Disclosed herein is the discovery that administration of the NRIF3
family of transcriptional coregulators (NRIF3 and related molecules)
to breast cancer cells induce rapid and profound apoptosis (nearly
100% cell death within 24 h). A novel death domain (DD1) was mapped
to a short 30 amino acid region common to all members of the NRIF3
family. Two other death domains (DD2 and DD3) were also found to
have effective breast cancer killing activities. Mechanistic studies
showed that DD1-induced apoptosis occurred through a novel caspase-2
mediated pathway that involved mitochondria membrane permeabilization
but did not require other caspases. Interestingly, cytotoxicity
of NRIF3 related molecules was cell-type specific, as they selectively
killed breast cancer or related cells but not other examined cells
of different origins, suggesting the presence in breast cancer cells
of a specific death switch that can be selectively triggered by
NRIF3 and related molecules. Also disclosed are strategies utilizing
NRIF3 related molecules and/or targeting this death switch for the
development of novel and more selective therapeutics against breast
What is claimed is:
1. A method for treating a patient suffering from breast cancer
comprising administering to a patient in need of such treatment
an amount effective to treat breast cancer of an agent selected
from NRIF3 related molecules and derivatives thereof.
2. The method of claim 1 wherein said agent causes apoptosis in
said cancer cell.
3. The method of claim 2 wherein said agent is selected from full-length
NRIF3, EnL, EnS, DD1, DD2, DD3 and mixture thereof.
4. The method of claim 1 wherein said derivatives are NRIF3 related
molecules linked to cell permeation peptide sequences.
5. The method of claim 4 wherein said cell permeation peptide sequences
are derived from the hydrophobic region of Kaposi fibroblast growth
6. The method of claim 4 wherein said cell permeation peptide sequences
are derived from the HIV tat protein.
7. The method of claim 1 wherein said derivative is an N-myristoylated
polypeptide selected from NRIF3 related molecules.
8. A pharmaceutical formulation for treating a mammal suffering
from breast cancer comprising an agent selected from NRIF3 related
molecules and derivatives thereof and a pharmaceutical acceptable
carrier or diluent.
15. The pharmaceutical formulation of claim 8 wherein said NRIF3
related molecule is selected from full-length NRIF3. DD1, DD2, DD3,
EnS, EnL and derivatives thereof.
16. A method a method for killing a breast cancer cell comprising
contacting said cell with an amount of an agent selected from NRIF3
related molecules and derivatives thereof effective to kill said
17. A method for treating a patient suffering from breast cancer
comprising administering to a patient in need of such treatment
an amount of a nucleic acid encoding NRIF3 related molecules effective
to kill said cancer cell.
18. The method of claim 17 wherein said nucleic acid is administered
in a viral vector.
19. The method of claim 17 wherein said nucleic acid comprises
a naked DNA plasmid.
CROSS REFERENCE TO RELATED APPLICATION
 This application claims priority under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Applications Ser. No. 60/548,758 filed Feb.
26, 2004 which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
 The present invention is broadly directed to the treatment
of breast cancer and more specifically to the use of NRIF3 related
molecules in methods for treating breast cancer in mammals and pharmaceutical
formulations for use in the methods.
BACKGROUND OF THE INVENTION
 Many anticancer drugs act by inducing apoptosis (20). The
rapid progress in understanding mechanisms underlying apoptosis
may present opportunities to harness the cellular death machinery
for the benefit of treating human diseases such as cancer (20, 35).
Ideally, therapeutic strategies targeting an apoptotic pathway(s)
should selectively kill cancer but not other cells. At present,
however, this remains a very challenging objective.
 Breast cancer is the second leading cause of cancer-related
deaths in women (23). Each year more than 180,000 women in the United
States are diagnosed with breast cancer. Currently, effective drug
treatment for breast cancer is somewhat limited. Since many early
stage breast tumors express the estrogen receptor (ER), and depend
on estrogen for their optimal growth, anti-estrogens (ER antagonists)
have been widely used in the treatment of ER+ tumors (5, 41). Anti-estrogens,
however, are not effective in ER-tumors. Also, tumors that are initially
ER+ may lose ER expression and become independent of estrogen for
their growth and refractory to anti-estrogen therapy.
 Currently, the primary treatment of localized breast cancer
is either breast-conserving surgery and radiation or mastectomy
with or without breast reconstruction. Systemic adjuvant therapies
are also employed to eradiate microscopic deposits of cancer cells
that may have spread or metastisized from the primary tumor. Systemic
adjuvant therapies include chemotherapy and hormonal therapy. Radiation
is also used as a local adjuvant treatment to eradicate cancer cells
in the chest wall or regional lymph nodes after mastectomy (reviewed
in 54). Major acute and long-term side effects of adjuvant treatments
include premature menopause, weight gain, mild memory loss and fatigue.
 Apoptosis or programmed cell death is a fundamental cellular
process where the affected cell dies by actively executing a coordinately
regulated death program (11, 18). For multicellular organisms (e.g.
mammals) apoptosis plays important roles in normal development,
tissue homeostasis, and in diverse pathological processes. Caspases
and mitochondria are two key cellular components involved in the
execution and regulation of apoptosis (18, 50). Caspases are a group
of cysteine-proteases that are ordinarily inactive in cells as pro-enzymes
but are activated upon appropriate apoptotic stimuli. Generally,
the initiator caspases (e.g. 2, 8, 9, and 10) are activated when
complexed with adaptor molecules, resulting in either autoprocessing
due to induced proximity or holoenzyme formation (9, 18, 26, 40).
The downstream effector caspases (e.g. 3, 6, and 7) are activated
through proteolytic cleavage by initiator caspase(s). Effector caspases
then cleave various cellular components, leading to the morphologic
and biochemical phenotypes characteristic of apoptosis (11, 18).
 Mitochondria also play an important role in apoptosis, as
various apoptotic stimuli converge on mitochondria and lead to mitochondrial
membrane permeabilization (MMP) (25, 38, 50). Upon MMP, mitochondria
release a number of factors that are involved in apoptosis initiation
and/or execution, such as cytochrome-c, Smac/Diablo, and AIF (Apoptosis
Inducing Factor) (8, 18, 38, 50). The released cytochrome-c interacts
with the adaptor protein Apaf-1 and pro-caspase-9 to form an activated
complex referred to as an apoptosome, which then cleaves and activates
downstream effector caspases (e.g. caspase-3) (18, 52). In contrast,
AIF released from mitochondria triggers apoptosis (e.g. by inducing
chromatin condensation and large-scale DNA fragmentation) independent
of effector caspases (8, 31, 46, 51). This caspase-independent apoptogenic
function of AIF is evolutionarily conserved, and plays an important
role both in normal development and in cell death processes whereby
caspases are minimally activated or inhibited (e.g. by chemical
inhibitors) (8, 38).
 There are two major apoptotic pathways in mammalian cells
(11, 18). The extrinsic pathway is initiated by the binding of transmembrane
death receptors (e.g. Fas, TNF-R1, and TRAIL receptors) with cognate
extracellular ligands. Liganded receptors recruit adaptor proteins
(e.g. FADD) which interact with and trigger the activation of caspase-8.
Activated caspase-8 then cleaves and activates downstream effector
caspases such as caspase-3. In contrast, the intrinsic pathway is
characterized by disruption of mitochondria membrane integrity when
cells are exposed to various stresses (e.g. DNA damaging agents).
Mitochondrial membrane permeabilization (MMP) triggers apoptosis
via both caspase-dependent (e.g. the cytochrome-c/caspase-9 pathway)
and caspase-independent (e.g. the AIF pathway) mechanisms. Crosstalk
exists between the extrinsic and intrinsic pathways, as activated
caspase-8 can cleave Bid to produce truncated Bid (tBid), which
then binds to mitochondria and promotes MMP (30, 32). The subsequent
release of cytochrome-c from mitochondria further facilitates the
 MMP is regulated by the Bcl-2 family of proteins, which
act upstream of mitochondria, and contain both anti-apoptotic (e.g.
