U.S. patent application number 13/175912 was filed with the patent office on 2012-05-17 for cell cycle arrest and apoptosis.
Invention is credited to Joshua L. Andersen, Vicente Planelles, Erik Zimmerman.
Application Number | 20120122770 13/175912 |
Document ID | / |
Family ID | 36228187 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122770 |
Kind Code |
A1 |
Planelles; Vicente ; et
al. |
May 17, 2012 |
Cell Cycle Arrest and Apoptosis
Abstract
The HIV-1 accessory gene vpr encodes a conserved 96-amino acid
protein that is necessary and sufficient for the HIV-1-induced
block of cellular proliferation and induction of apoptosis.
Expression of vpr in CD4.sup.+ lymphocytes results in G2 arrest,
followed by apoptosis. ATR, as a cellular factor that mediates
Vpr-induced cell cycle arrest, is required for activation of the
Breast Cancer-Associated Protein-1 (BRCA1). In addition, the Growth
Arrest and DNA Damage protein (GADD45) is upregulated by Vpr in an
ATR-dependent manner. Posttranscriptional silencing of either ATR
or GADD45 leads to nearly complete suppression of the pro-apoptotic
and/or cell cycle arrest effect of Vpr.
Inventors: |
Planelles; Vicente; (Salt
Lake City, UT) ; Andersen; Joshua L.; (Sandy, UT)
; Zimmerman; Erik; (Salt Lake City, UT) |
Family ID: |
36228187 |
Appl. No.: |
13/175912 |
Filed: |
July 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11665416 |
Aug 13, 2008 |
7973018 |
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PCT/US04/35464 |
Oct 21, 2004 |
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13175912 |
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Current U.S.
Class: |
514/4.8 ;
435/375; 506/9; 514/1.1; 514/18.9; 530/350 |
Current CPC
Class: |
A61K 38/10 20130101;
G01N 33/5008 20130101; A61P 43/00 20180101; A61P 31/00 20180101;
A61P 3/04 20180101; C12N 2310/14 20130101; G01N 2510/00 20130101;
A61K 35/13 20130101; C12N 15/113 20130101 |
Class at
Publication: |
514/4.8 ;
530/350; 514/18.9; 514/1.1; 435/375; 506/9 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 43/00 20060101 A61P043/00; C40B 30/04 20060101
C40B030/04; A61P 3/04 20060101 A61P003/04; C07K 19/00 20060101
C07K019/00; C12N 5/071 20100101 C12N005/071 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Work described herein was supported in part by National
Institute of Health Grant Nos. AI49057 and AI054188. The United
States Government may have certain rights in the invention.
Claims
1. A compound comprising Vpr or a functional fragment thereof
linked to an adipose tissue targeting moiety.
2. The compound of claim 1, wherein the targeting moiety comprises
SEQ ID NO:1.
3. A method of treating obesity in a subject, the method comprising
administering a compound according to claim 1 to the subject.
4. A method of inducing apoptosis in a subject, the method
comprising: introducing Vpr and BRAC1, or a functional fragment
thereof to a subject; and inducing apoptosis in the subject.
5. The method according to claim 4, wherein the subject has been
diagnosed as having a mutation in a BRAC1 gene.
6. A method of inducing GADD45, the method comprising administering
Vpr, ATR or a functional fragment thereof to a subject.
7. A method of activating BRAC1, the method comprising
administering Vpr, ATR or a functional fragment thereof to a
subject.
8. The method according to claim 4, wherein the subject is a
mammal.
9. The method according to claim 4, wherein the subject comprises a
cell culture system.
10. A method of inducing G2 cell cycle arrest, the method
comprising: administering Vpr, activated ATP, or a functional
fragment thereof to a subject; inducing activation of BRAC1 or
RAD17; and arresting cell cycle progression in G2.
11. A method of screening a compound for apoptotic activity, the
method comprising: administering a compound to a subject having an
ATR protein and a BRAC1 protein; assaying for ATR dependent
phosphorylation of BRAC1; and identifying the compound as inducing
or inhibiting apoptosis.
12. The method according to claim 11, wherein assaying for ATR
dependent phosphorylation of BRACT comprises assaying for
phosphorylation at serine 1423 of BRAC1.
13. The method according to claim 11, wherein assaying for ATR
dependent phosphorylation of BRAC1 comprises knocking down ATR
expression.
14. The method according to claim 12, further comprising culturing
the subject.
15. The method according to claim 11, wherein administering the
compound to the subject comprises administering the compound to an
animal model for a disease state.
16. The method according to claim 14, further comprising
introducing Vpr into the subject and screening the compound for
inhibition of apoptosis.
17. The method according to claim 14, further comprising comparing
the compound to Vpr-induced apoptosis.
18. The method according to claim 6, wherein the subject is a
mammal.
19. The method according to claim 7, wherein the subject is a
mammal.
20. The method according to claim 4, wherein the subject c
comprises a cell culture system.
Description
TECHNICAL FIELD
[0002] This invention relates generally to biotechnology, more
particularly, to compounds, compositions, and/or methods useful in
the regulation of apoptosis and/or G.sub.2 cell cycle arrest.
BACKGROUND
[0003] Human immunodeficiency virus type 1 (HIV-1) has four genes,
vif, vpr, vpu, and nef, termed "accessory genes," that are
dispensable for viral replication in vitro (14). Many important
functions related to HIV-1 pathogenesis have been ascribed to these
accessory genes. Specifically, Vpr has been implicated in long
terminal repeat transactivation, nuclear import of the
preintegration complex, induction of G.sub.2 cell cycle arrest, and
apoptosis. Recent studies identifying single amino acid changes in
Vpr in a cohort of HIV-1-infected long-term nonprogressors
substantiated the role of Vpr in HIV-1 pathogenesis in vivo (29,
43). Vpr induces G.sub.2 arrest,and apoptosis in infected CD4.sup.+
lymphocytes (18, 21, 35, 38). G.sub.2 arrest by Vpr is effected in
HeLa cells through activation of the ATR-dependent DNA damage
checkpoint pathway (40). This data supports previous work
demonstrating the inhibition of cyclin B1-p34.sup.cdc2 complexes by
Vpr (18) and establishes the identity of some of the upstream
regulators of Cdc2. ATR-dependent activation of Chk1 kinase leads
to the inhibition of Cdc25C phosphatase, which is normally required
to dephosphorylate and activate Cdc2 (40). The signaling pathway
downstream of ATR activation was recently reviewed in references 1
and 33.
[0004] While Vpr activates the ATR-specific checkpoint, the role of
other molecules required in the ATR pathway is not known. Activated
ATR can also phosphorylate proteins other than those required for
G.sub.2 arrest. One of these substrates is the histone 2A variant X
(H2AX). H2AX is deposited randomly throughout chromatin, comprising
approximately 10% of total nucleosomal histone H2A (34). H2AX has a
highly conserved serine residue at position 139 that is
phosphorylated by ATR and/or ATM in response to DNA damage (10, 37,
46). It is estimated that hundreds to thousands of H2AX molecules
are phosphorylated per double-stranded break (37). ATM-dependent
H2AX phosphorylation occurs in response to doublestranded DNA
breaks (10, 46, 47). In contrast, ATR phosphorylates H2AX under
circumstances of replication stress, such as stalled replication
forks (9). In the presence of DNA damage or replication stress,
H2AX molecules that are located in the vicinity of the DNA lesion
become phosphorylated in a highly specific localized manner (34).
Thus, immunofluorescence staining for phosphorylated H2AX (also
referred to as .gamma.-H2AX) following DNA damage produces a
staining pattern of distinct nuclear foci (34). .gamma.-H2AX is
thought to amplify the DNA damage signal by enhancing and
stabilizing the recruitment of DNA damage sensor proteins, such as
ATR, ATM, Rad17, and the 9-1-1 complex, and DNA repair proteins,
such as breast cancer susceptibility protein 1 (BRCA1), Nbs1,
Mre11, and Rad50, to sites of DNA damage (15). This action may
effectively "mark" the site of DNA damage, maintaining checkpoint
signaling at the damaged region until DNA repair is completed.
[0005] Another substrate of activated ATR is BRCA1. BRCA1 is
important for both checkpoint activation and DNA repair. BRCA1
colocalizes with DNA repair factors, such as Rad51, PCNA, and
Mre11-Rad5O-Nbs1 (15). It has been proposed that BRCA1 may
represent an essential link in coordinating cell cycle arrest with
genomic repair efforts (reviewed in reference 27) and with the
induction of apoptosis.
[0006] In addition to a role in cell cycle arrest, Vpr plays a role
in apoptosis. However, it is not possible to extrapolate the
findings relating to cell cycle arrest to apoptosis, as the
pathways do not completely overlap or follow one from the other.
Therefore, there is also a need to determine the role of Vpr in
apoptosis.
[0007] It has been suggested that apoptosis of infected cells may
play a significant role in the depletion of CD4+ lymphocytes in
vivo (62, 82, 56, 93). However, the mechanism by which Vpr induces
apoptosis was not understood. Muthumani et al. reported that
vpr-expressing cells undergo apoptosis via the intrinsic pathway
that involves loss of mitochondrial membrane potential (74). This
pathway of apoptosis is characterized by cytochrome C release, and
caspase 9 activation, and is triggered in the absence of death
receptor ligation (74). However, the initial event induced by Vpr
towards activation of the proapoptotic signaling cascade was not
elucidated.
[0008] To elucidate whether Vpr might directly promote the release
of pro-apoptotic mediators from the mitochondria, Veira et al., and
Jacotot et al. incubated recombinant Vpr with purified mitochondria
(88, 67). These two studies found that in a cell-free system, Vpr
interacts with the permeability transition pore complex (PTPC) to
cause ion permeability and swelling of mitochondria leading to
release of cytochrome C (88, 67). These results support a model in
which Vpr induces mitochondrial depolarization directly rather than
activating upstream stress signals (88, 67). The present invention
provides data that does not support the model of Jacotot et al. and
provides additional methods of activating apoptosis.
