U.S. patent application number 12/463666 was filed with the patent office on 2009-11-19 for non-steroidal anti-inflammatory drug activated gene with anti-tumorigenic properties.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human. Invention is credited to Seung Joon Baek, Thomas E. Eling.
Application Number | 20090286313 12/463666 |
Document ID | / |
Family ID | 22868380 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286313 |
Kind Code |
A1 |
Eling; Thomas E. ; et
al. |
November 19, 2009 |
NON-STEROIDAL ANTI-INFLAMMATORY DRUG ACTIVATED GENE WITH
ANTI-TUMORIGENIC PROPERTIES
Abstract
The present invention provides methods and compositions for drug
screens to identify and characterize agents that are agonistic or
antagonistic to activation of the promoter region of the NAG-1
gene. Activation of the NAG-1 gene is associated with the apoptotic
elimination of cancer cells both in vitro and in vivo. The
invention also provides novel promoter region sequences of the
NAG-1 gene.
Inventors: |
Eling; Thomas E.; (Raleigh,
NC) ; Baek; Seung Joon; (Knoxville, TN) |
Correspondence
Address: |
LEYDIG, VOIT & MAYER, LTD.
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and
Human
Bethesda
MD
Service
|
Family ID: |
22868380 |
Appl. No.: |
12/463666 |
Filed: |
May 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11563586 |
Nov 27, 2006 |
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12463666 |
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10363514 |
Aug 15, 2003 |
7141661 |
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PCT/US01/27544 |
Sep 6, 2001 |
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11563586 |
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60231246 |
Sep 8, 2000 |
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Current U.S.
Class: |
435/325 ;
530/387.9 |
Current CPC
Class: |
C07K 14/495 20130101;
C07K 14/4747 20130101; A01K 2217/05 20130101; G01N 33/5082
20130101; A61P 35/00 20180101; G01N 2500/10 20130101 |
Class at
Publication: |
435/325 ;
530/387.9 |
International
Class: |
C12N 5/00 20060101
C12N005/00; C07K 16/00 20060101 C07K016/00 |
Claims
1-14. (canceled)
15. An isolated or purified antibody reactive with a polypeptide
encoded by a nucleic acid sequence comprising SEQ ID NO: 2.
16. A composition comprising the isolated or purified antibody of
claim 15.
17. A host cell comprising the isolated or purified antibody of
claim 15.
Description
FIELD OF THE INVENTION
[0001] This invention generally relates to drug screens for agents
that are agonistic or antagonistic to activation of the promoter
region of the NAG-1 gene.
BACKGROUND OF THE INVENTION
[0002] Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely
used in the treatment of inflammatory disease. Their
anti-inflammatory effects are believed to result from their ability
to inhibit the formation of prostaglandins by prostaglandin H
synthase (COX). Two isoforms of prostaglandin H synthase, COX-1 and
COX-2 have been identified. COX-1 is constitutively expressed in
many tissues, while the expression of COX-2 is regulated by
mitogens, tumor promoters, and growth factors. High expression of
COX-2 has been reported in human colorectal tumors and tumors from
other tissues, suggesting a role for this enzyme in regulating
tumor growth. NSAIDs are effective in reducing human and rodent
colorectal and possibly, breast and lung cancer. In addition,
retrospective and prospective studies link NSAID usage to a reduced
risk for colorectal cancer death. NSAIDs are effective in reducing
the number and size of polyps in animal models, and epidemiological
studies indicate that use of NSAIDs provide a 40-50% reduction in
mortality from colorectal cancer.
[0003] Our understanding of the mechanisms by which NSAIDs exert
their anti-tumor effect is not clear. One possible mechanism is
altered arachidonic acid (AA) metabolism, since NSAIDs inhibit the
formation of prostaglandins by COX-1 and COX-2. Recent data suggest
that inhibition of COX by NSAIDs may increase the cellular pool of
AA, resulting in the hydrolysis of sphingomyelin to ceramide, which
promotes apoptosis. Some data Link NSAID chemoprevention in
colorectal cancer cells to prevention of angiogenesis and induction
of apoptosis. COX appears to regulate angiogenesis induced by colon
cancer cells and NSAIDs appear to limit tumor growth. In epithelial
cells of the intestinal crypt, elevated COX-2 expression appears to
attenuate apoptosis. Some evidence links NSAID-induced apoptosis to
inhibition of COX, but other data suggest a
prostaglandin-independent mechanism for the induction of apoptosis.