Bcl-2 and Bcl-xL) and pro-apoptotic members (e.g. Bak, Bax, Bid,
and Bad) (3, 7). The relative balance between the pro- and anti-apoptotic
members of the Bcl-2 family is critical in controlling MMP. Interestingly,
a number of recent studies have shown that caspase-2 acts upstream
of mitochondria and is required for MMP during stress-induced apoptosis
in certain cell types (17, 27, 42). While these studies implicate
caspase-2 as an initiator caspase in certain intrinsic pathway(s)
of apoptosis, the mechanistic interplay between caspase-2 and members
of the Bcl-2 family in controlling MMP is not yet clear (24).
 The present inventors have studied the role of nuclear receptors
in breast cancer cell proliferation (2). U.S. Pat. No. 6,639,064,
issued Oct. 28, 2003 discloses the cloning of a novel coregulator
(designated as NRIF3) which specifically interacts with and enhances
the activity of ligand-bound thyroid hormone receptors (TRs) and
retinoid X receptors (RXRs) (28, 29). However, no therapeutic role
was ascribed to the protein in these publications.
SUMMARY OF THE INVENTION
 It has now been unexpectedly discovered that expression
of NIRF3 and related molecules inhibits growth of breast cancer
cells independent of retinoid treatment. Further studies indicate
that this apparent growth inhibition resulted from the induction
of rapid and profound apoptosis in these cells by NRIF3 (virtually
100% cell death within 24 h). The apoptogenic function of NRIF3
was independent of its interaction with nuclear receptors, and was
mapped to a novel death domain (DD1) that is relatively small in
size (.about.30 amino acids). Mechanistic studies suggest that DD1-induced
apoptosis occurs through a novel caspase-2 mediated pathway that
involves MMP and AIF translocation but does not appear to require
other caspases. Cytotoxicity of NRIF3 and DD1 was cell-type specific,
as their expression led to efficient apoptosis in all the breast
cancer cell lines surveyed (ER+ T-47D, MCF-7, MDA-MB-231 -ER+ cells
and ER- MDA-MB-231 and MDA-MB-435 cells), but not in five other
cell types of different origins (HeLa, GH4C1, 293, UOK145, and Cos-1).
 One aspect of the present invention provides a method for
treating a patient suffering from breast cancer comprising administering
to a patient in need of such treatment an amount effective to treat
breast cancer of an agent selected from NRIF3 related molecules
and derivatives thereof.
 In another aspect, the present invention provides a pharmaceutical
formulation for treating a mammal suffering from breast cancer comprising
NRIF3 related molecules, derivatives thereof and a pharmaceutical
acceptable carrier or diluent.
 In yet another aspect, the present invention provides an
isolated polypeptide comprising an amino acid sequence consisting
 In still another aspect, the present invention provides
an isolated polypeptide comprising an amino acid sequence consisting
 In still a further aspect of the present invention provides
an isolated polypeptide comprising an amino acid sequence consisting
 In still another aspect, the present invention provides
an isolated nucleic acid comprising a nucleotide sequence consisting
 In still another aspect, the present invention provides
an isolated nucleic acid sequence consisting of DD2.
 In still another aspect the present invention provides an
isolated nucleic acid sequence consisting of DD3.
 In still another aspect, the present invention provides
a method for killing a breast cancer cell comprising contacting
said cell with an amount of an agent selected from NRIF3 related
molecules and derivatives thereof effective to kill said cell.
 In still another aspect, the present invention provides
a method for treating a patient suffering from breast cancer comprising
administering to a patient in need of such treatment an amount of
a nucleic acid encoding NRIF3 related molecule effective to kill
said cancer cell.
 These and other aspects of the present invention will be
apparent to those of ordinary skill in the art in light of the present
description claims and drawings.
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1. (A and B) NRIF3 induces apoptosis in T-47D cells.
(A) GFP-NRIF3 was expressed in T-47D cells by transient transfection.
Twenty-four h later the green fluorescent cells were collected after
sorting by flow-cytometry, and re-inoculated onto cover-slips. Cells
were then analyzed for apoptosis by Annexin V staining (red). Control
cells expressing GFP alone were negative for Annexin V staining
(not shown). (B) Representative fluorescent micrographs of T-47D
cells transfected with either GFPNLS or GFP-NRIF3. Cells were examined
for apoptosis by TUNEL assay (red). (C) Quantitative presentation
of the experiments described in (B). The percent of green fluorescent
cells that were TUNEL positive were scored for T-47D cells transfected
with either GFP-NRIF3 or GFPNLS.
 FIG. 2. (A and B) Cell death induced by EnS and EnL. (A)
Domain organization and functional motifs in NRIF3, EnS, and EnL.
NRIF3 contains two nuclear receptor interaction domains (RID1, residues
162-177; and RID2, residues 9-13) (28, 29). An activation domain
(AD1) co-resides with RID1. A transrepression domain (RepD1) maps
to residues 20-50 (29). Also shown are a cyclin A binding motif
RxL (residues 6-8), a coiled-coil dimerization domain (residues
86-112) that contains a leucine-zipper-like motif, and a nuclear
localization signal (NLS, residues 63-66) (29, 36). Ser28 in RepD1
is marked by an arrow. EnS and EnL share extensive identities with
NRIF3 and contain the same domains/motifs except for RID1/AD1. (B)
Representative fluorescent micrographs of T-47D cells transfected
with either GFP-EnS or GFP-EnL. Cells were examined for apoptosis
by TUNEL assay (red).
 FIG. 3. (A-C) NRIF3 contains a novel death domain (DD1).
(A) T-47D cells were transfected with each of the indicated constructs
expressing various regions of NRIF3 fused to GFP or GFPNLS. Ser28
in wild type DD1 is marked by an arrow, while Ala28 in the mutant
DD1 is marked by a star. These regions were expressed either as
a GFP-fusion, or for those lacking an intrinsic NLS, as a GFPNLS-fusion.
Green fluorescent cells were scored for apoptosis by TUNEL assay
or Annexin V staining or both. "+++" indicates profound
cell death, where nearly 100% of green cells displayed positive
staining for TUNEL and/or Annexin V, while "-" indicates
no apoptosis (less than 2% positive). (B) Representative fluorescent
micrographs of T-47D cells transfected with GFPNLS, wild type (WT)
GFPNLS-DD1 (residues 20-50 of NRIF3), or the GFPNLS-DD1 mutant (S28A).
Cells were examined for apoptosis by TUNEL assay (red). (C) The
Ser28 to Ala mutation severely compromises the apoptogenic function
of DD1. The percent of green fluorescent cells that were TUNEL positive
were scored for T-47D cells transfected with either wild type GFPNLS-DD1
or the mutant GFPNLS-DD1 S28A.
 FIG. 4. (A and B) Cell death mediated by NRIF3 or DD1 is
insensitive to zVAD-fmk. (A) T-47D cells were transfected with either
GFP-NRIF3 or GFPNLS-DD1 in the absence or presence of the broad-spectrum
caspase inhibitor zVAD-fmk. When present, the inhibitor was incubated
with the cells before and after transfection. Cells were examined
for apoptosis by TUNEL assay (red). Representative fluorescent micrographs
are shown for cells treated with zVAD-fmk. (B) Quantitative presentation
of the experiments in (A). The percent of green fluorescent cells
that were TUNEL positive were scored for T-47D cells transfected
with either GFP-NRIF3 or GFPNLS-DD1 in the absence or presence of
 FIG. 5. (A and B) DD1-mediated cell death involves MMP.
