
15 .mu.l of the eluted protein solution was used for 1D-SDS-PAGE
and western analysis. From a Coomassie blue-stained Tris-HCl gel,
bands running between the protein markers for 10 and 15 kDa were
collected. Proteins were reduced by DTT, alkylated by iodoacetamide
and digested by trypsin overnight. The tryptic peptides were
extracted from the gel by using 60% acetonitrile in 0.1% formic
acid. Recombinant Trx-tagged IFN.gamma.R2.sub.263-337 was used as
positive control for the LC-MS/MS analysis. The tryptic peptides
were analyzed by LC-MS/MS using a LTQ Orbitrap XL linear ion trap
mass spectrometer (Thermo Fisher Scientific, Waltham, Mass.)
coupled to an Ultimate 3000 HPLC system (Dionex) in the Case Center
for Proteomics. The LC-MS/MS analysis was performed as follows:
peptide solutions were injected into a reverse phase Aclaim PepMap
100 C18 column (3 mm, 100 .ANG., 150 mm.times.75 mm, Dionex
Corporation, Sunnyvale, Calif.). Mobil phases used were: 2%
acetonitrile, 0.1% formic acid in water (solvent A), and 80%
acetonitrile, 0.1% formic acid (solvent B). A linear gradient of
solvent B from 0% to 60% over a period of 60 min was used at a flow
rate of 300 mL/min. Three specific peptide ions that are expected
to be produced from IFN.gamma.R2 were selected with a mass window
of 3 amu and subjected to MS/MS analysis with a normalized
collision energy of 35%. These ions were: m/z 670.4 (z=2,
DPTQPILEALDK (SEQ ID NO: 11)), 868.9 (z=2, DDVWDSVSIISFPEK (SEQ ID
NO: 12)), and 754.4 (z=3, YWFHTPPSIPLQIEEYLK (SEQ ID NO: 13)). LTQ
injection time was set to 2 s and automatic gate control target was
10,000 ions. The results from the LC-MS/MS analysis were subjected
to an NCBI nr (version 20070216, containing 4626804 sequences)
database search using Mascot Daemon Version 2.2.0 with a mass
tolerance set to 2 Da for the precursor and 1 Da for the product
ions. In addition to the targeted analysis, the remaining digest
was also analyzed by data-dependent LC MS/MS.
The Cytoplasmic C-Terminal Portion of IFN.gamma.R2 is a Bax
Inhibitor
[0086] To perform a yeast-based functional screen for Bax
inhibitors, yeast expression cDNA libraries were generated from
purified mRNAs of human HeLa cells and mouse brain tissue using
pJG4-5 and pYES2 vectors, respectively. As previously reported, two
clones encoding the C-terminus of Ku70 were found as Bax
suppressors in this screening (FIG. 1A). In the same experiment we
also obtained a clone from the HeLa cell library encoding the
cytoplasmic domain of IFN.gamma.R2 (IFN.gamma.R2.sub.263-337; amino
acids 263-337 of IFN.gamma.R2) (FIGS. 1A and B) as a Bax
suppressor. IFN.gamma.R2.sub.263-337 contains a Jak2-binding domain
(amino acids 284-295). To determine the role of the Jak2-binding
domain in Bax inhibition, two IFN.gamma.R2 mutants were generated
(FIG. 1B) and tested for their anti-Bax activity in human cells as
described below. One mutant, IFN.gamma.R2.sub.296-337, encodes
amino acids 296-337 of IFN.gamma.R2 and does not contain the
Jak2-binding domain; the other mutant, IFN.gamma.R2.sub.1-295,
encodes amino acids 1-295, retaining the Jak2-binding domain but
not the C-terminal 41 amino acids of the receptor subunit.
[0087] IFN.gamma.R2 inhibits Bax-mediated apoptosis.
IFN.gamma.R2.sub.296-337 as well as IFN.gamma.R2wild type were able
to inhibit apoptosis induced by Bax overexpression in HEK293 cells
(FIG. 2A). On the other hand, IFN.gamma.R2.sub.1-295 could not
protect cells from Bax (FIG. 2A). These results suggest that the
Bax-inhibiting domain localizes to the 41 amino acid sequence of
the C-terminus of IFN.gamma.R2, and that Jak2-STAT1 signaling
activated by IFN.gamma. is not necessary for Bax inhibition. To
confirm that IFN.gamma.R2 does not require Jak2-mediated signaling
for Bax inhibition, human cell lines lacking IFN.gamma.R2 and Jak2
were examined. These cell lines were derived from the HT1080 human
fibrosarcoma cell line. In these experiments, etoposide, a DNA
topoisomerase II inhibitor, was used to induce apoptosis because
etoposide is known to activate the Bax-mediated intrinsic cell
death pathway. IFN.gamma.R2.sub.296-337 and IFN.gamma.R2wild type
were both able to inhibit etoposide-induced apoptosis in these
cells, but IFN.gamma.R2.sub.1-295 could not (FIGS. 2B-E). These
results support the hypothesis that IFN.gamma.R2 can rescue cells
from apoptosis independent of Jak2-mediated signal
transduction.