PPAR.gamma. agonists also have anti-tumorigenic activity and slate
apoptosis. For example, ligand activation of PPAR.gamma. inhibits
proliferation of breast cancer cells, and the addition of
PPAR.gamma. ligands to cancer cells induces apoptosis. PPAR.gamma.
agonists also inhibit the growth of transplantable tumors in a nude
mouse model.
[0004] As is evident from the foregoing, NSAIDs, and the pathways
they activate, have great potential for use in the war against
cancer. However, as is also evident from the above, insufficient
information has been available regarding the NSAID-induced or
associated anti-cancer pathways, to help identify agents that are
potentially advantageous as cancer treatments. What is needed are
novel reagents and methods that can be used to screen for compounds
that show promise as anti-cancer agents.
SUMMARY OF THE INVENTION
[0005] The present invention generally relates to compositions and
methods of identifying and testing NAG-1 gene-activating agonists
and antagonists. Activation of the NAG-1 gene by NSAIDs has
been-correlated with the apoptotic elimination of certain types of
cancer cells. Although not limited to any particular mechanism, one
embodiment of the present invention involves the use of the
promoter region of the NAG-1 gene to test for agents that are
agonistic or antagonistic to promoter activation. In this regard,
test kits also constitute an embodiment of the present invention.
In addition, the invention provides methods to identify other
members of the NAG-1 gene family, methods to identify homologs of
NAG-1 which are native to other tissue or cell types and methods to
generate reagents derived from the invention.
[0006] The present invention is not limited by the method of the
employed screen. In one embodiment, the present invention
contemplates screening suspected compounds in a system utilizing
transfected cell lines. in one preferred embodiment, the cells are
transfected transiently, while in another preferred embodiment, the
cells are stably transfected. In yet another preferred embodiment,
transgenic animals are generated. In yet another embodiment, high
throughput screens are contemplated. For example, the present
invention provides means to screen compound libraries, peptide
libraries and the like, in order to identify suitable antagonists
or agonists.
[0007] The present invention also contemplates the use of a
reporter gene construct operationally liked to the NAG-1 promoter.
The present invention is not limited to any particular reporter
gene construct, as many reporter gene constructs will find use with
the present invention For example, in a preferred embodiment, a
luciferase reporter gene is used. In other embodiments, genes
encoding fluorescent proteins (e.g., green fluorescent protein),
.beta.-galactosidase, or proteins that may be precipitated (e.g.,
by immunoprecipitation) are used.
[0008] It is further contemplated that the present invention will
find use in the identification of native activators or inhibitors
of the NAG-1 gene. For example, it is contemplated that peptide
libraries produced from native genes are screened by the present
invention. Non-natively occurring peptides are also be screened by
the present invention. In another embodiment, combinatorial
libraries are screened In yet another embodiment, mixtures of
compounds are screened. In some preferred embodiments, if a
compound mixture gives a positive test result, the mixture is then
subdivided into constitutory components and retested. In still
fisher embodiments, this procedure is repeated until the active
component or components are identified.
[0009] The present invention also be finds use in the
identification of new homologs of NAG-1, as well as genes that
share similarities in the promoter region or natural mutations
thereof. The present invention contemplates screening for homologs
using standard molecular procedures. In one preferred embodiment,
screens are conducted using Northern and Southern blotting.
[0010] Furthermore, as shown in Example 8 below, the NAG-1 peptide
has anti-tumor and pro-apoptotic effects. It is contemplated that
this peptide, or a portion thereof, will be effective in the
treatment of certain cancers when administered directly to tumors
or to cancer patients. In this regard, the present invention also
provides compositions and methods for the use of the NAG-1 peptide
for the treatment of cancers. The present invention further
provides the compositions and methods for use of modified or
derived variations of the NAG-1 peptide for the treatment of
cancers.
[0011] The present invention is not limited to the treatment of any
particular cancer as it is contemplated that all cancers may be
treated with the NAG-1 peptide. The present invention is also not
limited to any particular portion of the NAG-1 peptide for the
treatment of cancers, as it is contemplated that any suitable
portion of the NAG-1 peptide will find use in cancer treatment.