(A) Representative fluorescent micrographs of T-47D cells transfected
with either GFPNLS-DD1, or GFPNLS-DD1 and Bcl-2. Cells were examined
for apoptosis by TUNEL assay (red). (B) Quantitative presentation
of the experiments in (A). The percent of green fluorescent cells
that were TUNEL positive were scored for T-47D cells transfected
with either GFPNLS-DD1 or with GFPNLS-DD1 and Bcl-2. (C) T-47D cells
were transfected to express AIF-GFP along with either a control
vector or a vector expressing DD1. Approximately 5 h after transfection
the cells were fixed and subjected to TUNEL assay. The cells were
then examined by fluorescent microscopy for sub-cellular location
of AIF-GFP (green) and for apoptosis (red). Nuclei were stained
with Hoechst (blue). Representative fluorescent micrographs of cells
transfected with the control vector or DD1 are compared.
 FIG. 6. (A-C) Requirement for caspase-2 in DD1-mediated
apoptosis. (A) T-47D cells pre-treated with caspase-2 siRNA or mock-treated
control cells were transfected with GFPNLS-DD1. Cells were examined
for apoptosis by TUNEL assay (red). Representative fluorescent micrographs
of siRNA-treated or mock-treated cells are compared. (B) T-47D cells
pre-treated with caspase-2 siRNA or mock-treated control cells were
incubated with etoposide. Cells were examined for apoptosis by TUNEL
assay (red) while the nuclei were visualized by Hoechst staining
(blue). Representative fluorescent micrographs are shown for the
siRNA treated cells. Similar result is observed for the mock-treated
control cells (not shown). (C) Quantitative presentation of the
experiments in (A). The percent of green fluorescent cells (expressing
GFPNLS-DD1) that were TUNEL positive were scored for cells pre-treated
with caspase-2 siRNA or for mock-treated control cells.
 FIG. 7. (A and B) Cell-type specificity in cytotoxicity
mediated by NRIF3 and DD1. (A) Various breast cancer cell lines
and other cells were transfected with either GFP-NRIF3 or GFPNLS-DD1.
Cells were examined for apoptosis by TUNEL assay (red). Representative
fluorescent micrographs of transfected HeLa and MDA-MB-231 cells
are compared. (B) A summary of results from all cell lines examined
in (A). "+++" indicates profound apoptosis (>90% of
green fluorescent cells were TUNEL positive) while "-"
indicates no apoptosis (less than 2% positive).
 FIG. 8. A model for NRIF3- or DD1-induced apoptosis. Breast
cancer cells contain a specific death switch that can be selectively
triggered by NRIF3 or its death domain DD1. Triggering of this switch
by NRIF3 or DD1 leads to activation of caspase-2. Activated caspase-2
promotes MMP, which results in the release of AIF. The released
AIF then mediates effector caspase-independent cell death (which
is insensitive to zVAD-fmk). The anti-apoptotic factor Bcl-2 could
inhibit this pathway by acting either upstream of caspase-2 (to
prevent its activation) or downstream of caspase-2 (to prevent caspase-2-mediated
changes in MMP) (24). It is also possible that activated caspase-2
can directly elicit cell death (dashed line) in addition to the
depicted mitochondria-mediated pathway.
 FIG. 9. NRIF3 contains a second death domain (DD2). Representative
fluorescent micrographs are shown for T-47D cells transfected with
a vector expressing GFPNLS-DD2 (DD2 corresponds to amino acid residues
47-86 of NRIF3). Cells were examined for apoptosis by TUNEL assay
(red). In a control experiment, cells transfected with the GFPNLS
control vector were not TUNEL positive (data not shown, but representative
micrographs of cells transfected with GFPNLS are shown in FIG. 3B).
 FIG. 10. NRIF3 contains a third death domain (DD3). T-47D
cells were transfected to express GFPNLS, along with either a control
vector or a vector expressing DD3 (DD3 corresponds to amino acid
residues 112 to 177 of NRIF3). About 24 h after transfection, the
cells were fixed and subjected to TUNEL assay. The transfected cells
were then visualized by fluorescent microscopy (green), and at the
same time examined for apoptosis (red). Representative fluorescent
micrographs of cells transfected with the control vector or DD3
 FIG. 11. (A-C) depicts the DNA sequence for (A) full-length
NRIF3; (B) full-length EnL; (C) full-length EnS.
 FIG. 12. (A-C) depicts the DNA sequence for (A) DD1; (B)
DD2 and (C) DD3.
 FIG. 13. Nucleotide and deduced amino acid sequences of
NRIF3. Only part of the cDNA sequence is shown. A putative nuclear
localization signal (KRKK) is underlined. The putative LxxLL motif
is shown with a double underline. NRIF3 and the .beta.3-endonexin
long form (EnL) share 95% identity. They differ only in the C-terminus
where the last 16 amino acids (dot underlined) in NRIF3 are replaced
with 9 different amino acids (GQPQMSQPL) in the .beta.3-endonexin
long form. The short form of .beta.3-endonexin consists of 111 amino
acids and is 100% identical to the first 111 amino acids of NRIF3
or the .beta.3-endonexin long form. The relative positions of DD1,
DD2 and DD3 are shown.
DETAILED DESCRIPTION OF THE INVENTION
 The term about or approximately means within an acceptable
error range for the particular value as determined by one of ordinary
skill in the art, which will depend in part on how the value is
measured or determined, i.e., the limitations of the measurement
system, i.e., the degree of precision required for a particular
purpose, such as a pharmaceutical formulation. For example, about
can mean within 1 or more than 1 standard deviations, per the practice
in the art. Alternatively, about can mean a range of up to 20%,
preferably up to 10%, more preferably up to 5%, and more preferably
still up to 1% of a given value. Alternatively, particularly with
respect to biological systems or processes, the term can mean within
an order of magnitude, preferably within 5-fold, and more preferably
within 2-fold, of a value. Where particular values are described
in the application and claims, unless otherwise stated the term
"about" meaning within an acceptable error range for the
particular value should be assumed.
 "NRIF3 related molecules" are defined as peptides
consisting of full-length NRIF3, Death Domains 1-3 (DD1-DD3), EnS
(endonexin short form) and EnL (endonexin long form) and nucleic
acids encoding said peptides.
 "NRIF3 derivatives" are defined as NRIF3 and related
molecules (as defined above) that are either in the form of peptides
that have been modified by linkage to known cell permeation peptide
sequences derived from another protein or in the form of nucleic
acids that encode the peptides. Examples of known cell-permeation
peptide sequences include the hydrophobic region (h-region) of the
signal peptide of Kaposi fibroblast growth factor (as described
in Ye, H. et al., Nature 418:443-447, 2002; Yan Liu, X. et al.,
J. Biol. Chem. 275(22):16774-16778, 2000; Lin, Y. Z. et al., J.
Biol. Chem. 270(24):14255-14258, 1995) or a cell-permeation sequence
from the HIV tat protein (described in Goubaeva, F. et al., J. Biol.
Chem. 278(22):19634-19641, 2003). The hydrophobic sequence derived
from the Kaposi fibroblast growth factor signal sequence is as follows:
 The cell-permeation sequence derived from the HIV tat protein
is as follows:
 Other derivatives are N-myristoylated versions of the NRIF3-related
peptides. N-myristoylation is known to facilitate cell permeation
by the modified peptide (as disclosed in Goubaeva, F. et al. supra,
Eichholtz, T. et al., J. Biol. Chem. 268(3):1982-1986, 1993; Harris,
T. E. et al., Biochem. Biophys. Res. Commun. 232(3):648-651, 1997).
 "Sequence Conservative Variants" of a nucleotide
sequence are those in which a change of one or more nucleotide in
a given codon position results in no alteration in the amino acid
encoded at that position.