DISCLOSURE OF THE INVENTION
[0009] The invention relates to the induction of apoptosis and/or
cell cycle arrest in a subject. The invention also relates to the
induction of apoptosis and/or cell cycle arrest in a subject
lacking one or more functional ATR, BRAC1, RAD17 and/or GADD45
proteins. For example, the invention relates to the treatment of
breast cancer by introducing Vpr and BRAC1, or a functional
fragment thereof, into a breast cancer cell having a mutation in
BRAC1, wherein Vpr and BRAC1 induce apoptosis in a cancer cell.
[0010] Another aspect of the invention relates to one or more
vectors containing one or more Vpr, ATR, BRAC1, RAD17, HUS1 or
GADD45 encoding nucleic acid sequences. The nucleic acid in the
vector can be operatively linked to a promoter, for example, an
inducible or regulatable promoter that is capable of expressing or
overexpressing a protein, such as Vpr, ATR, BRAC1, RAD17, HUS1
and/or GADD45, or that is capable of expressing or overexpressing
the protein in a conditional manner. The vector may include one or
more of the following: a selectable marker, an origin of
replication, or other sequences known in the art. The nucleic acid
encoding a protein such as Vpr, ATR, BRAC1, RAD17, HUS1 and/or
GADD45, or a vector including such a nucleic acid, may be contained
in a cell, such as a bacterial, mammalian, or yeast cell. Another
aspect of the invention relates to host cells containing a vector
capable of directing expression of a protein, such as Vpr, ATR,
BRAC1, RAD17, HUS1 and/or GADD45.
[0011] The invention also relates to a method of increasing
apoptosis and/or G.sub.2 cell cycle arrest in a subject, such as a
cancer cell, by introducing one or more nucleic acid sequences
encoding one or more Vpr, ATR, BRAC1, RAD17, HUS1 or GADD45
proteins.
[0012] The invention also relates to a compound comprising Vpr or a
functional fragment thereof linked to a tissue targeting moiety,
such as an adipose tissue targeting moiety. For example, the
targeting moiety of SEQ ID NO:1.
[0013] The invention also relates to a method of treating obesity
in a subject, wherein Vpr linked to a targeting moiety is
administered to the subject.
[0014] The invention also relates to a method of inducing apoptosis
in a subject by introducing Vpr and BRAC1 and/or ATR, or a
functional fragment thereof to the subject and inducing apoptosis.
The subject is optionally believed to suffer from breast cancer due
to a mutation in a brac1 gene or a subject having a mutation in an
atr gene. In another aspect, the invention relates to a method of
inducing GADD45 by administering Vpr, ATR, or a functional fragment
thereof, to the subject.
[0015] The invention also relates to a method of inducing G2 cell
cycle arrest by administering Vpr, activated ATR, or a functional
fragment thereof to a subject, inducing activation of BRAC1, HUS1
and/or RAD17, and arresting cell cycle progression in G2.
[0016] The invention also relates to a method of screening a
compound for apoptotic activity, comprising administering a
compound to a subject having an ATR protein and a BRAC1 protein,
assaying for ATR dependent phosphorylation of BRAC1, and
identifying the compound as inducing or inhibiting apoptosis.
Optionally, ATR dependent phosphorylation of BRAC1 comprises
assaying for phosphorylation at serine 1423 of BRAC1. Optionally,
Vpr may be introduced into the subject. As will be recognized by a
person of ordinary skill in the art, introducing a protein includes
introducing a nucleic acid encoding the protein as well as
introducing the protein itself.
[0017] Optionally, the methods of the invention may comprise
knocking down ATR ATR, BRAC1, RAD17 and/or GADD45 expression in a
subject.
[0018] The invention also relates to a medicament and/or method of
manufacturing a medicament for the treatment of a disease in a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Rad17 inhibition by RNA interference relieves
Vpr-mediated G2 arrest. FIG. 1A is an immunoblot of total Rad17
(upper panel) or actin (lower panel). Cells were mock transfected
(lane 1), transfected with nonspecific siRNA (lane 2); or
transfected with Rad17-specific siRNA (lane 3). FIG. 1B illustrates
the results of cell cycle analysis of HeLa cells transfected as
indicated, and infected with lentivirus vector pHR-Vpr or pHR-GFP.
Cell cycle distributions were analyzed 48 h after infection. Left
peaks represent diploid cells in G1. Right peaks represent
tetraploid cells in G2/M.
[0020] FIG. 2. Hus1 is required for Vpr-mediated G2 arrest.
Hus1.sup.-/- p21.sup.-/- or Hus1.sup.+/+ p21.sup.-/- mouse
embryonic fibroblasts were infected with lentivirus vector pHR-Vpr
or pHR-GFP. Cell cycle distributions were analyzed at 48 h after
infection.
[0021] FIG. 3. Vpr induces .gamma.-H2AX and BRCA1 focus formation.
FIGS. 3A and 3B show HeLa cells that were transduced with
lentivirus vector pHR-Vpr or pHR-GFP, mock transduced, or treated
with 10 mM HU for 2 h, at 48 h after transduction, cells were
stained with .gamma.-H2AX- or BRCA1-specific antibodies and
visualized for .gamma.-H2AX (red) (3A) or BRCA1 (red) (3B) and GFP
(green) localization by confocal microscopy. FIG. 3C illustrates
.gamma.-H2AX- and BRCA1-positive and -negative cells that were
visually counted. Results represent averages for three fields with
approximately 50 cells per field. FIG. 3D shows human primary
thymocytes that were infected with HIV-1NL4-3 (bottom panel) or
mock infected (top panel) and, at 20 h postinfection, fixed and
stained for .gamma.-H2AX.
[0022] FIG. 4. ATM is not necessary for Vpr-mediated G2 arrest.
FIG. 4A illustrates an immunoblot of total ATM (upper panel), ATR
(middle panel), or actin (lower panel). Cells were mock transfected
(lane 1) or transfected with nonspecific siRNA (lane 2),
ATM-specific siRNA (lane 3), or ATR-specific siRNA (lane 4). FIG.
4B illustrates cell cycle analysis of HeLa cells transfected as
indicated and infected with lentivirus vector pHR-Vpr or pHR-GFP.
Cell cycle distributions were analyzed at 48 h after infection.
Left peaks represent diploid cells in GI. Right peaks represent
tetraploid cells in G2/M.
[0023] FIG. 5 shows a schematic representation of the ATR pathway.
ATR has eight known targets; only the ones with potential relevance
to HIV-1 Vpr are represented. Question marks denote functional
relationships that are expected but not confirmed. The asterisk
denotes the inhibition of Cdc25C reported by Goh et al., (17).
[0024] FIG. 6. siRNA-mediated knockdown of ATR and GADD45 abrogates
Vpr-induced apoptosis. FIG. 6A shows HeLa cells that were
transfected with non-specific (NS) siRNA or siRNA targeted to ATM,
CHK2, ATR or GADD45 then, 48 hours post-transfection, cells were
mock-transduced or transduced with pHR-VPR. FIG. 6B illustrates the
results from 3 independent experiments described in panel A that
were quantitated. FIG. 6C shows Western analysis of Hela cells that
were either mock treated or transduced with pHR-VPR in the presence
of non-specific siRNA or siRNA targeted to ATR, GADD45, ATM, or
CHK2. 48 hours post-transduction, cell lysates were harvested and
subject to Western blot analysis with anti-PARP antibodies that
recognize both full length PARD and caspase cleaved (89 kDa) PARP.
FIG. 6D shows cells that were treated with indicated siRNAs, lysed,
and each siRNA treatment was assayed by Western blot to verify
knockdown. FIG. 6E illustrates that the siRNA treatments did not
affect pHR-VPR expression, by showing lysates from each siRNA
treatment that were assayed by western blot for Vpr protein
levels.
[0025] FIG. 7. Vpr induces ATR-dependent phosphorylation of BRCA1
at serine 1423. HeLa cells were either mock treated or transduced
with pHR-VPR in the presence of either nonspecific siRNA or siRNA
targeted to ATR. Cell lysates were harvested 48 hours
posttransduction then subject to Western blot analysis with
anti-BRCA1 phospho-serine 1423 antibodies.
[0026] FIG. 8. Vpr activates BRCA1, as evidenced by BRCA1 nuclear
foci formation. HeLa cells were transduced with either pHR-VPR, or
pHR-GFP. As a positive control, HeLa cells were treated with HU. 2
hours post-HU treatment, and 48 hours post-transduction, cells were
subject to an immunofluorescence assay with BRCA1-specific
antibodies and analyzed by laser scanning confocal microscopy.
[0027] FIG. 9. Vpr upregulates GADD45 protein levels in both SupT1
cells, and primary human CD4+lymphocytes. (a) SupT1 cells were
transduced with pHR-VPR or pHR-GFP, then harvested 24, 48, and 72
hours post-transduction. Cell lysates from each timepoint were
subject to Western blot analysis with polyclonal antibodies against
GADD45. (b) Primary human CD4+ lymphocytes were transduced with
pHR-VPR or pHR-VPR(R80A), then harvested 48 and 72 hours
post-transduction. Primary cell lysates were subject to Western
blot analysis with polyclonal antibodies against GADD45.
Transduction efficiencies for both pHR-VPR and pHR-VPR(R80A) in
primary CD4+ lymphocytes were between 25% to 30%, a marked
reduction from efficiencies observed in. SupT1 cells which ranged
from 70% to 80%.
[0028] FIG. 10. Vpr-induced upregulation of GADD45 is
ATR-dependent. HeLa cells were transfected with either non-specific
siRNA, GADD45-specific siRNA, or ATR-specific siRNA, then
mock-transduced or transduced with pHR-VPR. The cells were lysed at
48 hours posttransduction and subject to Western blot with
polyclonal antibodies against GADD45.
[0029] FIG. 11. Vpr-induced upregulation of GADD45 does not result
in activation of JNK or p38 kinase. (a) SupT1 cells were transduced
with pHR-VPR. At 48 hours post-transduction, cells were lysed and
incubated with recombinant c-Jun. The relative levels of c-Jun
phosphorylation for each treatment were determined by western blot,
using phospho-specific antibodies against c-Jun. As a positive
control, cells were treated with Anisomycin to induce JNK
activation. (b) SupT1 cells were transduced with pHR-VPR then lysed
at 24, 48, and 72 hours posttransduction. VPR-induced
phosphorylation of p38 kinase was determined by western blot using
a phospho-specific antibody against p38 kinase. As a positive
control, cells were treated with Anisomycin to induce p38 kinase
activation.