Furthermore, the present invention is not limited to any particular
modification of the NAG-1 peptide for the treatment of cancer.
Indeed, any modification that changes the ability of the NAG-1
peptide to treat cancer is embodied in the present invention. Such
changes include, but are not limited to, increased stability,
increased solubility, increased tumor killing ability, decreased
toxicity, increased half-life before and after administration to
the patient etc. Also, the present invention is not limited to any
particular method of administration, as the NAG-1 peptide may be
administered by any suitable means including, but not limited to,
oral, nasal, intravenous, intramuscular, intrathecal and
subcutaneous modes of administration. Additionally, the NAG-1
peptide may be administered as an ointment, lotion or gel (i.e.,
for the treatment of skin and mucosal tumors).
[0012] The present invention further provides purified DNA having
an oligonucleotide sequence comprising SEQ ID NO:1, as well as
compositions comprising this sequence or portion(s) thereof. In
addition, the present invention provides expression vectors
comprising at least a portion of the oligonucleotide sequence of
SEQ ED NO: 1. Further still, the present invention provides
compositions comprising the translation product of at least a
portion of SEQ ID NO:1. The present invention further provides
compositions comprising antibodies reactive with at least a portion
of the translation product of SEQ ID NO:1. The present invention
also provides transgenic animals generated from an expression
vector comprising at least a portion of the oligonucleotide
sequence of SEQ ID NO:1.
[0013] The present invention provides methods for screening
compounds, comprising the steps of: a) providing in any order,
cells comprising a recombinant expression vector, wherein the
vector comprises at least a portion of the oligonucleotide sequence
of SEQ ID NO:1 and at least one reporter construct; and, at least
one compound; b) contacting the cells with the compound(s) to
produce treated cells; and, c) detecting the readout of the
reporter construct or constructs in the treated cells.
[0014] The present invention also provides methods for screening
compounds, comprising the steps of: a) providing in any order, at
least one transgenic animal generated from a recombinant expression
vector wherein, the vector comprises at least a portion of the
oligonucleotide sequence of SEQ ID NO:1 and at least one reporter
construct; and, at least one compound; b) exposing the transgenic
animal(s) to at least one compound to produce at least one treated
animal; and, c) detecting the readout of the reporter construct(s)
in the treated animal(s). The present invention further provides
methods wherein, at least one transgenic animal is repeatedly (i.e.
more than once) exposed to at least one compound. The present
invention also further provides methods wherein the treated animal
further comprises cancer cells and a decrease or increase in the
number of cancer cells present in the treated animal is
measured.
[0015] The present invention also provides methods comprising:
providing in any order, a patient with symptoms of cancer and, a
composition comprising at least a portion of the NAG-1 peptide; and
b) administering the composition to the patient The present
invention is not limited to any particular type of cancer as it is
contemplated that the present invention will find use with all
types of cancer. Additionally, the present invention is not limited
to any particular means of administration of NAG-1 to the patient,
as the NAG-1 peptide may be administered by any suitable means
including, but not limited to, oral, nasal, intravenous,
intramuscular, intrathecal and subcutaneous modes of
administration. Additionally, the NAG-1 peptide may be administered
as an ointment, lotion or gel (i.e., for the treatment of skin and
mucosal tumors).
DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the identification and expression of NAG-1 by
INDO. Panel A provides a schematic diagram for reported genes PLAB,
PTGFB, PDDF, MIC-1, and IP00269. The bar indicates the coding
region of cDNA with amino acids reported previously. Panel B
provides Northern and Western analysis results that indicate NAG-1
induction is time-dependent. Panel C is a graph showing the
apoptosis and cell cycle kinetics of INDO-treated -HCT-116 cells at
different time points. Panel D provides Northern and Western data
showing the concentration-dependent expression of NAG-1. Panel E is
a graph showing the apoptosis and cell-cycle kinetics of
INO-treated HCT-116 cells at different concentrations.
[0017] FIG. 2 shows NAG-1 induction and NSAID-induced apoptosis by
several NSAIDs. Panel A shows results for HCT-116 cells treated
with various NSAIDs.1 Panel B is a graph showing the correlation
between induction of apoptosis and increased expression of
NAG-1.