IFN.gamma.R2 Knock-Down Increases Apoptosis in HeLa Cells
[0088] To determine whether endogenously expressed IFN.gamma.R2 has
a physiological role in suppressing apoptosis, IFN.gamma.R2 was
knocked down by shRNA. HeLa cells were transfected with lentivirus
that expresses shRNA targeting IFN.gamma.R2 mRNA. HeLa cells
transfected with empty vector (pLKO1) or an shRNA targeting GFP
mRNA were used as controls (FIG. 3A). IFN.gamma.R2 knock-down
increased the sensitivity of HeLa cells to etoposide-induced
apoptosis (FIG. 3B). Importantly, the basal level of apoptosis was
also increased by IFN.gamma.R2 knock-down (FIG. 3A). These results
suggest that IFN.gamma.R2 has a significant role in determining the
cell-death sensitivity in HeLa cells.
IFN.gamma.R2 Inhibits Apoptosis Induced by Bim but not Bak
[0089] Bim is a BH3-only protein, which triggers Bax-mediated
apoptosis. IFN.gamma.R2wild type as well as
IFN.gamma.R2.sub.296-337 were able to inhibit apoptosis induced by
Bim overexpression (FIG. 4A). This result suggests that
IFN.gamma.R2 is able to suppress Bim-dependent Bax activation. On
the other hand, IFN.gamma.R2 could not rescue cells from apoptosis
induced by Bak overexpression (FIG. 4B), suggesting that
IFN.gamma.R2 specifically inhibits Bax-mediated apoptosis.
Subcellular Localization of IFN.gamma.R2-GFP
[0090] FIG. 5 shows HeLa (FIGS. 5A-H) and HEK293 (FIGS. 5I and J)
cells expressing IFN.gamma.R2-GFP fusion proteins. IFN.gamma.R2wild
type-GFP was detected in the plasma membrane, cytosol and a
mitochondrion-like structure (FIGS. 5C, D, I and J) as previously
reported. In HeLa cells expressing IFN.gamma.R2wild type-GFP, GFP
signal was detected mostly in the cytosol and plasma membrane
(FIGS. 5C and D), though a weak punctate pattern of GFP signal
suggestive of mitochondrial localization was also detected (the
image of this pattern was very difficult to capture due to the
strong GFP fluorescence in the cytosol and plasma membrane). In the
case of HEK293 cells, IFN.gamma.R2wild type-GFP localized to a more
definite mitochondrionlike structure that was captured in the image
(FIGS. 5I and J).
[0091] IFN.gamma.R2.sub.296-337-GFP was detected in the cytosol of
both HeLa (FIGS. 5E and F) and HEK293 cells (not shown).
IFN.gamma.R2.sub.1-295-GFP was detected in the cytosol, plasma
membrane and the mitochondria-like structures in HeLa (FIGS. 5G and
H) and HEK293 cells (not shown). In HeLa cells, GFP signal from the
mitochondria-like structure was more evident in cells expressing
IFN.gamma.R2.sub.1-295-GFP than cells expressing IFN.gamma.R2wild
type-GFP (FIGS. 5C, D and K).
Western Blot Analysis of IFN.gamma.R2-GFP
[0092] IFN.gamma.R2-GFP expression in IFN.gamma.R2-null (mutant
HT1080) cells was determined by western blot analysis using GFP
antibody (FIG. 6A). Estimated molecular weights of IFN.gamma.R2wild
type-GFP, IFN.gamma.R2.sub.1-295-GFP and
IFN.gamma.R2.sub.296-337-GFP, are approximately 67 kDa, 64 kDa and
34 kDa, respectively. Proteins that have similar molecular weights
were detected by GFP antibodies (FIG. 6A), suggesting that
IFN.gamma.R2-GFP were expressed in these cells. Interestingly, we
observed that IFN.gamma.R2.sub.1-295-GFP migrated slower than
IFN.gamma.R2wild type-GFP in every western blotting experiment
performed in this study (FIGS. 5A and C). Since the estimated
molecular weight of IFN.gamma.R2.sub.1-295-GFP is smaller than
IFN.gamma.R2wild type-GFP, this observation was unexpected. At
present, we do not know the exact reason for this phenomenon, but a
posttranslational modification such as glycosylation may be the
cause of the slower migration of this mutant protein in
SDS-PAGE.