BEST MODES FOR CARRYING OUT THE INVENTION
[0030] As used herein, "peptide," "polypeptide" and "protein"
include polymers of two or more amino acids of any length. No
distinction, based on length, is intended between a peptide, a
polypeptide or a protein.
[0031] In addition to initiating G.sub.2 arrest signaling through
Chk1, activated ATR also phosphorylates cellular proteins in
separate branches of the DNA damage response (see, Table 1). ATR is
a 2,644 amino acid protein with a C-terminal catalytic domain,
which is flanked by two loosely conserved domains In light of the
fact that ATR has at least eight cellular targets for
phosphorylation (1, 51), additional pathways controlled by ATR may
be affected by Vpr-dependent activation. Therefore, the activation
status of other known ATR targets has been investigated. The
present invention shows that both Rad17 and Hus1 are required for
Vpr-mediated G.sub.2 arrest. In addition, HIV-1 Vpr expression
leads to the formation of intense .gamma.-H2AX and BRCA1 nuclear
foci, characteristic markers of DNA damage. These results suggest a
role of Vpr in activating the ATR-dependent G.sub.2 checkpoint.
However, other aspects of HIV-1 pathogenesis, such as the induction
of apoptosis, remain speculative, at best;, based on this data
alone.
[0032] Rad17 is a replication factor C-related protein that, in a
complex with Rfc2, Rfc3, Rfc4, and Rfc5, loads the heterotrimeric
sliding clamp consisting of Rad9, Rad1, and Hus1 (9-1-1 complex) at
sites of DNA damage (54). ATR, Rad17, and the 9-1-1 complex
colocalize and activate one another to signal G.sub.2 checkpoint
activation (54). Rad17 and the 9-1-1 complex are necessary for
downstream signaling of G.sub.2 arrest through ATR activation
(54).
[0033] It is shown here that Vpr activates ATR and its downstream
signaling events in a manner that is similar to that of activation
by bona fide DNA damage (FIG. 5 shows a schematic diagram). Various
means of genetic analysis, including, RNA interference, knockout
cell lines, and/or dominant-negative constructs, may be used to
demonstrate Vpr-dependent activation of G2 arrest and apoptosis.
Important mechanistic details of the downstream signaling
consequences are now being elucidated.
[0034] Rad17 and Hus1 are required for signaling when ATR dependent
G.sub.2 arrest is induced in response to genotoxic stress. Upon
recognition of genotoxic stress, Rad17 is phosphorylated and is the
first target of ATR (54) (FIG. 5). This phosphorylation requires
the participation of Hus1 (and the larger complex of which Hus1 is
a part, 9-1-1) (54). Only after Rad17 is phosphorylated can ATR
modify its next target, Chk1. By down-regulating endogenous Rad17
protein levels via RNA interference, the present invention shows
that Rad17 function is also required for Vpr-induced G.sub.2
arrest. Additionally, the present invention shows that
Hus1-deficient cells are refractory to the effects of Vpr on the
cell cycle. Therefore, Rad17 and Hus1 are necessary components of
the G.sub.2 checkpoint response to Vpr expression.
[0035] Interestingly, Hus1 is known to be dispensable for ATR
mediated H2AX phosphorylation (46). Based on these findings, Ward
and Chen (46) suggested that ATR activation may lead to two types
of downstream events, which are Hus1 dependent and Hus1
independent. Hus1-dependent consequences of ATR activation (such as
Chk1 phosphorylation) may specifically induce cell cycle arrest,
while downstream events independent of Hus1 (such as H2AX
phosphorylation) may recruit members of the DNA repair machinery,
such as BRCA1, Nbs1, and Rad50 (15, 46).
[0036] The cyclin-dependent kinase inhibitor p21.sup.Waf1 was
previously shown to be transcriptionally upregulated in a
p53-dependent fashion in the context of Vpr expression (12). This
observation led the authors to formulate the hypothesis that
p21.sup.Waf1 may mediate Vpr-induced G.sub.2 arrest, although this
hypothesis was not tested (12). Here it is shown that
p21.sup.Waf1-/- mouse embryonic fibroblasts are able to activate
the G.sub.2 checkpoint when transfected with Vpr (FIG. 2). Without
wishing to be bound by theory, this observation suggests that
p21.sup.Waf1 does not play a major role in mediating G.sub.2 arrest
by Vpr.
[0037] It is shown here that Vpr induces .gamma.-H2AX and BRCA1
focus formation. Therefore, four targets of ATR that have been
tested (Table 1) play an active role in the response to Vpr
expression. Due to the present observation of a very specific
histone modification and directed recruitment of a known DNA repair
protein in response to Vpr, it is believed that Vpr-induced ATR
stimulation occurs at distinct sites throughout chromatin. This
specificity may be due to DNA sequence, chromatin modifications, or
replication- and expression-dependent DNA and/or chromatin
dynamics. Vpr-induced signaling through ATR may have cellular
effects other than G.sub.2 arrest, such as recruitment of DNA
repair proteins and/or initiation of apoptotic signaling
cascades.
TABLE-US-00001 TABLE 1 Phosphorylation targets of ATR and their
roles in activation of the G.sub.2 checkpoint Status in the
presence of Vpr ATR target (reference) (reference or source)a Chk1
(25) P, A (40) Rad17 (54) N (this work) H2AX (9, 10, 37, 46) P, F
(this work) BRCA1 (45) P, F (this work) Plk1 (13) ? p53 (24) D (41)
53BP1 (46, 48) ? E2F (11, 36) ? aP, phosphorylated; A, activated;
N, necessary for G2 checkpoint activation; F, focus formation; ?,
unknown; D, dispensable for G.sub.2 checkpoint activation.
[0038] The present invention demonstrates that primary human
CD4.sup.+ thymocytes, an in vivo target for HIV-1 (22, 23), display
.gamma.-H2AX foci when infected with full-length HIV-1. Therefore,
the findings with HeLa cells can be extended to primary CD4.sup.+
cells, one of the target cell types of HIV-1. More importantly,
this indicates that the host cell DNA damage response is activated
in the context of an HIV-1 infection.
[0039] These results may also be replicated in natural targets of
HIV-1 (such as primary CD4.sup.+ lymphocytes and macrophages).
However, human primary cells that are defective for genes in the
ATR pathway are rare or nonexistent. Hence, RNA interference
technology with primary cells may be used to test the necessity of
various mediators for activation of the G.sub.2 checkpoint by Vpr.
Although transfection of primary cells with RNA duplexes is
inefficient, construction of lentivirus vectors expressing short
hairpin RNAs (5, 6) offer an additional alternative or sorting of
the cell population based on co-transfection of a selection or
sorting marker.
[0040] The polo-like kinase (Plk1) has been described as a positive
regulator of the G.sub.2/M transition. This effect is thought to be
mediated by Plk1 kinase activity directed at cyclin B1 (13). Plk1
phosphorylation promotes nuclear accumulation of the cyclin B1-Cdc2
heterodimer, ultimately allowing progression into M phase (26). In
instances of DNA damage, Plk1 kinase activity is inhibited to
prevent advance into mitosis (FIG. 5) (42). It has been
demonstrated that this inhibition is dependent on the kinase
activity of ATR (13). Thus, inactivation of Cdc25C by Chk1 may not
be the sole contributor to inducing G.sub.2 arrest, and concerted
action by Plk1 may also be required.
[0041] The p53-binding protein, 53BP1, rapidly associates with
nuclear foci containing .gamma.-H2AX, ATR, and BRCA1 in response to
genotoxic stress (7, 46). This organization into foci occurs in an
ATR-dependent manner in response to replication stress (46, 48). If
Vpr directly causes DNA lesions, stalls replication forks to cause
double-stranded breaks, or somehow mimics DNA damage through DNA,
chromatin, or protein-protein interactions, then one would expect
53BP1 to be activated by ATR.
[0042] The transcriptional activator E2F1 is another target of ATR.
E2F1 is essential for promoting the G.sub.1/S transition and DNA
replication. E2F1 is also involved in several stress response
pathways, including apoptosis and DNA repair (reviewed in
references 11 and 36). For example, E2F1 is implicated in
p53-dependent apoptosis in response to DNA damage (19, 39). It has
also been shown that E2F1 recruits the DNA repair proteins Nbs1 and
Mre11 to origins, of replication (30). Therefore, E2F1
phosphorylation may play a role in the cellular response to
Vpr.
[0043] The tumor suppressor p53 can be a target for ATR as well as
for ATM, leading to the induction of cell cycle arrest and
apoptosis in response to environmental insults, including DNA
damage (reviewed in reference 24). Shostak et al. previously
examined the role of p53 in mediating the effects of Vpr and found
that p53 is dispensable for both checkpoint activation and
apoptosis induction (41). However, it is possible that the
activation of p53 by ATR may allow Vpr to modulate certain aspects
of infected cells via the transcriptional effects of p53. For
example, p53 is known to transcriptionally activate the
p53-dependent ribonucleotide reductase, p53R2, thrombospondin-1,
and aldehyde dehydrogenase-4, enzymes which participate in diverse
processes, such as DNA repair, inhibition of angiogenesis, and the
response to oxidative stress, respectively (for a review, see
reference 31).
[0044] The precise mechanism of ATR activation in the context of
HIV-1 Vpr has remained unclear (FIG. 5). ATR activation is thought
to be specific for DNA damage manifested as single-stranded DNA
through either processed double-stranded breaks or stalled
replication forks due to either replicational pausing or
single-stranded breaks; in contrast, the ATM response is thought to
be predominantly responsible for the immediate signaling of
unprocessed double- , stranded DNA breaks (1, 33). This pathway
specificity suggests that Vpr activates the DNA damage-induced
G.sub.2 checkpoint in a manner that resembles or causes the
accumulation of single-stranded DNA. Therefore, there are several
possible mechanisms by which Vpr may activate ATR. One possibility
is that. Vpr directly causes DNA lesions through intrinsic nuclease
activity. This possibility seems unlikely, as Vpr shares no
sequence homology or known structural motifs with any known
nucleases. Another plausible explanation is that Vpr
inappropriately recruits ATR or other DNA damage-sensing proteins
to undamaged DNA through DNA-protein and protein-protein
interactions. Alternatively, Vpr could interact with proteins or
DNA in a manner that causes DNA damage. One possible mode of
indirectly inducing DNA damage would be the recruitment of an
endonuclease which would enzymatically induce single-stranded or
double-stranded DNA breaks which, once processed into
single-stranded DNA, would activate ATR.