 The present invention is based on the unexpected discovery
that NRIF3 related molecules and derivatives thereof selectively
kill breast cancer cells by inducing apoptosis in the recipient
cell. The human NRIF3 gene encodes several different proteins as
a result of alternative splicing, including NRIF3, EnS (endonexin
short form) and EnL (endonexin long form). Both EnS and EnL share
extensive identity with NRIF3 at the amino acid level. EnS is identical
to the first 111 amino acids of NRIF3. NRIF3 and EnL share 95% identity
and differ only in the C-terminus where the last 16 amino acid residues
in NRIF3 are replaced with 9 different amino acids in EnL (See FIG.
 NRIF3 related molecules and derivatives thereof can be used
to treat mammals suffering from breast cancer. Such treatments would
comprise administering to a patient in need of such treatment a
breast-cancer-cell-killing effective amount of NRIF3 related molecules
and derivatives thereof. For proteins or peptides or their derivatives,
such effective amounts would broadly range between about 0.1 mg/kg
body weight of the recipient and about 100 mg/kg body weight of
the recipient. Such effective amounts can be optimized using various
systems such as an in vitro cell-based testing system, which uses
cultured breast cancer cell lines such as T-47D cells (available
from the American Type Culture Collection, Manassas, Va. as ATCC
Accession No. HTB-133), and into which NRIF3 related molecules and
derivatives thereof are added at desired concentrations. The cells
are then examined to see if the treated cells undergo apoptosis
(by an appropriate apoptopic assay such as the TUNEL assay described
below). In this way it is possible to determine the effective "killing"
concentrations of the tested peptides. In addition, an animal-based
testing system such as the well known nude mouse model (Brunner
N. et al., Breast Cancer Res. Treat. 10(3):229-242, 1987; Brodie
A., Semin. Oncol. 30(4 Suppl 14):12-22, 2003) can be used to test
the effective amounts of an NRIF3-related agent. Nude mice are naturally
immunodeficient and can be inoculated to grow human breast cancer
cells. Thus, the anti-tumor or therapeutic effect of the peptides
and nucleic acids of the present invention can be studied. From
such a study one can determine the effective doses that would suppress
tumor growth and/or eradicate tumors, as well as possible toxicity
 Pursuant to the present invention, NRIF3 related molecules
and derivatives thereof may be administered to a mammal in need
of such treatment in effective amounts to treat breast cancer parenterally,
e.g., intramuscularly, intraperitoneally, subcutaneously and preferably
intravenously or by direct injection into the tumor.
 One of the advantages of using NRIF3 related molecules and
derivatives thereof is that they only cause apoptosis in breast
cancer cells. All of the other standard breast cancer treatments,
i.e., chemotherapy and radiation cause extensive toxicity to normal
cells of the recipient.
 All of the peptides of the present invention are derived
from NRIF3. These are full-length NRIF3, EnL, EnS and the common
death domains shared by all three of them (DD1, amino acid residues
20-50; DD2, amino acid residues 47-86, as well as DD3 amino acid
 The NRIF3 related molecules can be obtained using techniques
well known to those of ordinary skill in the arts after expression
of the constructs described herein below in the Materials and Methods
section of the Examples. The peptides can be obtained after expression
in suitable eukaryotic or prokaryotic cells well known to those
of ordinary skill in the art, or by chemical synthesis. Expressed
peptides can be purified directly using conventional chromatography
techniques, or purified via suitable affinity chromatography when
they are expressed as a fusion to an appropriate affinity tag (as
described in Current Protocols in Protein Science, and Current Protocols
in Molecular Biology; John Wiley & Sons). In all cases, a further
purification using HPLC can also be applied. The methodology involved
for producing such peptides is generally known in the art.
 FIG. 11 depicts the DNA sequence for full-length NRIF3,
full-length EnL and full-length EnS. FIG. 12 depicts the DNA sequence
coding for DD1, DD2 and DD3. FIG. 13 depicts the DNA and protein
sequence of full-length NIRF3 and shows the relative positions of
DD1, DD2 and DD3.
 The present invention also includes sequence conservative
variants of the NRIF3 related molecules of the present invention
as defined above.
 Amino Acid Sequence of Death Domain 1 (DD1):
2 P S K I T R K K S V I T Y S P T T G T C Q M S L F A S P T S S
 Amino Acid Sequence of Death Domain 2 (DD2):
3 P T S S E E Q K H R N G L S N E K R K K L N H P S L T E S K E
S T T K D N D E F
 Sequence of Death Domain 3 (DD3):
4 A L E G S R E L E N L I G I S C A S H F L K R E M Q K T K E L
M T K V N K Q K L F E K S T G L P H K A S R H L D S Y E F L K A
I L N
 Another embodiment of the present invention is directed
to pharmaceutical formulations and dosage forms for treating patients
suffering from breast cancer. When formulated in a pharmaceutical
formulation, NRIF3 related molecules and/or derivatives thereof
can be admixed with a pharmaceutically acceptable carrier or excipient.
The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are "generally regarded as safe",
e.g., that are physiologically tolerable and do not typically produce
an allergic or similar untoward reaction such as gastric upset,
dizziness and the like, when administered to a human. Preferably,
as used herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a State
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and, more particularly,
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous saline solutions and aqueous dextrose
and glycerol solutions are preferably employed as carriers, particularly
for injectable solutions. Suitable pharmaceutical carriers are described
in "Remington's Pharmaceutical Sciences" by E. W. Martin.
 The pharmaceutical formulation of the present invention
need not contain an amount effective for treating breast cancer
as such effective amount can be attained by administering a plurality
of the formulations.
 In an alternative preferred embodiment of the present invention,
the breast cancer killing properties of the NRIF3 related molecules
can be utilized in a gene therapy format. For example, instead of
administering a therapeutic peptide, the corresponding nucleic acid(s)
(e.g., DNA), can be introduced into breast cancer cells. In this
way, the breast cancer cells would synthesize the peptides which
would result in the death of the cancer cell. The Examples below
demonstrate that this approach is effective in killing the recipient
breast cancer cell.
 In this embodiment, effective amounts of the NRIF3 related
molecules of the present invention effective for killing breast
cancer cells are synthesized using the DNA encoding such molecules.
The DNA can be administered in a viral vector, a DNA plasmid or
as "naked" DNA as described further below. The effective
amounts can be determined by routine experimentation using for example,
the nude mouse model in combination with known human breast cancer
 The nucleic acids encoding the NRIF3 related molecules of
the present invention can be delivered to cancer cells in a retroviral
vector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;
Mann et al., Cell 1983, 33:153; U.S. Pat. Nos. 4,650,764, 4,980,289,
and 5,124,263; Markowitz et al., J. Virol. 1988, 62:1120; Temin
et al., U.S. Pat. No.; EP 453242, EP178220; Bernstein et al. Genet.
Eng. 1985, 7:235; McCormick, BioTechnology 1985, 3:689; PCT; and
Kuo et al., Blood 1993, 82:845. These vectors can be constructed
from different types of retrovirus, such as, HIV; MoMuLV ("murine
Moloney leukaemia virus"); MSV ("murine Moloney sarcoma
virus"); HaSV ("Harvey sarcoma virus"); SNV ("spleen
necrosis virus"); RSV ("Rous sarcoma virus") and
Friend virus. Suitable packaging cell lines have been described
in the prior art, in particular the cell line PA317 (U.S. Pat. No.
4,861,719); the PsiCRIP cell line (PCT Publication No. WO 90/02806)
and the GP+envAm-12 cell line (PCT Publication No. WO 89/07150).
Retrovirus vectors can also be introduced by DNA viruses, which
permits one cycle of retroviral replication and amplifies transfection
efficiency (see PCT Publication Nos. WO 95/22617, WO 95/26411, WO
96/39036, WO 97/19182).