[0093] Western blot analysis of IFN.gamma.R2-GFP expression was
also performed using HEK293 cells (FIGS. 6B-D). Although
IFN.gamma.R2.sub.296-337-GFP was detected at its estimated
molecular weight (FIG. 6B), IFN.gamma.R2wild type-GFP and
IFN.gamma.R2.sub.1-295-GFP could not be detected in a simple
western blot using GFP antibodies in HEK293 cell lysates. To verify
the expression of IFN.gamma.R2-GFP fusion proteins (both wt and
mutants), cell lysates were subjected to GFP immunoprecipitation
and samples were further analyzed by IFN.gamma.R2 antibodies (FIGS.
6C and D). After enrichment of the GFP-tagged proteins, expression
of IFN.gamma.R2wild type-GFP and IFN.gamma.R2.sub.1-295-GFP was
confirmed by monoclonal antibody recognizing the N-terminus of
IFN.gamma.R2 (FIG. 6C), and IFN.gamma.R2.sub.296-337-GFP expression
was confirmed by antibodies detecting the C-terminus of
IFN.gamma.R2-GFP (FIG. 6D).
[0094] There were GFP-antibody-positive bands with slightly higher
and lower molecular weight than GFP (29 kDa) in cells transfected
with pEGFP C2-IFN.gamma.R2wild type and pEGFP
C2-IFN.gamma.R2.sub.1-295 (bands marked with * in FIGS. 6A and B).
We speculate that protease-dependent cleavage of IFN.gamma.R2-GFP
fusion proteins produced these fragments. Protease inhibitors were
present in the cell lysis buffer; therefore, it is likely that this
cleavage occurred in the cells prior to preparation of the cell
lysate, though further careful study will be needed to reveal the
reasons for the appearance of these bands.
IFN.gamma.R2 Inhibits Bax Activation
[0095] Bax activation involves exposure of the protein's N-terminus
by a conformational change followed by Bax translocation from the
cytosol to mitochondria. Exposure of the N-terminus of Bax can be
monitored by immunohistochemistry using 6A7 Bax monoclonal antibody
(6A7 Ab) recognizing an epitope in the N-terminus of Bax.
Staurosporine (STS), a pan-kinase inhibitor, 30 was used to induce
the Bax conformational change. STS treatment (100 nM, 3 h) induced
Bax activation that was detected by 6A7 Ab as shown in FIG. 7A. GFP
expression itself did not inhibit Bax activation (FIG. 7A).
IFN.gamma.R2.sub.296-337-GFP as well as IFN.gamma.R2wild type-GFP
(FIGS. 7C and D) inhibited STS-induced Bax activation. On the other
hand, IFN.gamma.R2.sub.1-295-GFP did not inhibit Bax activation
(FIG. 7B). The percentages of 6A7 Ab-positive cells in GFP-positive
cells were calculated and the results are shown in FIG. 7E. The
inhibition of Bax activation by IFN.gamma.R2.sub.296-337-GFP as
well as IFN.gamma.R2 wild type-GFP was statistically significant
(FIG. 7E).
IFN.gamma.R2 Directly Interacts with Bax
[0096] To examine whether IFN.gamma.R2 can bind Bax, we performed
co-immunoprecipitation of endogenously expressed Bax and
IFN.gamma.R2 in HEK293T cells (FIG. 8). It is known that certain
detergents such as NP40 artificially activate Bax whereas CHAPS
does not. Interestingly, Bax and IFN.gamma.R2 were
co-immunoprecipitated by anti-Bax antibody in buffers containing
either NP40 or CHAPS (FIG. 8A). This interaction was also observed
when anti-IFN.gamma.R2 was used for immunoprecipitation and
anti-Bax was used for Bax detection in western blot (FIG. 8B).
Furthermore, the direct interaction of purified recombinant
proteins of Bax.DELTA.TM (in which the c-terminal transmembrane
(TM) domain is deleted) and IFN.gamma.R2263-337 (tagged with
thioredoxin (rTrx)) was confirmed. These results suggest that the
C-terminus of IFN.gamma.R2 directly binds Bax.