[0045] Vpr may also interact with DNA or proteins present at sites
of DNA replication in a manner that inhibits replication fork
progression. It has been proposed that abnormally long, replication
protein A-bound single-stranded DNA at stalled replication forks
allows for ATR recruitment via an ATR-interacting protein (55).
Additionally, if halted forks are not stabilized and resolved, then
their eventual collapse can activate DNA damage sensors (1, 33).
However, if this were a highly potent, nonspecific effect of Vpr,
then one would expect a global inhibition of replication manifested
as early S-phase arrest, instead of the conspicuous G.sub.2 arrest.
Vpr could directly interact with DNA in a fashion that causes or
resembles damaged DNA or stalled replication forks. It has been
shown that the C-terminal alpha helix of Vpr binds DNA in vitro and
that Vpr is detected in chromatin and nuclear matrix fractions in
vivo (28, 52).
[0046] An alternative model suggests that Vpr may directly interact
with ATR or other components of the checkpoint signaling pathway
independent of DNA or chromatin localization. Coprecipitation
experiments for ATR and Vpr using conventional methods have been
unable to demonstrate any binding between these proteins. However,
the use of cross-linking agents to stabilize a potentially weak
interaction or a protein complex with multiple proteins bridging
ATR and Vpr may demonstrate interaction.
[0047] A recent study indicated that Vpr interacts directly with
Cdc25C and inhibits Cdc25C phosphatase activity (17). Inhibition of
Cdc25C then prevents activation of the cyclin B1-p34.sup.cdc2
complex. Although this finding does not explain why ATR, Rad17,
Hus1, and, Chk1 are required for Vpr-induced G.sub.2 arrest (FIG.
5), it is plausible that Vpr induces G.sub.2 arrest in a redundant
manner, both by signaling DNA damage and by inhibiting downstream
mediators of cell cycle progression, such as Cdc25C (17). It is
also formally possible that Cdc25C inhibition has an unforeseen
effect on the activation or expression of upstream proteins in the
ATR signaling cascade. Regardless of the mechanism of action, Vpr
is shown to induce G.sub.2 a rest and may be used to induce such an
arrest in target cells. For example, Vpr or a functional fragment
thereof may be introduced into cancer cells or a subject to produce
a desired G.sub.2 arrest.
[0048] The cytopathic effects of HIV-1 infection are thought to be
multiple and related to the expression of several viral genes. The
present invention demonstrates that Vpr has at least two discrete
functions, it exerts a potent antiproliferative effect due to
G.sub.2 arrest and also produces proapoptotic effects. However, the
interrelationship of G.sub.2 arrest and apoptosis is not clear,
since Rad17 is important for checkpoint activation and BRCA1 is
related to DNA damage and may be potentially proapoptotic (FIG.
5).
[0049] Since. Vpr was found to be sufficient to induce apoptosis.
(86), the functional relationship between induction of apoptosis
and that of G.sub.2 arrest has been controversial. While G2 arrest
plateaus at approximately 36 hours post-transduction with pHR
vectors, apoptosis appears to be maximal at 48-72 hours. In
addition, alleviation of cell cycle arrest with drugs such as
caffeine largely eliminated induction of apoptosis (5). On the
other hand, mutants of Vpr have been described, which are able to
partially dissociate both phenotypes (89, 75, 29, 43). The present
invention provides a different model, where G.sub.2 arrest and
apoptosis are induced concomitantly, since both are dependent on
activation of the same kinase, ATR. However, for reasons that were
not understood, apoptosis and G.sub.2 arrest develop with different
kinetics, such that G.sub.2 arrest peaks first. Without wishing to
be bound by theory, two different targets of ATR may initiate the
G.sub.2 arrest and apoptotic responses. These targets are believed
to be CHK1 and BRCA1/GADD45, respectively. In support of this
model, the data demonstrates that siRNA-mediated knockdown of ATR
effectively abrogates both responses.
[0050] The present invention demonstrates that, in contrast to the
studies by Veira et al. and Jacotot et al. (67, 88), the treatment
of vpr-infected cells with caffeine, which inhibits the DNA
damage-signaling proteins ATM and ATR, significantly reduces
Vpr-induced apoptosis (53). This observation indicates that Vpr
first induces stress signals that are similar or identical to those
induced by certain forms of genotoxic stress, and then these
signals activate a proapoptotic signaling cascade. It has recently
been found that ATR is the mediator of Vpr-induced DNA damage-like
signals (40). It was reasoned that if Vpr induces apoptosis by
directly binding and controlling the PTPC, then signaling through
ATR would still be necessary for induction of G.sub.2 arrest, but
would be dispensable for induction of apoptosis. Conversely, if ATR
activation was required for induction of apoptosis, then
examination of potential pro-apoptotic phosphorylation targets of
ATR should identify a specific target or set of targets, that would
mediate the signaling events between ATR activation and
apoptosis.
[0051] ATR targets initiate signaling cascades that may result in
three global effects: cell cycle blockade, recruitment of DNA
repair/transcription factors, and induction of apoptosis. The
present invention shows that both RAD17 and H2AX are targets of
Vpr. Because p53 has been previously ruled out as a mediator of
apoptosis induced by Vpr (41, 87), another possible pro-apoptotic
target of ATR, BRCA1 was examined.
[0052] In response to genotoxic insults, BRCA1 is recruited to
sites of DNA damage and is phosphorylated by both. ATM and ATR (45,
58). BRCA1 has been proposed to play a distinct role in DNA repair
and apoptosis as a transcriptional regulator of genes including
Cyclin B1, p53R2, MDM2, and p53 (72). Recently, GADD45 was
identified as a transcriptional target of BRCA1 (65).
[0053] GADD45 was originally identified in Chinese hamster cell
lines as one of several genes rapidly induced by `UV radiation
(61). GADD45 is induced by a variety of genotoxic stresses
including ionizing radiation (IR), medium starvation, and methyl
methanesulfonate (MMS) (69, 78), and has been shown to play roles
in both G.sub.2/M arrest and apoptosis following DNA damage (68,
94). Harkin et al. demonstrated that BRCA1-induced upregulation of
GADD45 resulted in. JNK/SAPK-dependent apoptosis (65). In the
present invention, targets of ATR with possible roles in
Vpr-induced apoptosis were examined and Vpr-induced apoptosis was
found to be signaled through the DNA damage signaling protein ATR,
which initiates a pathway that involves activation of BRCA1 and
upregulation of GADD45. Hence, both ATR and GADD45 are required for
Vpr-induced apoptosis.
[0054] Loss of CD4.sup.+ lymphocytes over the course of an HIV-1
infection plays a central role in disease, progression and immune
suppression in AIDS patients (reviewed in ref. (66)). However, the
mechanism by which CD4.sup.+ T cells are lost is poorly understood.
Several mechanisms have been proposed to explain the loss of
CD4.sup.+ T cells in HIV1-infected patients, including direct
killing by HIV-1 infection, CD8.sup.+ T cell-mediated killing of
infected CD4+ phocytes, and apoptosis of uninfected "bystander"
cells. In addition, the HIV-1 proteins Tat, Rev, Vpu, Nef and Vpr
have been implicated in the apoptosis of infected and/or bystander
cells (reviewed in ref. (79)).
[0055] Previous reports have demonstrated in vitro binding of Vpr
to the PTPC, which resulted in the release of cytochrome C from
fractionated mitochondria (88, 67). These observations suggest that
Vpr induces mitochondrial depolarization directly rather than
activating upstream stress receptors, such as ATR. However, the
model proposed by Jacotot et al. does not explain the observation
that Vpr-expressing cells undergo apoptosis in a cell
cycle-dependent manner, specifically from G.sub.2. The observations
of Jacotot et al. would also suggest that Vpr induces apoptosis
rapidly after being expressed (67), in contrast, observations made
with virus infection indicate that apoptosis induced by Vpr is
maximal at day 3 post-infection.
[0056] The present invention demonstrates that Vpr induces
formation of distinct BRCA1 foci within the nucleus of
vpr-expressing cells, concomitant with Vpr-induced apoptosis and
G.sub.2 arrest. Vpr also induces ATR-dependent phosphorylation of
BRCA1 at serine 1423, which is indicative of BRCA1 activation
following genotxic stress (45). It has been suggested that BRCA1
plays a role in transcriptional regulation of genes involved in
cell cycle arrest, apoptosis, and DNA repair (71, 72).
Specifically, overexpression of BRCA1 resulted in transcriptional
upregulation of GADD45 (65, 72). Data presented herein shows that
activation of BRCA1 is concomitant with upregulation of GADD45.
Upregulation of GADD45, by Vpr, may require BRCA1, which is tested
by knockdown of BRAC1 or use of BRAC1.sup.-/- subjects. BRCA1
C-terminus acts as a transactivation domain that has been suggested
to play a critical role in cancer development.
[0057] Interestingly, upregulation of GADD45 by Vpr does not result
in activation of the MAP Kinases p38 or INK (65, 73). In the
context of reports from Harkin et al. demonstrating that
overexpressed BRCA1 results in GADD45 upregulation and
JNK-dependent apoptosis (65), the present results suggest that a
JNK- and p38-independent pathway is active in Vpr-induced
apoptosis. Wang et al. demonstrated that GADD45-deficient
fibroblasts are capable of JNK activation following DNA damage, and
wild-type fibroblasts, in response to UV radiation, showed JNK
activation prior to GADD45 upregulation (90). These data suggest
that the proapoptotic effects of GADD45 may be signaled by a
pathway that circumvents activation of the MAP Kinases, p38 and
INK. GADD45 is able to associate with several other proteins,
including p21.sup.Waf1, CDC2, and the proliferating subject nuclear
antigen (PCNA) (57,70, 83, 94). These. GADD45 partners are assayed
for a role in apoptosis induced by Vpr.