 DNA viral vectors, including an attenuated or defective
DNA virus, such as but not limited to herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated
virus (AAV), and the like can also be used to deliver the NRIF3
related molecules of the present invention. Defective viruses, which
entirely or almost entirely lack viral genes, are preferred. Defective
virus is not infective after introduction into a cell. Use of defective
viral vectors allows for administration to cells in a specific,
localized area, without concern that the vector can infect other
cells. Thus, a specific tissue can be specifically targeted. Examples
of particular vectors include, but are not limited to, a defective
herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.
1991, 2:320-330), defective herpes virus vector lacking a glyco-protein
L gene (Patent Publication RD 371005 A), or other defective herpes
virus vectors (PCT Publication Nos. WO 94/21807 and WO 92/05263);
an attenuated adenovirus vector, such as the vector described by
Stratford-Perricaudet et al. (J. Clin. Invest. 1992, 90:626-630;
see also La Salle et al., Science 1993, 259:988-990; various replication
defective adenovirus and minimum adenovirus vectors have been described
in PCT Publication Nos. WO 94/26914, WO 95/02697, WO 94/28938, WO
94/28152, WO 94/12649, WO 95/02697, and WO 96/22378); and a defective
adeno-associated virus vector (Samulski et al., J. Virol. 1987,
61:3096-3101; Samulski et al., J. Virol. 1989, 63:3822-3828; Lebkowski
et al., Mol. Cell. Biol. 1988, 8:3988-3996; PCT Publication Nos.
WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368 and 5,139,941;
European Publication No. EP 488 528).
 Various companies produce viral vectors commercially, including
but by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors),
Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors,
and lentiviral vectors), Clontech (retroviral and baculoviral vectors),
Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec
(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral
vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes
viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,
United Kingdom; lentiviral vectors), and Transgene (Strasbourg,
France; adenoviral, vaccinia, retroviral, and lentiviral vectors).
 In another embodiment, the vector can be non-viral. Such
vectors include "naked" DNA, and transfection facilitating
agents (peptides, polymers, etc.). Synthetic cationic lipids can
be used to prepare liposomes for transfection of a gene encoding
a desired molecule (Felgner, et al., Proc. Natl. Acad. Sci. U.S.A.
1987, 84:7413-7417; Felgner and Ringold, Science 1989, 337:387-388;
see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031;
Ulmer et al., Science 1993, 259:1745-1748). Useful lipid compounds
and compositions for transfer of nucleic acids are described in
International Patent Publications WO95/18863 and WO96/17823, and
in U.S. Pat. No. 5,459,127. Lipids may be chemically coupled to
other molecules for the purpose of targeting (see Mackey, et. al.,
supra). Targeted peptides, e.g., hormones and proteins such as antibodies,
or non-peptide molecules could be coupled to liposomes chemically.
Other molecules are also useful for facilitating transfection of
a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International
Patent Publication WO95/21931), peptides derived from DNA binding
proteins (e.g., International Patent Publication WO96/25508), or
a cationic polymer (e.g., International Patent Publication WO95/21931).
 In another embodiment the vector comprises a naked DNA plasmid.
Naked DNA vectors for gene therapy can be introduced into the desired
host cells by methods known in the art, e.g., electroporation, microinjection,
cell fusion, DEAE dextran, calcium phosphate precipitation, use
of a gene gun, or use of a DNA vector transporter (see, e.g., Wu
et al., J. Biol. Chem. 1992, 267:963-967; Wu and Wu, J. Biol. Chem.
1988, 263:14621-14624; Hartmut et al., Canadian Patent Application
No. 2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl.
Acad. Sci. USA 1991, 88:2726-2730). Receptor-mediated DNA delivery
approaches can also be used (Curiel et al., Hum. Gene Ther. 1992,
3:147-154; Wu and Wu, J. Biol. Chem. 1987, 262:4429-4432). U.S.
Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous
DNA sequences, free of transfection facilitating agents, in a mammal.
 The present invention is described below in working examples
which are intended to further describe the invention without limiting
 In the examples below the following materials and methods
 Plasmids. Plasmids expressing GFP-NRIF3, GFP-EnS, GFP-EnL,
the GFP control, wild type NRIF3, Gal4-DD1, Gal4-DD3 and the Gal4
control, have been described (28, 29). The GFPNLS vector expresses
nuclear-localized GFP, where the NLS is derived from the SV40 T
antigen (1). Constructs expressing GFP- or GFPNLS-fusion of various
regions of NRIF3 were generated by PCR-based cloning. Briefly, DNA
fragments encoding a desired NRIF3 region was produced by PCR amplification,
digested with XhoI and Acc65I, and cloned into either the original
GFP vector (for regions corresponding to residues 1-86 and 20-86)
or a GFPNLS vector (for wild type DD1 and the S28A mutant DD1, as
well as DD2) digested with the same pair of enzymes. All GFP-fusion
constructs were confirmed by sequence analysis. The AIF-GFP plasmid
was kindly provided by Dr. Guido Kroemer (31). Vectors expressing
Bcl-2 and Bcl-xL were gifts from Dr. Honglin Li.
 Cell culture and transfection conditions. All breast cancer
cell lines were maintained in DMEM (GIBCO-BRL, Life Technologies)
supplemented with 10% fetal bovine serum (FBS). Other cells were
cultured in DMEM supplemented with either 10% FBS or 10% Hyclone
defined/supplemented bovine calf serum. For most of the transient
transfections, T-47D cells were plated at a density of 3.times.10.sup.4
cells/well on coverslips in 24-well tissue culture plates. About
20 h later, the cells were transfected with indicated plasmid(s)
by Genefect transfection reagent (Molecula, USA) according to manufacturer's
protocol. Generally, the amount of plasmids used in transfections
was the following: GFP or GFP-fusion 300-500 ng, Bcl-2 or Bcl-xL
1-1.5 .mu.g, AIF-GFP 500 ng, Gal4-DD1, GA14-DD3 or Gal4 control
0.5-1 .mu.g. After transfection, cells were incubated in DMEM/10%
FBS medium for 5 h to 24 h before being harvested and processed
for appropriate analyses. Transient transfection of other breast
cancer cell lines were carried out similarly, except that the transfection
reagent used was Lipofectamine 2000 (Life technologies). All other
cells were transfected using Geneporter 2 (Gene Therapy Systems).
When indicated, the following compounds were included in the medium:
all-trans retinoic acid (100 nM), etopside (100 .mu.M), zVAD-fmk
(100 .mu.M), zVDVAD-fmk (20 .mu.M), TNF.alpha. (10 ng/ml) and cycloheximide
 Flow cytometry and cell sorting analysis. Cells transfected
with an appropriate GFP construct were processed for flow cytometry
analysis as previously described (2). Flow cytometric analysis for
GFP and propidium iodide was performed using four-color FACscan
(Becton-Dickinson Immunocytometry System, San Jose, Calif., USA).
GFP positive or negative cells were analyzed for changes in cell
cycle distribution. For re-inoculation studies, cells were plated
at a density of 3.times.10.sup.6 cells/plate in a 100 cm.sup.2 tissue
culture plate and transfected with 50 .mu.g of indicated GFP or
GFP-fusion constructs using Genefect. About 24 h after transfection,
cells were harvested and sorted by flow cytometry. GFP positive
or negative cells were collected, and re-plated at 3.times.10.sup.4
cells/well in 24-well plates. Cells were monitored over a period
of 48-72 h for attachment, growth, and morphological changes. In
some cases, the collected cells were re-plated onto cover-slips
and subsequently processed for Annexin V staining.
 siRNA studies. A small interference RNA (siRNA) duplex that
efficiently silences human caspase-2 expression has been previously
described (27), and was purchased from Dharmacon. The siRNA was
dissolved at 20 pmol/.mu.l in H.sub.2O. T-47D cells were plated
at the density of 1.5.times.10.sup.5 cells/well in 6-well plates
the day before being transfected with siRNA. Transfection was carried
out using Oligofectamine (Invitrogen), with 12 .mu.l of dissolved
siRNA and 12 .mu.l of Oligofectamine reagent. Cells were fed with
additional DMEM/10% FBS the second day, and harvested about 42 h
after siRNA transfection. Mock-transfected cells were treated similarly
but did not receive caspase-2 siRNA. The harvested cells were then
re-plated on cover-slips in 24-well plates as described earlier,
and incubated for a few hours to let attachment occur. Cells were
then transfected with the indicated GFP or GFP-fusion vectors as
described earlier or treated with etoposide (100 .mu.M). Cells were
harvested about 20-24 h later and processed for appropriate assays.