Bcl-2 Competed with IFN.gamma.R2 to Bind Bax In Vitro
[0097] Since it is known that Bcl-2 binds and inhibits Bax, we
examined whether Bcl-2 has any influence on the IFN.gamma.R2-Bax
interaction in vitro. Interestingly, addition of recombinant Bcl-2
protein (a truncated form without the C-terminal transmembrane
domain to increase solubility in the buffer) to the HEK293T cell
lysate interferes with the interaction of Bax and IFN.gamma.R2. In
this experiment, NP40-based buffer was used because the Bcl-2-Bax
interaction is known to be observed in this buffer. This result
suggests that Bcl-2 and IFN.gamma.R2 recognize the same domain of
Bax. We also examined the effects of Bax inhibiting peptide (BIP)
designed from the Bax-binding domain of Ku70. Because Ku70 and BIP
are known to bind the inactive form of Bax in CHAPS-based buffer,
we used CHAPS-based buffer to examine the effects of BIP. As shown
in FIG. 8E, BIP did not cause a significant inhibition of the
Bax-IFN.gamma.R2 interaction. Three independent experiments were
performed, and we observed results similar to that in FIG. 8E in
two of the experiments. In one experiment, BIP caused a detectable
reduction in the amount of IFN.gamma.R2 protein pulled down by Bax
antibody (data not shown); however, this effect was not
reproducible.
Expression of Cytoplasmic IFN.gamma.R2 in Transformed Cell
Lines
[0098] IFN.gamma.R2 C20 antibody (C20 Ab) recognizes the C-terminal
20 amino acids of IFN.gamma.R2 as an epitope. This
antibody-detected a small fragment (approximately 10 kDa) in
western blot analysis of cell lysates prepared from megakaryocytic
cancer cells (DAMI) and SV40-transformed kidney cells (HEK293T),
but not from normal primary cultured cells (HUVECs) (FIG. 9A). This
small fragment was enriched by immunoprecipitation (FIG. 9B),
digested by trypsin and its identity was determined by targeted
LC-MS/MS analysis. As a result, it was confirmed that a tryptic
peptide derived from the C-terminus of IFN.gamma.R2, DPTQPILEALDK
(SEQ ID NO: 11), was present in the sample. Expression of this
C-terminal fragment was also detected in two cancer cell lines,
MDA-MD468 (breast cancer cell line) and PC3 (prostate cancer cell
line) (FIG. 9C). Interestingly, the C-terminal fragment of
IFN.gamma.R2 was not detected in normal mammary epithelial cells
(4A 100) or in non tumorigenic immortalized breast (HME-1) and
prostate (RWPE-1) cells. These results suggest that a certain
protease expressed in malignant tumorigenic cells may produce the
antiapoptotic cytoplasmic fragment derived from IFN.gamma.R2.
Intracellular Concentrations of IFN.gamma.R2 and Bax
[0099] The approximate intracellular protein concentrations of
IFN.gamma.R2 and Bax were determined by densitometric analysis of
western blots using purified recombinant proteins as standards
(FIGS. 10A and B). For IFN.gamma.R2, densitometric analysis was
performed on a band corresponding to non-glycosilated full-length
INF.gamma.R2 (approximately 37 kDa). For Bax, the density of a band
corresponding to the full length of Bax (approximately 21 kDa) was
measured. As there are other forms (glycosylated, truncated, etc.)
of IFN.gamma.R2 and Bax, the estimated protein concentration in
this experiment may underestimate the actual total expression
levels of these proteins in cells.
[0100] However, our attempt to obtain an estimate of the
stoichiometry of Bax and IFN.gamma.R2 will help determine our
working hypothesis of how IFN.gamma.R2 regulates Bax-mediated
apoptosis in the cell. First, the concentrations of Bax and
IFN.gamma.R2 in total cell lysate (i.e., no fractionation) were
measured. The ratio of Bax to IFN.gamma.R2 was approximately 1:1,
3:1 and 2:1 in HUVECs, DAMIcells and HEK293T cells, respectively.
Next, the concentrations of Bax and IFN.gamma.R2 in the cytosol,
nucleus and heavy membrane (mitochondria-rich fraction) were
measured using DAMI cells and HEK293T cells. The Bax:IFN.gamma.R2
ratio in the cytosolic fraction was 2:1 and 3.5:1 in DAMI cells and
HEK293T cells, respectively. The ratio in the heavy membrane
fraction of DAMI cells and HEK293T cells was approximately 1.5:1
and 6:1, respectively. Interestingly, Bax and IFN.gamma.R2 were
also detected in the nuclear fraction (FIG. 10). Because the
nuclear fraction contains ER membranes attached to the cytosolic
surface of the nucleus, the estimated concentration of Bax and
IFN.gamma.R2 in the nuclear fraction is expected to be higher than
the actual concentration in the nucleus.
[0101] From the above description of the invention, those skilled
in the art will perceive improvements, changes and modifications.