[0018] FIG. 3 shows NAG-1 induction by INDO and aspirin in
different cell lines. Panel A provides Northern. blot results for
A549 lung carcinoma cells, while Panel B provides Northern blot
results for MCF-7 breast carcinoma cells, Panel C provides Northern
blot results for U937 leukemia cells, and Panel D provides Northern
blot results for PC-3 prostate cells.
[0019] FIG. 4 shows PPAR.gamma. ligands also increase NRAG-1
expression. Panel A provides results for 15dPGJ.sub.2 and TGZ.
Panel B provides Northern and Western results indicating the
time-dependent expression of NAG-1 mRNA and protein in HCT-116
cells. Panel C is a graph showing the apoptosis and cell cycle
kinetics of TGZ-treated HCT-116 cells at different times.
[0020] FIG. 5 shows the genomic structure and promoter analysis of
NAG-1 in HCT-116 cells. Panel A provides a restriction map of the
NAG-1 gene. Panel B is a deletion analysis of the NAG-1 promoter.
Panel C is a graph showing the luciferase activity of 2021 bp NAG-1
promoter in the presence of TGZ and various NSAIDs.
[0021] FIG. 6 shows ectopic expression of NAG-1 induces apoptosis
in vitro. Panel A provides Western analysis of ectopic expression
of NAG-1 in HCT-116 cells. Panel B is a graph showing the results
for stably transfected HCT-116 cells treated with a vehicle or 100
.mu.M INDO.
[0022] FIG. 7 shows that NAG-1 reduces growth rate of tumors in
nude mice. Panel A, is a Western blot showing the results for
transfected with HCT-116 cells probed with anti-NAG-1 antibody.
Panel B is a graph showing the tumor size determined over time.
Panel C provides H&E stained and TUNEL tissue sections of the
tumor area.
[0023] FIG. 8 provides tie NAG-1 promoter region. In particular,
the 9 kb nucleotide sequence promoter region (SEQ ID NO:1) of the
NAG-1 gene (SEQ ID NO:2) is shown. In this Figure, lower case
letters represent previously published sequences. The region from
5368 to 8821 is the 3.5 kb region referred to as SEQ ID NO:3.
Position 8821 is the translational start point. Position 8788 is
the transcriptional start point.
[0024] FIG. 9 provides the NAG-1 gene sequence (SEQ ID NO:2;
GenBank database accession no. AF019770).
[0025] To facilitate understanding of the invention, a number of
terms are defined below.
[0026] As used herein, the terms "purified" and "to purity" refer
to the removal of one or more (undesired) components from a sample.
For example, where recombinant polypeptides are expressed in
bacterial host cells, the polypeptides are purified by the removal
of host cell proteins thereby increasing the percent of recombinant
polypeptides in the sample.
[0027] As used herein, the term "partially purified" refers to the
removal of a moderate portion of the contaminants of a sample to
the extent that the substance of interest is recognizable by
techniques known to those skilled in the art as accounting for a
measurable amount of the mixture.
[0028] As used herein, the term "substantially purified" refers to
molecules, (e.g., nucleic or amino acid sequences) that are removed
from their natural environment, isolated or separated, and are at
least 60% free, preferably 75% free, and more preferably 90% free
from other components with which they are naturally associated.
Furthermore, an "isolated polynucleotide" encompasses a
substantially purified polynucleotide.
[0029] As used herein "agent," "compound," and "drug," are used
herein to denote a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials such as
bacteria, plants, fungi, or animal (particularly mammalian) cells
or tissues that are suspected of having therapeutic properties. The
compound, agent or drug may be purified, substantially purified or
partially purified Additionally, an "agent," "compound" or "drug"
may be substantially pure (i.e., in some cases, comprised of
essentially one component).
[0030] As used herein "agonist" refers to molecules or compounds
which mimic the action of a "native" or "natural" compound. The
present invention encompasses agonists that are homologous to these
natural compounds, in respect to conformation, charge or other
characteristics, as well as compounds that are not homologous.
Thus, agonists may or may not be recognized by, for example,
receptors expressed on cell surfaces. In any event, regardless of
whether the agonist is recognized by a natural compound in a manner
similar to a "natural" compound or molecule, in some cases the
agonist causes physiologic and/or biochemical changes within the
cell (i.e., such that the cell reacts to the presence of the
agonist) in the same manner as if the natural compound was
present.