To document caspase-2 knock down, total lysates from siRNA transfected
or control cells were quantified for protein concentrations. Equal
amount of proteins were then subjected to SDS-PAGE, followed by
Western analysis using a monoclonal antibody against caspase-2 (11B4,
 Apoptosis assays. Cells plated on cover-slips in 24-well
tissue culture plates were transfected and/or treated with appropriate
compounds as indicated. Generally, cells were harvested within 20-24
h and processed for TUNEL and/or Annexin V assays. For Annexin V
assay, cells were washed 3.times. with PBS and assayed using ApoAlert
Nitric Oxide/Annexin V Dual Sensor Kit (BD Biosciences, USA) according
to manufacturer's protocol. Cells were then mounted on slides using
Dako Fluorescent Mounting Media, (DAKO Corporation, USA) and examined
by fluorescent microscopy. For TUNEL assays, cells were washed 3.times.
with PBS, fixed in 3.7% formaldehyde, and assayed using the "In
Situ Cell Death Detection Kit" (TMR red) (Roche Diagnostics
GmbH, Germany) according to manufacturer's protocol. In some cases,
cells were also stained with Hoechst dye to visualize nuclei. Cells
were then mounted on slides and examined by fluorescent microscopy.
For quantitative analysis, fields consisting of at least several
hundred cells were scanned, and a number of representative fields
were photographed using GFP and rhodamine filters. Green and red
cells from the same field were then counted. Generally, about one
hundred of total green cells were counted for each data point. The
percent of green cells counted that are also red were then calculated.
 NRIF3 induces rapid and profound cell death in T-47D cells.
We previously identified NRIF3 as a co-activator for certain members
of the nuclear hormone receptor superfamily, including TR and RXR
(28, 29). A unique feature of RXR is that it serves as the common
heterodimeric partner for many other members of the nuclear receptor
superfamily (33). Thus, a heterodimer composed of the retinoic acid
receptor (RAR) and RXR is the functional unit that transduces retinoid
signaling in vivo (21, 22). Retinoid signaling plays important roles
in both development and homeostasis (21, 22). In addition, retinoids
are known to inhibit the proliferation of certain breast cancer
cells (2, 15), although the underlying molecular mechanism(s) have
not been fully defined.
 Our identification of NRIF3 as a co-activator for RXR prompted
us to test whether NRIF3 would enhance the anti-proliferative effect
of retinoids in responsive breast cancer cells. We and others have
previously shown that retinoid treatment inhibits the proliferation
of T-47D and MCF-7 breast cancer cell lines (2, 39, 43). This results
in an increase in distribution of cells in the G0-G 1 phase of the
cell cycle and a concomitant decrease in the number of cells in
S phase (2). To test the effect of NRIF3, we transfected T-47D cells
with a vector expressing GFP or GFP-NRIF3 and incubated the cells
with or without all-trans-retinoic acid (tRA). Green fluorescent
and non-green cells were then sorted by flow cytometry and analyzed
for cell cycle distribution. Intriguingly, we found that expression
of GFP-NRIF3 (but not the GFP control) was sufficient to inhibit
the proliferation of T-47D cells, whether or not the cells were
incubated with tRA (data not shown). This result suggested that
NRIF3 mediates an anti-proliferative effect on T-47D cells independent
of retinoid treatment.
 To examine this further, we collected sorted green fluorescent
cells expressing either GFP-NRIF3 or GFP and re-plated them in culture
dishes to monitor cell growth. While GFP expressing cells attached
and grew normally, cells expressing GFP-NRIF3 attached to the dish
inefficiently and failed to divide. Microscopy examination revealed
that the GFP-NRIF3 expressing cells displayed morphological changes
suggestive of apoptosis (rounded-up cell shape and cytoplasmic shrinkage).
Thus, we examined these cells for apoptosis using an Annexin V assay
(19, 49), and found that virtually all cells expressing GFP-NRIF3
were positive for Annexin V staining while the control cells expressing
GFP were negative (FIG. 1A, and data not shown).
 To further confirm that NRIF3 induces apoptosis, T-47D cells
expressing GFP-NRIF3 or GFP fused to a nuclear localization signal
(GFPNLS) were examined by a TUNEL assay (16). GFPNLS was included
as a control since NRIF3 is a nuclear protein (28). Initial experiments
suggested that the number of green fluorescent cells is maximal
20 to 24 h after transfection. Therefore, in most of our studies,
a TUNEL assay was carried out about 20 h after transfection. We
found that GFP-NRIF3 expressing cells were TUNEL positive while
GFPNLS expressing cells were TUNEL negative (FIG. 1B). Quantitative
analyses revealed that nearly 100% of GFP-NRIF3 expressing cells
were TUNEL positive within 24 h after transfection, compared with
little or no TUNEL reaction for GFPNLS expressing cells (FIG. 1C).
To rule out the possibility that cell death mediated by GFP-NRIF3
results from the fusion of GFP and NRIF3 instead of NRIF3 itself,
we also transfected T-47D cells with a vector expressing full-length
wild-type NRIF3 (not as a GFP fusion) and found that the transfected
cells also underwent apoptosis (data not shown). Taken together,
our results indicate that expression of NRIF3 induces rapid and
profound death in T-47D cells via apoptosis or an apoptosis-like
 Other members of the NRIF3 family induce apoptosis in T-47D
cells. The human NRIF3 gene encodes several different proteins as
a result of alternative splicing, including NRIF3, EnS (endonexin
short form), and EnL (endonexin long form) (28, 29, 44). Both EnS
and EnL share extensive identity with NRIF3 at the amino acid level
(28, 29) (FIG. 2A). EnS is identical to the first 111 amino acids
of NRIF3, and thus can be viewed as a naturally occurring truncation
of NRIF3. Like NRIF3, EnS and EnL are primarily nuclear-localized,
and together with NRIF3 constitute a new family of transcriptional
coregulators (29). Given the similarity among the NRIF3 family members,
we asked whether EnS and EnL also induce apoptosis in T-47D cells.
To this end, T-47D cells were transfected with vectors expressing
GFP-EnS or GFP-EnL, and 20 h later, were analyzed for expression
of the GFP-fusion proteins and for apoptosis. We found that expression
of GFP-EnS or GFP-EnL resulted in profound apoptosis (nearly 100%
of green cells were TUNEL positive), indicating that the N-terminal
portion of NRIF3 (residues 1-111) is sufficient to induce death
in T-47D cells (FIG. 2B and data not shown).
 NRIF3 contains a novel death domain (DD1). The region comprising
the first 111 amino acids of NRIF3 (equivalent to EnS), which is
sufficient to induce apoptosis, contains a number of structural
and functional features identified in previous studies (29, 36).