RNAi
[0058] The RNAi pathway consists of the presentation of a
"triggering" dsRNA that is subsequently processed into siRNAs by an
RNaseIII-like enzyme, for example, Dicer (Zamore, P. D. et al.,
RNAi: double-stranded RNA directs the ATP-dependent cleavage of
mRNA at 21 to 25 nucleotide intervals, 101 Cell 25(2000);
Hutvagner, G. and Zamore, P. D., RNAi: nature abhors a
double-strand, 12 Curr. Opin. Genet. Dev. 225 (2002)). This siRNA
species, which may be about 19 to about 25 by in length, is then
incorporated into a multi-subunit RNA-induced silencing complex,
which targets the unique cellular RNA transcript for enzymatic
degradation. RNA hydrolysis occurs within the region of homology
directed by the original siRNA (Fibashir, S. M. et al., RNA
interference is mediated by 21 and 22 nucleotide RNAs, 15 Genes
Dev. 188 (2001)), thereby selectively inhibiting target gene
expression.
[0059] dsRNA activates a normal cellular process leading to a
highly specific RNA degradation, and a cell-to-cell spreading of
this gene silencing effect in several RNAi models. (Shuey, et al,
RNAi: gene-silencing in therapeutic intervention, 7(20) Drug
Discovery Today 1040 (2002)). Injection of dsRNA, for example, acts
systemically to cause post-transcriptional depletion of the
homologous endogenous RNA in C. elegans (U.S. Pat. Appl. Pub. No.
2003/0084471 A1). This depletion of endogenous RNA causes effects
similar to a conditional gene `knock out,` revealing the phenotype
caused by the lack of a particular gene function. C. elegans
nematodes can, for example, be fed with bacteria engineered to
express dsRNA corresponding to a C. elegans target gene. Nematodes
fed with engineered bacteria show a phenotype similar to mutants
containing a mutation in the target gene (1998 Nature 395: 854).
Likewise, RNAi may be used in other subjects.
[0060] To circumvent the limitations of transfection efficiency,
while retaining desirable sustained RNAi expression, a selection
marker may be incorporated. This method allows for the selection of
cells having the RNAi molecule, using the selection marker to sort
such cells. Briefly, the method comprises introducing an RNAi
molecule or molecule capable of producing the RNAi and a selection
marker or molecule capable of producing the selection marker, and
sorting the cells based on the presence of the separation marker.
Hence, such RNAi technology may be used to enrich a population of
cells transfected with RNAi, such as BRAC1 RNAi, and the selectable
marker, thereby compensating for low transfection efficiency.
[0061] Proteins and/or peptides disclosed herein may be synthesized
using D-amino acids or other amino acid modifications known in the
art. For example, Vpr may be produced using one or more D-amino
acids to reduce proteolysis and/or degradation.
[0062] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems may be
used to provide a recombinant protein or protein fragment. The
methods of transformation, transfection or transduction, and the
choice of expression vehicle (vector), will depend on the host
system selected. Transformation and transfection methods are
described, e.g., in Ausubel, F. M. et al. (eds) (1997) CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons.; expression
vehicles may be chosen from those provided, for example, in Cloning
Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp.
1987) or known in the art.
[0063] Constructs of the invention may be prepared for introduction
into a prokaryotic or eukaryotic host and may comprise a
replication system recognized by the host, including the intended
polynucleotide fragment encoding the desired polypeptide, and will
preferably also include transcription and translational initiation
regulatory sequences operably linked to the polypeptide encoding
segment. The choice of vector will often depend on the host cell
into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, a viral or phage vector, a
transposable element, an integrating vector or an extrachromosomal
element, such as a minichromosome or an artificial chromosome. Such
vectors may be prepared by means of standard recombinant techniques
well known in the art. See for example, see Ausubel (1992);
Sambrook and Russell (2001); and U.S. Pat. No. 5,837,492.
[0064] The proteins of the invention may be cotranslationally,
post-translationally or spontaneously modified, for example, by
acetylation, famesylation, glycosylation, myristoylation,
methylation, prenylation, phosphorylation, palmitoylation,
sulfation, ubiquitination and the like. See, Wold, F. (1981), Annu.
Rev. Biochem. 50:783-814.
[0065] The present invention allows for the treatment of cancer,
for example, familial breast cancer, by introducing Vpr and BRAC1,
or a functional fragment thereof, into such a subject having or
thought to have ATR activity. Since the absence of ATR prevents
activation of BRAC1 and is required for Vpr-induced apoptosis, the
invention allows for the treatment of cancerous cells lacking one
or more of the functional forms of a required proteins. As will be
evident to a person of skill in the art in light of the present
invention, the disease state and known or postulated mutations may
be appropriately matched to the proteins disclosed herein and the
treatment tailored to the particular disease. Hence, the invention
provides a method of treating a cancerous cell lacking ATR, RAD17,
HUS1, BRAC1 and/or GADD45 function by introducing Vpr and the
appropriate protein, or a functional fragment thereof, into the
cancerous cell, thereby inducing G.sub.2 cell cycle arrest and/or
apoptosis.
[0066] Obesity is an increasingly prevalent human condition and,
although recent progress has been made in understanding the
underlying mechanism, no safe and effective treatment exists on the
market. The present invention provides a compound and/or method of
inducing GADD45 and/or activating BRAC1 in adipose tissue or
adipose tissue supporting vasculature of a subject, comprising a
targeting motif, such as a CKGGRAKDC (SEQ ID NO:1) peptide and/or
one or more peptides as disclosed in U.S. Patent Publication
20040170955, published Sep. 2, 2004, linked to Vpr and/or a
functional fragment of Vpr, wherein a functional fragment is a
fragment capable of activating BRAC1 and/or inducing GADD45 in a
subject. In another embodiment, a method of activating BRAC1 and/or
inducing GADD45 is provided, wherein Vpr or a functional fragment
thereof is administered to a subject.
[0067] Other tissue targeting moieties include, but are not limited
to, a molecule which is bound by a receptor and transported into a
cell by a receptor-mediated process, such as glucose, galactose,
mannose, mannose 6-phosphate, transferrin, asialoglycoprotein,
.alpha.-2-macroglobulins; insulin, a peptide growth factor,
cobalamin, folic acid or derivatives, biotin or derivatives,
YEE(GalNAcAH).sub.3 or derivatives, albumin, texaphyrin,
metallotexaphyrin, porphyrin, any vitamin, any coenzyme, an
antibody, an antibody fragment (e.g., Fab) and a single chain
antibody variable region (scFv), cobalamin and/or cobalamin
analogues or derivative. For example, studies have shown that the
absorption of physiological amounts of vitamin B.sub.12 by the gut
requires that it be complexed with a naturally occurring transport
protein known as intrinsic factor (IF). (Castle, 1953; Fox and
Castle, Allen and Majerus. 1972b). Folic acid, folinic acid,
pteropolyglutamic acid, and folate receptor-binding pteridines such
as teirahydropterins, dihydrofolates, tetrahydrofolates and their
deaza and dideaza analogs are useful as targeting molecules in
accordance with the present invention. The terms "deaza" and
"dideaza" analogs refer to the art-recognized analogs having a
carbon atom substituted for one or two nitrogen atoms in the
naturally-occurring folic acid structure. For example, the deaza
analogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and
10-deaza analogs. The dideaza analogs include, for example,
1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs.
The foregoing folic acid derivatives are conventionally termed
"folates," reflecting their capacity to bind with folate-receptors,
and such ligands when complexed with exogenous molecules are
effective to enhance trans-membrane transport. Other folates useful
as complex forming ligands for this invention are the folate
receptor binding analogs aminopterin, amethopterin (methotrexate).
N.sup.10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such
as 1-deazamethopterin or 3-deazamethopterin, and
3'5'-dichloro4-amino-4-deoxy-N.sup.10-methylpteroyl-glutamic acid
(dichloromethotrexate). In addition, biotin analogs such as
biocytin, biotin sulfoxide, oxybiotin and other biotin
receptor-binding compounds are ligands that may also be used as
suitable targeting molecules to promote the trans-membrane
transport of exogenous molecules, such as the proteins described
herein. Other suitable ligands capable of binding to receptors to
initiate receptor-mediated endocytotic transport of the complex
include anti-idiotypic antibodies to the folate receptor. An
exogenous molecule in complex with an anti-idiotypic antibody to a
receptor is used to trigger trans-membrane transport of the
complex. Such molecules are used in accordance with the present
invention as a targeting molecule (see, U.S. Pat. No. 6,315,978).
Any of these targeting moieties may be linked to a protein or
functional fragment thereof, such as Vpr, ATR, RAD17, HUS1, BRAC1,
GADD45; thereby allowing the targeting of the protein or functional
fragment thereof to a desired cell type.
[0068] Linker molecules are known in the art and include, but are
not limited to, organic molecules, such as one or more amino acids
or other hydrocarbon chains, or one or more carbohydrate molecules,
such a sugar unit, which may be modified such that the modified
sugar and/or linker is resistant to cleavage. The sugars of a
linker may be modified by methods known in the art, for example, to
achieve resistance to nuclease cleavage. Examples of modified
sugars include, but are not limited to, 2'-O-alkyl riboses, such as
2'-O-methyl ribose, and 2'-O-allyl ribose. The sugar units may be
joined by phosphate linkers. The linker may comprise a hydrogen,
and/or a straight or branced, substituted or unsubstituted, alky,
aryl, alkene, alkyne, alkylaryl, and combinations thereof, wherein
the linker does not abolish biologically activity, unless such
abolition is at least partially relieved upon cleavage. Preferable,
such cleavage is produced in a subject, more preferably in a target
tissue in the subject.
[0069] Subjects contemplated by the invention include, but are not
limited to, bacteria, cells, cell culture systems, plants, fungi,
animals, such as an animal disease model, nematodes, insects,
and/or mammals, such as humans.
[0070] The peptides of the invention may be formulated as a
pharmaceutically acceptable compound or composition. Excipients,
diluents and/or carriers are known in the art, for example, see
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa.) and GOODMAN AND GILMAN'S, THE
PHARMACOLOGICAL BASIS OF THERAPEUTICS (10.sup.th ed. 2001).
[0071] Apoptotic function, or apoptosis may be screened using any
of the methods known in the art or described herein.