[0031] As used herein "antagonist" refers to molecules or compounds
which inhibit the action of a "native" or "natural" compound. As
used herein, "antagonist" also encompasses compounds that are
homologous to these natural compounds in respect to conformation,
charge or other characteristics, as well as, compounds that are not
homologous. Thus, antagonists are recognized by the same or
different receptors or molecules as recognized by an agonist
Antagonists may have allosteric effects which prevent the action of
an agonist (e.g., by modifying a DNA adduct). In addition, in some
cases, antagonists prevent the function of the agonist (e.g., by
blocking a DNA repair molecule).
[0032] "Patient" as used herein, refers to a human or other animal,
such as a guinea pig, mouse, rat, etc. In some preferred
embodiments, a patient is treated using the methods and
compositions of the present invention.
[0033] "Host" as used herein, refers to a recipient cell or
organism.
[0034] "Transgenic animal" as used herein, refers to a non-human
animal having a genetically engineered genotype. In particularly
preferred embodiments, transgenic animals are produced by
experimental manipulation of the genome of the germline of the
non-human animal. Genetically engineered non-human animals may be
produced by several methods including the introduction of a
"transgene" comprising nucleic acid (usually DNA) into an embryonal
target cell or integration into a chromosome of the somatic and/or
germ line cells of a non-human animal by way of human intervention,
such as by the methods described herein. Non-human animals
containing at least one a transgene are referred to as "transgenic
non-human animals." Thus, a transgenic animal is an animal whose
genome has been altered by the introduction of a transgene. Such
non-human ails include vertebrates such as rodents, non-human
primates, ovines, bovines, ruminants, lagomorphs, porcines,
caprines, equines, canines, felines, aves, etc. Preferred non-human
animals are selected-from the order Rodentia which includes murines
(e.g., rats and mice), most preferably mice.
[0035] "Apoptosis" as used herein, refers to the generally
recognized term for the morphological changes that are observed in
a cell as the cell undergoes a non-accidental death.
[0036] "Programmed cell death" as used herein, refers to the
genetically controlled process that is executed in a cell that has
been induced to undergo apoptosis.
[0037] "Antibody" as used herein, refers a glycoprotein produced by
B cells and plasma cells that binds with high specificity to an
antigen (usually, but not always, a peptide) or a structurally
similar antigen, that generated its production. Antibodies may be
produced by any of the known methodologies and may be either
polyclonal or monoclonal.
[0038] "Wild-type," as used herein, refers to a gene or gene
product which has the characteristics of hat gene or gene product
when isolated from a naturally occurring source. A wild-type gene
is that which is most frequently observed in a population and is
thus arbitrarily designed the "normal" or "wild-type" form of the
gene.
[0039] "Mutant," as used herein, refers to any changes made to a
wild-type nucleotide sequence, either naturally or artificially,
that produces a translation product that functions with enhanced or
decreased efficiency in at least one of a number of ways including,
but not limited to, specificity for various interactive molecules,
rate of reaction and longevity of the mutant molecule.
[0040] "Staining," as used herein, refers to any number of
processes known to those in the field that are used to better
visualize a specific component(s) and/or feature(s) of a cell or
cells.
[0041] "TUNEL" as used herein, refers to terminal deoxynucleotidyl
transferase (TdT)-mediated FITC-dUTP nick end labeling, a technique
to quantitate apoptosis known to those in the field.
[0042] The terms "cancerous" and "cancer cell" refer to a cell
undergoing early, intermediate or advanced stages of multi-step
neoplastic progression as known in the art (See e.g., Pitot, in
Fundamentals of Oncology, Marcel Dekker (Ed.), New York, pp. 15-28
[1978]). The microscopic features of early, intermediate and
advanced stages of neoplastic progression have been described.