Such improvements, changes and modifications within the skill of
the art are intended to be covered by the appended claims. All
references, publications, and patents cited in the present
application are herein incorporated by reference in their entirety.
Sequence CWU 1
1
13142PRTHomo sapiens 1Lys Asp Pro Thr Gln Pro Ile Leu Glu Ala Leu
Asp Lys Asp Ser Ser 1 5 10 15 Pro Lys Asp Asp Val Trp Asp Ser Val
Ser Ile Ile Ser Phe Pro Glu 20 25 30 Lys Glu Gln Glu Asp Val Leu
Gln Thr Leu 35 40 25PRTArtificial SequenceSynthetic Peptide 2Pro
Ile Leu Glu Ala 1 5 3337PRTHomo sapiens 3Met Arg Pro Thr Leu Leu
Trp Ser Leu Leu Leu Leu Leu Gly Val Phe 1 5 10 15 Ala Ala Ala Ala
Ala Ala Pro Pro Asp Pro Leu Ser Gln Leu Pro Ala 20 25 30 Pro Gln
His Pro Lys Ile Arg Leu Tyr Asn Ala Glu Gln Val Leu Ser 35 40 45
Trp Glu Pro Val Ala Leu Ser Asn Ser Thr Arg Pro Val Val Tyr Gln 50
55 60 Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe Thr Ala Asp Ile
Met 65 70 75 80 Ser Ile Gly Val Asn Cys Thr Gln Ile Thr Ala Thr Glu
Cys Asp Phe 85 90 95 Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met
Asp Phe Asn Val Thr 100 105 110 Leu Arg Leu Arg Ala Glu Leu Gly Ala
Leu His Ser Ala Trp Val Thr 115 120 125 Met Pro Trp Phe Gln His Tyr
Arg Asn Val Thr Val Gly Pro Pro Glu 130 135 140 Asn Ile Glu Val Thr
Pro Gly Glu Gly Ser Leu Ile Ile Arg Phe Ser 145 150 155 160 Ser Pro
Phe Asp Ile Ala Asp Thr Ser Thr Ala Phe Phe Cys Tyr Tyr 165 170 175
Val His Tyr Trp Glu Lys Gly Gly Ile Gln Gln Val Lys Gly Pro Phe 180
185 190 Arg Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys Pro Ser Arg Val
Tyr 195 200 205 Cys Leu Gln Val Gln Ala Gln Leu Leu Trp Asn Lys Ser
Asn Ile Phe 210 215 220 Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr
Glu Thr Met Ala Asp 225 230 235 240 Ala Ser Thr Glu Leu Gln Gln Val
Ile Leu Ile Ser Val Gly Thr Phe 245 250 255 Ser Leu Leu Ser Val Leu
Ala Gly Ala Cys Phe Phe Leu Val Leu Lys 260 265 270 Tyr Arg Gly Leu
Ile Lys Tyr Trp Phe His Thr Pro Pro Ser Ile Pro 275 280 285 Leu Gln
Ile Glu Glu Tyr Leu Lys Asp Pro Thr Gln Pro Ile Leu Glu 290 295 300
Ala Leu Asp Lys Asp Ser Ser Pro Lys Asp Asp Val Trp Asp Ser Val 305
310 315 320 Ser Ile Ile Ser Phe Pro Glu Lys Glu Gln Glu Asp Val Leu
Gln Thr 325 330 335 Leu 46PRTArtificial SequenceSynthetic peptide
4Xaa Pro Xaa Leu Xaa Xaa 1 5 56PRTArtificial SequenceSynthetic
peptide 5Val Pro Met Leu Lys Glu 1 5 65PRTArtificial
SequenceSynthetic peptide 6Val Pro Met Leu Lys 1 5 75PRTArtificial
SequenceSynthetic peptide 7Pro Met Leu Lys Glu 1 5 84PRTArtificial
SequenceSynthetic peptide 8Pro Met Leu Lys 1 95PRTArtificial
SequenceSynthetic peptide 9Val Pro Thr Leu Lys 1 5 105PRTArtificial
SequenceSynthetic peptide 10Val Pro Ala Leu Arg 1 5
1112PRTArtificial SequenceSynthetic peptide 11Asp Pro Thr Gln Pro
Ile Leu Glu Ala Leu Asp Lys 1 5 10 1215PRTArtificial
SequenceSynthetic peptide 12Asp Asp Val Trp Asp Ser Val Ser Ile Ile
Ser Phe Pro Glu Lys 1 5 10 15 1318PRTArtificial SequenceSynthetic
peptide 13Tyr Trp Phe His Thr Pro Pro Ser Ile Pro Leu Gln Ile Glu
Glu Tyr 1 5 10 15 Leu Lys
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