Materials and Methods
[0072] Cell lines and primary cells. The human cervical cancer cell
line, HeLa, was maintained in Dulbecco's modified Eagle's medium
(DMEM) (BioWhittaker, Walkersville, Md.) supplemented with 10%
fetal calf serum (FCS), with or without 1%
penicillin-streptomycin-L-glutamate (PSG) (Invitrogen, Carlsbad,
Calif.). The human T-cell line SupT1 was propagated in RPMI 1640
(BioWhittaker, Walkersville, Md.) supplemented with 10% FCS.
Primary human CD4.sup.+ lymphocytes were first isolated in buffy
coats using vacutainer cell preparation tubes according to the
manufacturer's protocol (Becton Dickinson, Franklin Lakes, N.J.).
Buffy coats were then purified further using a CD4.sup.+ isolation
kit (Dynal Biotechnology, Olso, Norway,) according to the
manufacturer's instructions. Isolated lymphocytes were cultured in
RPMI 1640 supplemented with 100 u/ml IL-2 (National Institute of
Health, AIDS research and reference reagent program, Rockville,
Md.), 6 .mu.g/ml Phytohemaglutinin (Sigma Aldrich, St. Louis, Mo.;
L-9017), and 10% FCS, for a period of 4 days prior to transduction.
Following transduction, primary lymphocytes were cultured in RPMI
1640 supplemented with 10% FCS and 100 u/ml IL-2. Hus1.sup.-/-
p21.sup.-/- and Hus1.sup.+/+ p21.sup.-/- mouse embryonic
fibroblasts were cultured on glycerin-coated plates with RPMI
medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS),
1% PSG, and 10 mM nonessential amino acids (Invitrogen). Primary
human thymic cultures were prepared as previously described
(44).
[0073] RNA interference. All siRNA treatments were performed with
Dharmacon smart pool siRNA duplexes: GADD45 (Dharmacon, Lafeyette,
Colo.; M-003893-00), ATR (Dharmacon, Lafeyette, Colo.;
M-003202-01), ATM (Dharmacon, Lafeyette, Colo.; M-003201-01), CHK2
(Dharmacon, Lafeyette, Colo.; M-003256-03), and Scrambled siRNA
(Dharmacon, Lafeyette, Colo.; D-001206-13-05). Smart pool siRNAs
were transfected at a final concentration of 100 nm into
exponentially growing. HeLa cells with Lipofectamine 2000 or
Oligofectamine (Invitrogen, Carlsbad, Calif.), all according to
manufacturers' protocols.
[0074] Immunofluorescence staining. HeLa cells were harvested 48 h
posttransduction by trypsinization. Single-cell suspensions of
minced thymic tissue were prepared for immunostaining 20 h after
HIV-1NL4-3 infection. Cells were fixed with 2% parafomialdehyde in
phosphate-buffered saline (PBS) for 35 min at 4.degree. C. and then
washed three times for 5 min each time in PBS. All subsequent steps
were carried out at room temperature. Samples were blocked and
permeabilized for 20 min in blocking buffer (3% bovine serum
albumin (BSA), 0.2% Triton X-100, and 0.01% skim milk in PBS).
Primary antibody (rabbit anti-.gamma.-H2AX (48) or rabbit
anti-BRCA1 (Bethyl Laboratories, Montgomery, Tex.)) was diluted
1:400 in incubation buffer (1% BSA and 0.02% Triton X-100 in PBS)
and incubated with cells for 45 min. Cells were washed with PBS,
after which secondary antibody (goat anti-rabbit immunoglobulin G
(IgG)-AlexaFluor568 conjugate (Molecular Probes, Eugene, Oreg.))
diluted 1:500 in incubation buffer was applied for 35 min. Cells
were washed with PBS as before and mounted on glass slides by using
FluorSave reagent (CalBiochem, San Diego, Calif.). Cells were
visualized for .gamma.-H2AX or BRCA1 immunostaining and green
fluorescent protein (GFP) expression by scanning fluorescence
confocal microscopy (Fluo View FV300; Olympus, Melville, N.Y.).
[0075] Cell cycle analysis. At 48 h after infection, cells were
detached by trypsinization, washed with fluorescence-activated cell
sorting (FACS) buffer (2% FBS and 0.02% sodium azide in PBS), fixed
with 2% paraformaldehyde in PBS, and permeabilized with 0.01%
Triton X-100 in PBS for 15 min. Cells were washed again with FACS
buffer, incubated in DNA staining buffer (10 .mu.g of propidium
iodide/ml and 11.25 kU of RNase A/ml in FACS buffer) for 15 min,
and analyzed by FACScan flow cytometry for GFP expression or DNA
content (Beckton Dickinson, Franklin Lakes, N.J.). In experiments
involving transduction with lentivirus vectors, experiments with
90% transduction efficiency or higher were analyzed. Cell cycle
profiles were modeled by using ModFit software (Verity Software,
Topsham, Me.).
[0076] Lentivirus vectors. Lentivirus vectors were produced by
transient transfection of HEK293T cells. For defective lentivirus
vector production, plasmids pHRGFP and pHR-Vpr were cotransfected
with pCMV.DELTA.R8.2.DELTA.Vpr (4) and pHCMV-VSVG (3) by calcium
phosphate-mediated transfection (53). Virus supernatants were
collected at 48, 72, and 96 h posttransfection. Harvested
supernatants were cleared by centrifugation at 2,000 rpm. Cleared
supernatants were concentrated by ultracentrifugation at 25,000 rpm
for 1.5 h at 4.degree. C. Concentrated virus was allowed to
resuspend overnight at 4.degree. C., and the suspension was frozen
at -80.degree. C. for storage. Vector titers were measured by
infection of HeLa cells as described herein, followed by flow
cytometric analysis of cells that were positive for the reporter
molecule, GFP. Vector titers were calculated with the equation
[(F.times.C.sub.0)/V].times.D, where F is the frequency of
GFP-positive cells found by flow oytometry, C.sub.0 is the total
number of target cells at the time of infection, V is the volume of
inoculum, and D is the virus dilution factor. The virus dilution
factor used for titrations was 10. The total number of target cells
at the time of was 10.sup.6. Infections were performed at a
multiplicity of infection (MOI) of 2 or 2.5 with 10 .mu.g of
Polybrene/ml for 3 h. Infections of siRNA treated cells were
performed 48 h after siRNA transfection.
[0077] HIV-1 infection. HIV-1.sub.NL4-3 stocks were prepared as
previously described (20), diluted in Iscove's medium supplemented
with 2% FBS and used to infect primary thymocytes at an MOI of
1.0.
[0078] Drug treatment. Cells were incubated with 10 mM hydroxyurea
(HU) for 2 h before immunostaining was done.
[0079] Transduction methods. All transductions were carried out
using a multiplicity of infection of 2. SupT1 and primary CD4+
lymphocytes were transduced with virus diluted in cell culture
media with 8 .mu.g/ml polybrene. Transduction was performed as
previously described (51). HeLa cells were transduced in 6 well
plates with virus diluted into cell culture media with 10 .mu.g/ml
polybrene. After 6 hours, virus was replaced with fresh culture
media. Transduction efficiencies were verified by flow cytometry
for each experiment to ensure that efficiencies were similar
between treatments.
[0080] Western blotting procedures. All western blots were
performed using the BioRad Criterion gel system (BioRad, Hercules,
Calif.). Antibodies used were GADD45 (Santa Cruz Biotechnology,
Santa Cruz, Calif.; sc-797), Actin (Santa Cruz Biotechnologies,
Santa Cruz, Calif.; sc-797), CHK2 (Santa Cruz Biotechnology, Santa
Cruz, Calif.; sc-8813), ATM (Novus Biologicals, Littleton, Colo.;
catalog #100-104H1), phospho-JNK-Tyr183 (Cell Signaling Technology,
Beverly, Mass.; catalog #9255S), phospho-p38 kinase (Promega,
Madison; 15823207), JNK (Cell Signaling Technology, Beverly, Mass.;
9252), ATR (obtained from Dr. Paul Nghiem, Harvard), anti-rabbit
secondary-HRP (Santa Cruz Biotechnology, Santa Cruz, Calif.;
sc-2030), anti-goat secondary-HRP (Santa Cruz Biotechnology, Santa
Cruz, Calif.; se-2033), PARP. (Cell Signaling Technology, Beverly,
Mass.), BRCA1 phospho ser1423 (Bethyl Laboratories, Montgomery,
Tex.) Rabbit primary antibodies against Rad17 (Santa Cruz
Biotechnology, Santa Cruz, Calif.), and were applied for 90 min at
room temperature. Blots were washed three times in TPBS (0.1% Tween
20 in PBS) for 10 min each time at room temperature.
[0081] Secondary horseradish peroxidase-conjugated goat anti-rabbit
IgG antibodies were applied for 45 min at room temperature. Blots
were washed again three times in TPBS before protein detection with
enhanced chemiluminescence reagent (Amersham). HAtagged Vpr protein
was detected with anti-HA antibodies. SupT1 cells were treated with
Anisomycin (Sigma Aldrich, St. Louis, Mo.) at a concentration of 25
.mu.g/ml for 30 minutes, then lysed immediately. Changes in protein
levels observed by Western blot were assessed by densitometry
scanning.
[0082] Cells were detached at the time of cell cycle analysis and
lysed in Laemmli sodium dodecyl sulfate (SDS) sample buffer (60 mM
Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue, and 14.4 mM
2-mercaptoethanol in double-distilled H.sub.2O) at a concentration
of 5.times.10.sup.5 cells/100 .mu.l of buffer. Lysates were boiled
for 5 min prior to being loaded on SDS-10% polyacrylamide gels for
electrophoretic separation. Proteins were transferred to
polyvinylidene difluoride membranes by a semidry transfer method
(Bio-Rad, Hercules, Calif.) and then blocked for 45 min at room
temperature in blocking solution (5% skim milk and 0.1% Tween 20 in
PBS).