Cancer cells at each of the three stages of neoplastic progression
generally have abnormal Iaryotypes, including translocations,
inversion, deletions, isochromosomes, monosomies, and extra
chromosomes. A cell in the early stages of malignant progression is
referred to as "hyperplastic cell" and is characterized by dividing
without control and/or at a greater rate than a normal cell of the
same cell type in the same tissue. Proliferation may be slow or
rapid but continues unabated. A cell in the intermediate stages of
neoplastic progression is referred to as a "dysplastic cell." A
dysplastic cell resembles an immature epithelial cell, is generally
spatially disorganized within the tissue and has lost its
specialized structures and functions. For example, during the
intermediate stages of neoplastic progression, an increasing
percentage of the epithelium becomes composed of dysplastic cells,
"Hyperplastic" and "dysplastic" cells are referred to as
"pre-neoplastic" cells. In the advanced stages of neoplastic
progression a dysplastic cell become a "neoplastic" cell.
Neoplastic cells are typically invasive. Thus, they either invade
adjacent tissues, or are shed from the primary site and circulate
through the blood and lymph to other locations in the body where
they initiate secondary cancers. The term "cancer" or "neoplasia"
refers to a plurality of cancer cells.
[0043] "Nucleic acid sequence," "nucleotide sequence" and
"polynucleotide sequence" as used herein, refer to an
oligonucleotide or polynucleotide, and fragments or portions
thereof, and to DNA or RNA of genomic or synthetic origin which may
be single, or double-stranded, and represent the sense or antisense
strand.
[0044] As used herein, the terms "oligonucleotides" and "oligomers"
refer to a nucleic acid sequence of at least about 10 nucleotides
and as many as about 60 nucleotides, preferably about 15 to 30
nucleotides, and more preferably about 20-25 nucleotides, which can
be used as a probe or amplimer.
[0045] The term "nucleotide sequence of interest" refers to any
nucleotide sequence, the manipulation of which may be deemed
desirable for any reason, by one of ordinary skill in the art. Such
nucleotide sequences include, but are not limited to, coding
sequences of structural genes (e.g., reporter genes, selection
marker genes, oncogenes, drug resistance genes, growth factors,
etc.), and non-coding regulatory sequences that do not encode an
mRNA or protein product (e.g., promoter sequences, enhancer
sequences, polyadenylation sequences, termination sequences,
etc.).
[0046] "Amino acid sequence," "polypeptide sequence," "peptide
sequence," and "peptide" are used interchangeably herein to refer
to a sequence of amino acids.
[0047] A "variant" of a nucleotide sequence is defined as a
nucleotide sequence which differs from the referenced, parent or
wild type nucleotide sequence (e.g., by having one or more
deletions, insertions, or substitutions that may be detected using
hybridization assays or using DNA sequencing). Included within this
definition is the detection of alterations to the genomic sequence
of the nucleotide sequence. For example, hybridization assays may
be used to detect alterations in: (1) the pattern of restriction
enzyme fragments capable of hybridizing to a genomic sequence of
the first nucleotide sequence (ie., RFLP analysis); (2) the
inability of a selected portion of the first nucleotide sequence to
hybridize to a sample of genomic DNA which contains the first
nucleotide sequence (e.g., using allele-specific oligonucleotide
probes); and (3) improper or unexpected hybridization, such as
hybridization to a locus other than the normal chromosomal locus
for the first nucleotide sequence (e.g. using fluorescent in situ
hybridization (FISH) to metaphase chromosomes spreads, etc.). One
example, of a variant is a mutated wild type sequence.
[0048] The term "portion" when used in reference to a nucleotide
sequence refers to fragments of that nucleotide sequence. The
fragments may range in size from 5 nucleotide residues to the
entire nucleotide sequence minus one nucleic acid residue.
[0049] DNA molecules are said to have "5' ends" and "3' ends"
because mononucleotides are reacted to make oligonucleotides in a
manner such that the 5' phosphate of one mononucleotide pentose
ring is attached to the 3' oxygen of its neighbor in one direction
via a phosphodiester linkage. Therefore, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring. An
end of an oligonucleotide is referred to as the "3' end" if its 3'
oxygen is not linked to a 5' phosphate of another mononucleotide
pentose ring. As used herein, a nucleic acid sequence, even if
internal to a larger oligonucleotide, also may be said to have 5'
and 3' ends. In either a linear or circular DNA molecule, discrete
elements are referred to as being "upstream" or 5' of the
"downstream" or 3' elements. This terminology reflects that
transcription proceeds in a 5' to 3' direction along the DNA stand.