These include a coiled-coil domain (residues 84-112) that mediates
protein-protein interactions, a putative nuclear localization signal
(residues 63-66), a transrepression domain (residues 20-50, RepD1),
an LxxLL motif (residues 9-13) that plays a role in interaction
with certain nuclear receptors, and an RxL motif (residues 6-8)
that binds cyclin A and mediates interaction with cyclin A/Cdk2
(see FIG. 2A). To test whether any of these known domains/motifs
are required for NRIF3-mediated apoptosis and to further map the
functional death domain in NRIF3, we generated a series of GFP-vectors
expressing various regions of the N-terminal 111 amino acids of
NRIF3. These GFP-fusion constructs were then individually expressed
in T-47D cells to examine induction of apoptosis (FIG. 3A).
 Expression of regions comprising amino acids 1-86 or 20-86
were found to efficiently induce apoptosis in T-47D cells, indicating
that the coiled-coil domain (residues 84-112), the cyclin A binding
motif (residues 6-8), and the LxxLL motif (residues 9-13) are all
dispensable for the apoptogenic effect of NRIF3 (FIG. 3A). The region
comprising residues 20-86 of NRIF3 contains a previously identified
transrepression domain RepD1 (residues 20-50) (29). Thus, we further
examined RepD1 and found that its expression efficiently induced
death in T-47D cells (FIGS. 3A and 3B). Taken together, these results
identify a novel death domain (residues 20-50, designated here as
DD1) in the NRIF3 family, which interestingly, co-resides with RepD1
(FIG. 3A). We detected no homology for DD1 with other known death
domains in the database.
 Our previous study in other cell lines identified a putative
phosphorylation site (Ser28) in RepD1 (29). Phosphorylation of Ser28
appears to be essential for the transcriptional repression function
of RepD1 as a change of Ser28 to Ala (S28A) abolishes repression
(29). To test the potential role of Ser28 phosphorylation in apoptosis,
we examined the DD1/RepD1 S28A mutant and found that this mutation
markedly reduced the ability of DD1 to induce apoptosis in T-47D
cells (FIGS. 3B and 3C), suggesting that phosphorylation of Ser28
in vivo is important for the apoptogenic function of DD1.
 NRIF3- and DD1-mediated apoptosis is insensitive to zVAD-fmk.
Since activation of caspases is central to many cell death programs,
we tested whether zVAD-fmk, a broad-spectrum irreversible caspase
inhibitor (14, 47), has any effect on NRIF3- and DD1-mediated apoptosis
in T-47D cells. For these studies, T-47D cells were incubated with
100 uM zVAD-fmk prior to and after transfection with appropriate
GFP fusion constructs. We found that zVAD-fmk did not inhibit apoptosis
mediated by GFP-NRIF3 or GFPNLS-DD1 (FIGS. 4A and 4B). zVAD-fmk
alone did not cause cell death (data not shown). The same of dose
of zVAD-fmk was found to significantly inhibit apoptosis of HeLa
cells treated with TNF.alpha. and cycloheximide (data not shown),
where the apoptotic process is dependent on caspase-8 and downstream
effector caspase(s) (10, 18, 34). Taken together, our results indicate
that NRIF3- and DD1-mediated apoptosis in T-47D is insensitive to
 Role of mitochondria in DD1-mediated cell death. MMP is
a critical event in the intrinsic apoptosis pathway as it results
in the release of a number of death-promoting molecules such as
cytochrome-c and AIF (38, 50). Members of the Bcl-2 family regulate
mitochondria-mediated cell death by controlling MMP, which is determined
by the relative balance of pro-apoptotic (e.g. Bak, Bax, Bad and
Bid) and anti-apoptotic (e.g. Bcl-2 and Bcl-xL) members of the family
(3, 7). To assess whether DD1-induced cell death involves a mitochondria-mediated
pathway, we examined whether the apoptogenic effect of DD1 was inhibited
by Bcl-2. We found that co-expression of Bcl-2 significantly inhibited
DD1-mediated apoptosis in T-47D cells (FIGS. 5A and 5B). Similar
inhibition was found with Bcl-xL (data not shown). These results
suggest that MMP plays a role in DD1-induced apoptosis.
 To further document that DD1 induces MMP, we examined whether
AIF is translocated from the mitochondria to the nucleus during
DD1-mediated cell death, using a vector expressing AIF-GFP (31).
Consistent with a previous study (31), expression of AIF-GFP alone
resulted in a mitochondrial pattern of distribution (FIG. 5C). Interestingly,
co-expression of DD1 resulted in the translocation of AIF-GFP from
mitochondria to the nucleus as early as 5 h after transfection (FIG.
5C). Cells containing nuclear localized AIF-GFP also underwent apoptosis
as assessed by TUNEL assay (FIG. 5C). Taken together, our results
in FIG. 5 support a model whereby the DD1 of NRIF3 promotes apoptosis
in T-47D cells through a mitochondria-mediated pathway that is regulated
by Bcl-2 and involves the translocation of AIF. Since AIF promotes
effector caspase-independent apoptosis (8, 38), its translocation
during DD-induced cell death is consistent with our earlier finding
that this death program is insensitive to zVAD-fmk.
 Requirement for caspase-2 in DD1-mediated apoptosis. A number
of recent studies suggest an important role for caspase-2 in the
intrinsic (stress-induced) apoptosis pathway in certain cell types
where its activity is required for promoting MMP (17, 24, 27, 42).
Although caspase-2 was reported to be present in a number of cellular
compartments (for a review, see reference 48), recent studies suggest
that it is mainly a nuclear protein and that it can trigger MMP
and apoptosis from the nucleus without redistribution to the cytoplasm
(4, 12, 37, 45). The fact that members of the NRIF3 family are nuclear
proteins raises the possibility that the DD1 of NRIF3 might act
through activation of nuclear localized caspase-2. The finding that
DD1-induced cell death is not inhibited by zVAD-fmk does not exclude
a potential role for caspase-2, as caspase-2 is several orders of
magnitude more resistant to zVAD-fmk than other caspases (14).
 To explore this, we used an siRNA that had been previously
shown to efficiently silence human caspase-2 expression in transfected
cells (27). T-47D cells were first transfected with this caspase-2
siRNA. Forty-eight h later the cells were then harvested, re-plated,
and transfected with GFPNLS-DD1 to monitor apoptosis induced by
DD1. We found that DD1-mediated apoptosis was dramatically reduced
in cells treated with the caspase-2 siRNA (FIGS. 6A and 6C). In
contrast, mock-treated cells that did not receive caspase-2 siRNA
underwent rapid apoptosis upon expression of DD1 (FIGS. 6A and 6C).
Western analysis indicated that the level of caspase-2 protein was
reduced by more than 5- fold in specific siRNA treated cells, demonstrating
the effectiveness of the siRNA technique (data not shown). Consistent
with the siRNA study, we found that zVDVAD-fmk, a chemical inhibitor
of caspase-2, also efficiently blocked DD1-induced apoptosis in
T-47D cells, suggesting a requirement for caspase-2 activity (data
not shown). Interestingly, pre-treatment with the same caspase-2
siRNA had no effect on etoposide-induced apoptosis in T-47D cells
(FIG. 6B), indicating that caspase-2 is not universally required
for initiating apoptosis per se in these cells. The result of our
etopside study is reminiscent of the finding by Lassus et al. showing
that MCF-7 cells did not require caspase-2 for DNA damage-induced
release of cytochrome-c (27). Thus, while caspase-2 is not essential
for every intrinsic apoptotic program, our study here identifies
a specific role for caspase-2 in DD1-mediated apoptosis in T-47D
 Cell-type specificity in cytotoxicity mediated by NRIF3
and DD1. We previously reported that expression of NRIF3 enhances
ligand-dependent transactivation by TR or RXR in HeLa cells (28).
Thus, the finding of an apoptogenic function for NRIF3 in T-47D
cells was unexpected. One possibility is that the apoptogenic effect
of NRIF3 or DD1 is cell-type specific. In support of this notion,
we found that expression of GFP-NRIF3 or GFPNLS-DD1 did not lead
to apoptosis in HeLa cells (FIG. 7A).