[0083] Apoptosis assays. Cells were fixed in 2% paraformaldehyde
(in PBS) for 15 minutes at room temperature. Fixed cells were then
permeabilized in 0.1% Triton X-100 (in PBS) for 15 minutes at room
temperature, then washed 2 times in PBS and incubated in 0.5
.mu.g/ml 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)
(Molecular Probes, Eugene, Oreg.) dissolved in PBS, for 45 minutes
at 37.degree. C. DAPI-treated cells were then analyzed by
fluorescence microscopy. Random fields were chosen throughout the
dish and apoptotic cells were marked by the presence of fragmented
nuclei. Total apoptotic cells from 3 independent experiments were
counted and divided by the total cell number to obtain a percentage
of apoptotic cells for each treatment with standard deviations. A
minimum of 1000 cells were counted per treatment/per experiment.
PARP cleavage was assayed by Western blot as described above.
[0084] In vitro kinase assay. SupT1 cells were infected with
pHR-VPR and as a VPR-minus control, pHR-GFP, then lysed at 48 hours
post-transduction with supplied lysis buffer. JNK kinase activity
was measured with the SAPK/JNK non-radioactive assay kit (Cell
Signaling Technology, Beverly, Mass.), according to the
manufacturer's protocol.
EXAMPLE I
[0085] Rad17 is necessary for Vpr-mediated G.sub.2 arrest. To
examine the role of Rad17 in Vpr-mediated G.sub.2 arrest, RNA
interference was used to reduce endogenous Rad17 levels.
Transfected siRNA duplex oligonucleotides targeted at Rad17 mRNA
(54) were used to knock down Rad17, in parallel, siRNA with a
nonspecific target sequence and mock transfection was used as
controls. In these experiments, endogenous Rad17 protein levels
were reduced by approximately 85%, relative to those of
mock-transfected or nonspecific siRNA-transfected cells (FIG. 1A).
Following transfection, cells were transduced with lentivirus
vectors expressing either Vpr and GFP cDNAs separated by an
internal ribosome entry site (pHR-Vpr) or GFP alone (pHRGFP) (40)
(79). At 48 h after transduction, cells were analyzed by flow
cytometry for infection efficiency and DNA content, as reported by
GFP expression and propidium iodide staining, respectively. Cell
cycle distributions of the various experimental cell populations
were analyzed after electronic gating of GFP-positive (transduced)
and GFP-negative (untransduced) cells. Infection with pHR-GFP did
not affect the cell cycle profile of any of the transfected
populations. Infection with pHR-Vpr induced a marked accumulation
of cells in G.sub.2 phase (FIG. 1B). When cells were pretreated by
transfection with a Rad17-specific siRNA (but not with nonspecific
siRNA), Vpr-induced accumulation in G.sub.2 was dramatically
relieved (FIG. 1B). Therefore, Rad17 is required for activation of
the G.sub.2 checkpoint by Vpr.
EXAMPLE II
[0086] Hus1 is necessary for Vpr-mediated G.sub.2 arrest. Finding
that Rad17 is necessary for Vpr-induced G.sub.2 arrest, another
constituent of the ATR signaling pathway, Hus1, was examined. To
examine the role of Hus1 in Vpr-induced G.sub.2 arrest,
Hus1.sup.-/- p21.sup.-/- mouse embryonic fibroblasts (49) were
used. Both Hus1.sup.-/- p21.sup.-/- cells and Hus1.sup.+/+
p21.sup.-/- cells exhibited normal cell cycle distributions when
mock infected or infected with pHR-GFP (FIG. 2). Hus1.sup.-/-
p21.sup.-/- cells, how however, failed to arrest in G.sub.2 after
infection with pHR-Vpr, whereas their Hus1.sup.+/+ counterparts
exhibited robust G.sub.2 arrest (FIG. 2). These experiments
illustrate a requirement for Hus1 in Vpr-induced G.sub.2
arrest.
[0087] Taken together, the observations indicate that HIV-1 Vpr
activates the G.sub.2 checkpoint in a manner that is
mechanistically similar to that of certain genotoxic agents
(specifically, HU) that cause replication inhibition. Because
recognition of DNA damage via ATR can lead to dramatic cellular
changes, other than checkpoint activation, the consequences of ATR
activation were examined. For example, the effect of Vpr on
.gamma.-H2AX was examined, as well as, BRCA1, because they have
been reported to recruit DNA repair factors and induce
apoptosis.
EXAMPLE III
[0088] Vpr expression induces .gamma.-H2AX and BRCA1 focus
formation. It was possible that Vpr, through ATR activation, would
induce .gamma.-H2AX (9) and. BRCA1 (45) focus formation. To test
this hypothesis, HeLa cells were infected with pHR-Vpr or pHR-GFP
and, 48 h later, immunostained with .gamma.-H2AX- or BRCA1-specific
antibodies. As a positive control, nontransduced cells were treated
for 1 h with 10 mM HU 10 min prior to immunostaining (46). Samples
then were visualized by fluorescence scanning confocal microscopy
(FIGS. 3A and B). Cells with multiple (10 or more), intense nuclear
foci were manually counted. These quantitations are presented in
FIG. 3C. Approximately 93 or 69% of Vpr-expressing cells exhibited
significant .gamma.-H2AX or BRCA1 focal staining, respectively,
whereas only 9 or 16% of pHR-GFPinfected cells exhibited any
.gamma.-H2AX or BRCA1 foci, respectively. Less than 8% of
mock-infected cells exhibited .gamma.-H2AX or BRCA1 foci.
Approximately 94 or 65% of HU-treated cells exhibited .gamma.-H2AX
or BRCA1 foci, respectively. Therefore, it is concluded that Vpr
expression leads to .gamma.-H2AX and BRCA1 focus formation.
However, the phosphorylation status of BRCA1 was not formally
proven by the above experiments, because recognition by the
BRCA1-specific antibody used in this experiment was not dependent
on BRCA1 phosphorylation.
[0089] Phosphorylation of BRCA1 at serine 1423. by HIV-1 Vpr is
ATR-dependent. Following DNA damage, ATR phosphorylates BRCA1 at
serine 1423 (45, 63). To determine whether Vpr induced the
phosphorylation of BRCA1 at serine 1423 in an ATR-dependent manner,
HeLa cells were infected with pHR-VPR and examined for the
phosphorylation of BRCA1 at serine 1423, in the presence of either
non-specific or ATR-specific siRNA. Vpr induced phosphorylation of
BRCA1 at serine 1423 (FIG. 7A). Treatment of HeLa cells with
ATR-specific siRNA prior to transduction relieved Vpr-induced
phosphorylation of BRCA1, which indicated the phosphorylation was
ATR-dependent (FIG. 7A).
EXAMPLE IV
[0090] HIV-1 infection induces .gamma.-H2AX foci in primary
CD4.sup.+ thymocytes. The G.sub.2 arrest effect of Vpr is identical
in many human cell lines tested and in primary lymphocytes (21, 41,
53). Therefore, HeLa cells, although not a target for HIV-1,
constitute model cells in which to study the mechanism of G.sub.2
arrest by Vpr. Nonetheless, to confirm our observations in HeLa
cells, primary human CD4.sup.+ cells infected with full-length
HIV-1 were tested. Primary human CD4.sup.+ thymocytes were infected
with full-length HTV-1.sub.NL4-3 (2) or mock infected. At 20 h
after infection, cells were immunostained for .gamma.-H2AX (FIG.
3D). NL4-3 infection caused a staining pattern of distinct
.gamma.-H2AX nuclear foci that was not observed in mock-infected
cells. These data indicate that full-length HIV-1 induces
.gamma.-H2AX focus formation in primary CD4.sup.+ cells and confirm
the applicability of HeLa cells.
EXAMPLE V
[0091] ATR, but not ATM, is necessary for Vpr-induced G.sub.2
arrest. ATR is primarily responsible for G.sub.2 checkpoint
activation via Chk1 phosphorylation (25). However, it has been
shown that ATM, which, acts primarily on Chk2, can play a minor,
more transient role in Chk1 phosphorylation (1). Although Bartz and
colleagues demonstrated that ATM.sup.-/- cells were able to arrest
in G.sub.2 in response to Vpr (8), a partial role for ATM would be
formally possible. Specifically, two observations prompted
reexamination of the role of ATM. First, suppression of ATR or Chk1
by RNA interference is typically unable to completely relieve
Vpr-induced G.sub.2 arrest (40). It is possible that residual ATR
and/or Chk1 levels were responsible for the partial accumulation of
cells in G.sub.2. Alternatively, the low level of G.sub.2 arrest in
the context of ATR- and/or Chk1-specific inhibition could have been
attributable to ATM. The second finding that prompted reexamination
of the role of ATM was that caffeine, an inhibitor of both ATR and
ATM, was able to completely relieve Vpr-induced G.sub.2 arrest (40,
53).
[0092] To test the potential contribution of ATM activity to
Vpr-induced G.sub.2 arrest, siRNA directed at ATR and ATM were
transfected, in combination or separately, the cells were then
infected with pHR-VPR or control vectors. As expected, pretreatment
with ATR-specific siRNA produced a marked, although incomplete,
alleviation of G.sub.2 arrest by Vpr (FIG. 4). Pretreatment with.
ATM-specific siRNA, which reduced ATM protein levels by 85%,
relative to those in mock-treated cells (FIG. 4A), produced no
change in cell cycle arrest by Vpr compared with the results seen
in cells transfected with nonspecific siRNA or no siRNA (FIG. 4B).
In addition, simultaneous suppression of ATR and ATM did not
produce any additional relief of G.sub.2 arrest (data not shown).
Therefore, these results indicate that ATM is dispensable for
Vpr-induced G.sub.2 checkpoint activation (8).
EXAMPLE VI
[0093] ATR is required for apoptosis induced by HIV-1 Vpr.