The promoter and enhancer elements which direct transcription of a
linked gene are generally located 5' or upstream of the coding
region. However, enhancer elements can exert their effect even when
located 3' of the promoter element and the coding region.
Transcription termination and polyadenylation signals are located
3' or downstream of the coding region.
[0050] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule which is comprised of segments of DNA joined
together by means of molecular biological techniques.
[0051] The term "recombinant protein" or "recombinant polypeptide"
as used herein refers to a protein molecule which is expressed
using a recombinant DNA molecule.
[0052] As used herein, the terms "vector" and "vehicle" are used
interchangeably in reference to nucleic acid molecules that
transfer DNA segment(s) from one cell to another.
[0053] The terms "expression vector," "expression construct,"
"expression cassette" and "plasmid," as used herein refer to a
recombinant nucleic acid molecule containing a desired coding
sequence and appropriate nucleic acid sequences necessary for the
expression of the operably linked coding sequence in a particular
host organism. The sequences may be either double or
single-stranded. Nucleic acid sequences necessary for expression in
prokaryotes usually include a promoter, an operator (optional), and
a ribosome binding site, often along with other sequences.
Eukaryotic cells are known to utilize promoters, enhancers, and
termination and polyadenylation signals.
[0054] The terms "in operable combination," "in operable order,"
and "operably liked" as used herein refer to the linkage of nucleic
acid sequences in such a manner that a nucleic acid molecule
capable of directing the transcription of a given gene and/or the
synthesis of a desired protein molecule is produced. The terms also
refer to the linkage of amino acid sequences in such a manner so
that a functional protein is produced.
[0055] "Reporter construct," "reporter gene," and "reporter
protein," as used herein, refer to nucleic acid or amino acid
sequences, as appropriate, that, when expressed in a host cell or
organism, may be detected measured or quantitated.
[0056] The term "transfection" as used herein refers to the
introduction of foreign nucleic acid (e.g., DNA) into cells.
Transfection may be accomplished by a variety of means known to the
art including calcium phosphate-DNA co-precipitation,
DEAE-dextran-mediated transfection, polybrene-mediated
transfection, electroporation, microinjection, liposome fusion,
lipofection, protoplast fusion, retroviral infection, biolistics
(i.e., particle bombardment) and the like.
[0057] As used herein, the terms "complementary" or
"complementarity" are used in reference to "polynucleotides" and
"oligonucleotides" (which are interchangeable terms that refer to a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "5'-CAGT-3'," is complementary to the
sequence "5'-ACTG-3'."Complementarity can be "partial" or "total."
"Partial" complementarity is where one or more nucleic acid bases
is not matched according to the base pairing rules. "Total" or
"complete" complementarity between nucleic acids is where each and
every nucleic acid base is matched with another base under the base
pairing rules. The degree of complementarity between nucleic acid
strands may have significant effects on the efficiency and strength
of hybridization between nucleic acid strands. This may be of
particular importance in amplification reactions, as well as
detection methods which depend upon binding between nucleic
acids.
[0058] The terms "homology" and "homologous" as used herein in
reference to nucleotide sequences refer to a degree of
complementarity with other nucleotide sequences. There may be
partial homology or complete homology (i.e., identity). A
nucleotide sequence which is partially complementary (i.e.,
"substantially homologous") to a nucleic acid sequence is one that
at least partially inhibits a completely complementary sequence
from hybridizing to a target nucleic acid sequence. The inhibition
of hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous sequence to a target
sequence under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
identity); in the absence of non-specific binding the probe will
not hybridize to the second non-complementary target.
[0059] In preferred embodiments, an oligonucleotide sequence which
is a "homolog" of a first nucleotide sequence is an oligonucleotide
sequence which exhibits greater than or equal to 50% identity, and
more preferably greater than or equal to 70% identity, to the first
nucleotide sequence, when sequences having a length of 10 bp or
larger are compared.
[0060] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids using any
process by which a strand of nucleic acid joins with a
complementary stand through base pawing to form a hybridization
complex. Hybridization and the strength of hybridization (i.e., the
strength of the association between the nucleic acids) is impacted
by such actors as the degree of complementarity between the nucleic
acids, stringency of the conditions involved, the T.sub.m of the
formed hybrid, and the G:C ratio within the nucleic acids.