 The dramatic difference in cellular response to the expression
of NRIF3 in T-47D cells (which exhibit 100% apoptosis) and HeLa
cells (which show no apoptosis) suggests cell-type specificity in
the cytotoxic effect mediated by NRIF3 and DD1. To further explore
this, we examined 11 cell lines for the induction of apoptosis upon
expression of NRIF3 or DD1. Five were breast cancer cell lines,
including ER+ T-47D and MCF-7 cells, ER- MB-MDA-231 and MB-MDA-435
cells, as well as an ER+ derivative of MB-MDA-231 (2). We also examined
HBL100 cells, a non-malignant but immortalized breast epithelial
cell line (13). Remarkably, we found that expression of NRIF3 or
DD1 resulted in efficient apoptosis in all 5 breast cancer cell
lines, as well as in the immortalized HBL100 cells (FIGS. 7A and
7B, and data not shown).
 In contrast, expression of NRIF3 or DD1 did not lead to
apoptosis in any of the other 5 examined tumor cell lines (HeLa,
293, COS-1, UOK-145, GH4C1) which are not derived from breast epithelium
(FIG. 7B). Thus, NRIF3 and DD1 appear to selectively induce apoptosis
in breast cancer or related cells but not in the other cell types
examined. This finding suggests that breast cancer cells contain
a novel "death switch" that is specifically triggered
by NRIF3 or DD1 (FIG. 8). We propose that triggering of this switch
by NRIF3 (or DD1) results in activation of caspase-2, which in turn
leads to further downstream events of apoptosis (FIG. 8).
 NRIF3 contains two other death domains (DD2 and DD3). In
addition to the death domain DD1 that is described in Example 3,
we uncovered two additional death domains within the NRIF3 molecule.
In one experiment, a region comprising amino acid residues 47 to
86 of NRIF3 (termed DD2) was fused in-frame with GFPNLS. The resulting
vector expressing GFPNLS-DD2 was transfected into T-47D cells to
examine the induction of apoptosis. As shown in FIG. 9, we found
that green cells expressing GFPNLS-DD2 were TUNEL positive, indicating
cell death. As a control, cells transfected with the GFPNLS control
vector were not TUNEL positive (data not shown).
 In another experiment, we transfected T-47D cells with vectors
expressing a variety regions of NRIF3 fused in frame with the DNA
binding domain of Gal4, and examined the transfected cells for induction
of apoptosis. In each case, a vector expressing GFPNLS was cotransfected
to visualize transfected cells (shown as green cells). Not surprisingly,
we found that expression of Gal4-NRIF3, or Gal4-EnS, or Gal4-DD1
all induced rapid apoptosis in T-47D cells (data not shown), while
expression of the Gal4 control did not result in cell death (FIG.
10). Interestingly, expression of a region comprising amino acid
residues 112 to 177 of NRIF3 (termed DD3) as a Gal4-fusion resulted
in efficient induction of apoptosis, as shown by the TUNEL assay
(FIG. 10). Taken together, our results in FIG. 9 and FIG. 10 indicate
that the NRIF3 molecule contains two additional death domains, a
DD2 which resides from amino acid residues 47 to 86; and a DD3 which
resides from amino acid residues 112 to 177.
 In this study we present a novel finding that expression
of members of the NRIF3 family of co-regulators leads to rapid and
profound apoptosis in a number of different breast cancer cell lines.
Deletion analysis showed that apoptogenic effect of the NRIF3 family
is mediated by a novel death domain (DD1) (residues 20-50, see FIG.
3). Interestingly, cytotoxicity of NRIF3 and DD1 appears to be specific
to breast cancer or related cells, as their expression did not lead
to apoptosis in other cell types such as HeLa cells (FIG. 7). Consistent
with this, previous studies have shown that NRIF3 acts as a co-activator
for TR and RXR in HeLa cells (28).
 Apoptosis plays important roles in both normal tissue homeostasis
and pathological processes such as tumorgenesis. Disruption of cellular
apoptotic pathways often accompanies tumorgenesis and likely confers
survival advantage to tumor cells (20). Since many anticancer drugs
kill by inducing apoptosis, dysregulation of apoptosis could also
lead to drug resistance (20). Although the progress in understanding
mechanisms underlying apoptosis and its regulation/dysregulation
in cancer cells presents opportunities to utilize the cellular death
machinery for treating human diseases such as cancer (20, 35), how
to deliver tumor-specific cytotoxicity without killing other innocent
cells remains an important challenge.
 The data presented herein demonstrate that cytotoxicity
can be selectively induced in a specific cancer, as NRIF3, DD1 and
related molecules kill breast cancer but not other cells (FIG. 7).
Since DD1 is devoid of regions involved in interaction with nuclear
receptors (28, 29), it is unlikely that apoptosis mediated by NRIF3
or DD1 results from perturbation of nuclear receptor functions.
DD1-induced apoptosis is inhibited by co-expression of Bcl-2 or
Bcl-xL (FIGS. 5A and 5B, and data not shown), and is associated
with the translocation of AIF from mitochondria to the nucleus (FIG.
5C), suggesting that cell death is mediated by a mitochondrial pathway
(FIG. 8). Since AIF is capable of triggering apoptosis independent
of effector caspases (8, 31, 46, 51), its rapid translocation from
mitochondria to the nucleus is consistent with the finding that
the broad-spectrum caspase inhibitor zVAD-fmk failed to block NRIF3-
or DD1-induced cell death (FIGS. 4A and 4B).
 The results of the siRNA study indicate that DD1-mediated
apoptosis requires caspase-2 (FIG. 6). The same caspase-2 siRNA
has no effect on etopside-induced apoptosis in T-47D cells (FIG.
6B), suggesting that its effect on DD1-mediated cell death is specific.
The requirement for caspase-2 is not inconsistent with the zVAD-fmk
results, as caspase-2 is much more resistant to zVAD-fmk than other
caspases (14). Interestingly, several recent studies have identified
a novel apical initiator role for caspase-2 during stress-induced
apoptosis, where it acts upstream of mitochondria and is required
for MMP (17, 27, 42). Without wishing to be bound by theory, it
is believed that expression of NRIF3 or DD1 in breast cancer cells
triggers activation of caspase-2, which then promotes MMP, leading
to the release of AIF and subsequent downstream events of apoptosis
(see FIG. 8). Interestingly, expression of caspase-2-GFP in T-47D
cells resulted in nuclear localization of the GFP signal (Dr. Honglin
Li, personal communication). This finding is consistent with the
model above, as members of the NRIF3 family and DD1 trigger apoptosis
in breast cancer cells while being primarily (if not exclusively)
localized to the nucleus (FIGS. 1B, 2B, and 3B).
 Although caspase-2 functions upstream of mitochondria in
certain intrinsic apoptotic pathways, the molecular mechanism(s)
underlying activation of caspase-2 (upon apoptotic stimuli) is not
yet understood (24, 48). Activation of other initiator caspases
such as caspase-8 and -9 is thought be to be mediated by a dimerization
mechanism that does not require proteolytic cleavage (6). It is
conceivable that caspase-2 could be activated similarly by complex
formation and/or dimerization (9, 40, 48). In this respect, NRIF3
or DD 1 may function in breast cancer cells by regulating and/or
participating in the process of caspase-2 activation. Interestingly,
Western blot analysis showed that caspase-2 is expressed at similar
levels in T-47D, MDA-MB-23 1, HeLa, and 293 cells (unpublished observations),
despite the fact that NRIF3 or DD1 only induces cytotoxicity in
T-47D and MDA-MB-231, but not in HeLa and 293 cells (FIG. 7). Thus,
a simple mechanism such as direct activation of caspase-2 by NRIF3
or DD1 via a bilateral protein-protein interaction seems unlikely.