Vpr-induced G.sub.2 arrest is signaled via the ATR DNA damage
pathway. To investigate ATR in the context of Vpr-induced
apoptosis, HeLa cells were transfected with short-interfering RNA
(siRNA) duplexes directed at ATR or non-specific siRNA and then
cells were transduced with lentiviral vectors expressing either
HIV-1NL4-3 Vpr and GFP (pHR-VPR), or GFP alone (pHR-GFP). The
construct, pHR-VPR, expresses Vpr and GFP from a dicistronic mRNA
that uses an intervening internal ribosome entry site (IRES) (77,
79). To examine Vpr-induced apoptosis, cells were treated with the
nuclear stain, 4'6-diamidino-2-phenylindole dihydrochloride (DAPI),
and the nuclear morphology (FIG. 6A) was examined. Treatment of
pHR-VPR-transduced cells with ATR-specific siRNA resulted in a 67%
decrease in apoptosis (FIG. 6B). This reduction in apoptosis
correlated with a reduction in G2 arrest. As a control, siRNAs
against ATM or CHK2 were used. ATM is a close relative of ATR that
is dispensable for Vpr-induced G2 arrest (see, Example V). CHK2 is
a checkpoint kinase that is activated by ATM. Knockdown of ATM or
CHK2 produced no appreciable changes in the level of apoptosis
induced by Vpr (FIG. 6B). None of the siRNA treatments had a
significant effect on apoptosis in mock-treated or
pHR-GFP-transduced cells (FIG. 1b). In addition to measuring
apoptosis by DAPI staining, the results were confirmed by measuring
caspase-induced cleavage of poly(ADP-ribose) polymerase (PARP).
PARP cleavage produces an 89 kDa fragment that is an early result
of caspase activation which precedes DNA cleavage (84, 85), and is
essential for progression into apoptosis (reviewed in (56)). ATR
knockdown resulted in a marked decrease in PARP cleavage compared
to nonspecific, ATM, and CHK2 siRNA treatments (FIG. 6C, compare
lanes 2,3,5,6). Knockdown of the corresponding proteins by each of
the siRNAs was evaluated by Western blot (FIG. 6D). To rule out the
possibility that ATR- or GADD45-specific siRNA treatments may
relieve the effects of Vpr by disrupting expression of Vpr itself
rather than affecting the function of ATR or GADD45, Vpr protein
levels were analyzed by Western blot analysis (FIG. 6E). None of
the siRNA treatments had any, appreciable effect on Vpr protein
levels.
EXAMPLE VII
[0094] Knockdown of GADD45 relieves Vpr-induced apoptosis. Recent
reports have suggested that GADD45 is a transcriptional target of
BRCA1 (65, 71, 72), involved in the induction of apoptosis (65).
Based on these reports, and the present observation that BRCA1 is
activated in response to Vpr, GADD45 was examined for a role in
Vpr-induced apoptosis. To examine whether GADD45 is required for
Vpr-induced apoptosis, HeLa cells were treated with non-specific
siRNA and GADD45-specific siRNA, transduced with vpr- or
gfp-expressing viruses, and apoptosis measured by DAPI staining
(FIG. 6A) Efficient knockdown of GADD45 resulted in a 70% decrease
in Vpr-induced apoptosis (FIG. 6B). ATM, CHK2, and non-specific
siRNA treatments had any appreciable effect on Vpr-induced
apoptosis (FIG. 6B). PARP cleavage was then assayed to verify the
results from our DAPI experiments. As observed with ATR knockdown,
GADD45 knockdown prior to pHR-VPR transduction resulted in a marked
reduction in PARP cleavage (FIG. 6C, compare lanes 2, 4-6).
EXAMPLE VIII
[0095] HIV-1 Vpr upregulates GADD45 protein levels in primary
CD4.sup.+ lymphocytes and SupT1 cells. Based on the observation
that GADD45 was required for Vpr-induced apoptosis, it was
hypothesized that vpr expression, would lead to upregulation of
GADD45. To determine whether Vpr expression resulted in
upregulation of GADD45 protein, SupT1 cells (a CD4.sup.+ lymphocyte
cell line) and HeLa cells (HeLa cell data not shown) were
transduced with pHR viruses. Cells were lysed at 24, 48 and 72
hours post-transduction and GADD45 protein levels were measured by
Western blot analysis. A 3-fold upregulation of GADD45 protein was
detected at 48 hours post-transduction in pHR-VPR-transduced cell
lysates, in comparison to lysates from cells transduced with
pHR-GFP (FIG. 9A). These results prompted the examination of
Vpr-induce upregulation of GADD45 in primary human CD4.sup.+
lymphocytes, a physiologically relevant target of HIV-1. As an
additional negative control, a viral vector that expressed vpr with
the mutation R80A (64) was used. A previous report established that
Vpr(R80A) is unable to induce G.sub.2 arrest and apoptosis (64).
Therefore, if an increase in GADD45 expression was the principal
mediator of Vpr-induced apoptosis, then Vpr(R80A) should not be
capable of such an increase. Similar to our results in cell lines,
transduction of primary human CD4.sup.+ lymphocytes with pHR-VPR
resulted in a 4-fold upregulation of GADD45 protein (FIG. 9B). In
comparison, transduction with pHRVPR(R80A) did not induce GADD45
upregulation above levels observed in mock-transduced lysates (FIG.
9B).
EXAMPLE IX
[0096] Induction of GADD45 by Vpr is ATR-dependent. Although
overexpression of BRCA1, a known target, of ATR, has been shown to
transcriptionally upregulate GADD45 (65), no functional
relationship has previously been established between ATR and
GADD45. To determine whether GADD45 induction by Vpr was dependent
on signaling via ATR, a knockdown of ATR was used to assay
impairment of upregulation of GADD45. HeLa cells were transfected
with ATR siRNA or scrambled siRNA, and then transduced with pHR
viruses, as previously described. At 48 hours post-transduction,
GADD45 protein levels were measured by Western blot (FIG. 10).
Knockdown of ATR resulted in abrogation of Vpr-induced GADD45
upregulation. As a control experiment, knockdown of GADD45 with
siRNA did not reduce. ATR protein levels (FIG. 6D). This confirms
that Vpr upregulates GADD45 via ATR.
EXAMPLE X
[0097] Induction of apoptosis by Vpr is not mediated by activation
of the MAP kinases, JNK or p38. Considering earlier reports that
GADD45 activates a mitogen-activated Protein kinase (MAPK) cascade
culminating in Jun N-terminal kinase (JNK) activation and apoptosis
(65), Vpr-induced apoptosis was examined for association with
activation of JNK. Phosphorylation of c-Jun, a target of JNK, was
measured in response to Vpr. As a positive control for JNK
activation, cells were treated with anisomycin. Transduction of
SupT1 cells with pHR-VPR did not result in any detectable
phosphorylation of c-Jun (FIG. 11A). It remained possible that Vpr
was activating JNK in a manner that does not result in
phosphorylation of c-Jun, therefore, to measure JNK activation in a
more direct manner, SupT1 cells were transduced with pHR-VPR,
harvested and lysed at 24, 48 and 72 hours post-transduction. Cell
lysates from each time point were subjected to Western blot with
phosphor-specific antibodies against JNK. In agreement with the
previous data, it was determined that JNK was not activated in
response to Vpr (data not shown).
[0098] In view of the negative results concerning the role of JNK
downstream from GADD45, p38 kinase, another member of the MAPK
family implicated in apoptosis (77), was examined. SupT1 cells were
transduced with pHR-VPR and harvested at 24, 48 and 72 hours
post-infection, then assayed for the presence of activated,
phosphorylated p38 kinase by Western blot. It was found that, like
JNK, p38 kinase was not activated as result of Vpr-induced
upregulation of GADD45 (FIG. 11B).
EXAMPLE XI
[0099] Functional fragments of a protein, for example, Vpr, ATR,
RAD17, BRAC1 and/or GADD45 retain the desired function. For
example, a functional fragment of Vpr, wherein the function of
interest is cell cycle arrest, is a fragment that retains the
ability to produce a G.sub.2 cell cycle arrest. A G.sub.2 cell
cycle arrest fragment of Vpr is identified by introducing one or
more amino acid changes, or deletions into Vpr and assaying for
G.sub.2 arrest, for example, by FACs analysis.
EXAMPLE XII
[0100] A compound may be screened for cell cycle arrest and/or
apoptotic activity, for example, by administering a compound to a
subject having an ATR protein and a BRAC1 protein, such as HeLa
cells, SupT1 cells, primary cells and/or a mouse or rat, assaying
for ATR dependent phosphorylation of BRAC1, and identifying a
compound that either induces or inhibits G.sub.2 cell cycle arrest
and/or apoptosis.
[0101] SupT1 cells are cultured in a 96-well plate, a test compound
is introduced into the desired wells, and cultured with the cells
for an appropriate period of time. The cells are then transduced
with pHR-VPR and assayed at 24, 48 and 72 hours post-infection for
induction of apoptosis and/or G.sub.2 cell cycle arrest. Compounds
inhibiting apoptosis and/or G.sub.2 cell cycle arrest are
identified.
[0102] HeLa cells are cultured in a 96-well plate, a test compound
is introduced into the desired wells, and cultured with the cells
for an appropriate period of time. Control cells are transduced
with pHR-VPR in the absence of a test compound. The cells may be
harvested and assayed at 24, 48 and 72 hours post-infection for
phosphorylation of BRAC1 or induction of apoptosis. Compounds
capable of inducing apoptosis are identified as inducing a
sufficient level of BRAC1 phosphorylation or inducing apoptosis, as
compared to the control cells. Phosphorylation of BRAC1 is assayed
for phosphorylation at serine 1423 using a phosphorylation specific
antibody.
EXAMPLE XIII
[0103] Functional fragments of a protein, for example, Vpr, ATR,
RAD17, BRAC1 and/or GADD45 retain the desired function. For
example, a functional fragment of Vpr, wherein the function of
interest is inducing apoptosis similar to the mechanism induced by
genotoxic agents. An apoptosis inducing fragment of Vpr is
identified by introducing one or more amino acid changes, or
deletions into Vpr and assaying for induction of apoptosis, for
example, by treating cells exposed to the Vpr fragment with a
nuclear stain, such as 4'6-diamidino-2-phenylindole dihydrochloride
(DAPI), and examining the nuclear morphology of the cells.
[0104] While this invention has been described in certain
embodiments, the present invention can be further modified within
the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the invention using its general principle. Further, this
application is intended to cover such departures from the present
disclosure as come within known or customary practice in the art to
which this invention, pertains and which fall within the limits of
the appended claims.
[0105] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
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Sequence CWU 1
1
119PRTArtificialtargeting peptide for adipose tissue 1Cys Lys Gly
Gly Arg Ala Lys Asp Cys1 5
* * * * *