[0061] As used herein the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bounds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized to a solid support (e.g., a nylon membrane or
a nitrocellulose filter as employed in Southern and Northern
blotting, dot blotting or a glass slide as employed in in situ
hybridization, including FISH (fluorescent in situ
hybridization)).
[0062] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41 (% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations which take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0063] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. "Stringency" typically occurs in a
range from about T.sub.m.degree. C. to about 20.degree. C. to
25.degree. C. below T.sub.m. As will be understood by those of
skill in the art, a stringent hybridization can be used to identify
or detect identical polynucleotide sequences or to identify or
detect similar or related polynucleotide sequences. Under
"stringent conditions" the nucleotide sequence of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or portions
thereof, will hybridize to its exact complement and closely related
sequences.
[0064] Low stringency conditions comprise conditions equivalent to
binding or hybridization at 68.degree. C. in a solution consisting
of 5.times. SSPE (43.8 g/l NaCl, 6:9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times. Denhardt's reagent (50.times. Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma))
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 2.0.times. SSPE, 0.1% SDS at room temperature
when a probe of about 100 to about 1000 nucleotides in length is
employed.
[0065] It is well known in the art that numerous equivalent
conditions may be employed to comprise low stringency conditions;
factors such as the length and nature (DNA, RNA, base composition)
of the probe and nature of the target (DNA, RNA, base composition,
present in solution or immobilized, etc.) and the concentration of
the salts and other components (e.g., the presence or absence of
formamide, dextran sulfate, polyethylene glycol), as well as
components of the hybridization solution may be varied to generate
conditions of low stringency hybridization different from, but
equivalent to, the above listed conditions. In addition, conditions
which promote hybridization under conditions of high stringency
(e.g., increasing the temperature of the hybridization and/or wash
steps, the use of formamide in the hybridization solution, etc.)
are well known in the art. High stringency conditions, when used in
reference to nucleic acid hybridization, comprise conditions
equivalent to binding or hybridization at 68.degree. C. in a
solution consisting of 5.times. SSPE, 1% SDS, 5.times. Denhardt's
reagent and 100 .mu.g/ml denatured salmon sperm DNA followed by
washing in a solution comprising 0.1.times. SSPE and 0.1% SDS at
68.degree. C. when a probe of about 100 to about 1000 nucleotides
in length is employed.
[0066] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe which can hybridize either
partially or completely to either or both strands of the
double-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0067] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
which can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0068] The term "heterologous nucleic acid sequence" or
"heterologous DNA" are used interchangeably to refer to a
nucleotide sequence which is ligated to a nucleic acid sequence to
which it is not ligated in nature, or to which it is ligated at a
different location in nature. Heterologous DNA is not endogenous to
the cell into which it is introduced, but has been obtained from
another cell. Generally, although not necessarily, such
heterologous DNA encodes RNA and proteins that are not normally
produced by the cell into which it is expressed. Examples of
heterologous DNA include reporter genes, transcriptional and
translational regulatory sequences, selectable marker proteins
(e.g., proteins which confer drug resistance), etc.
[0069] "Amplification" is defined herein as the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction technologies well known
in the art (See e.g., Dieffenbach and Dveksler, PCR Primer, a
Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
[1995]). As used herein the term "polymerase chain reaction"
("PCR") refers to the methods of U.S. Pat. Nos. 4,683,195.,
4,683,202, and 4,965,188, all of which are hereby incorporated by
reference, and which describe a method for increasing the
concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. The length of the
amplified segment of the desired target sequence is determined by
the relative positions of two oligonucleotide primers with respect
to each other, and therefore, this length is a controllable
parameter. By virtue of the repeating aspect of the process, the
method is referred to as the "polymerase chain reaction"
(hereinafter "PCR"). Because the desired amplified segments of the
target sequence become the predominant sequences (in terms of
concentration) in the mixture, they are said to be "PCR
amplified."
[0070] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (e.g., hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide sequence can be amplified with the appropriate set
of primer molecules. In particular, the amplified segments created
by the PCR process itself are, themselves, efficient templates for
subsequent PCR amplifications.
[0071] The terms "reverse transcription polymerase chain reaction"
and "RT-PCR" refer to a method for reverse transcription of an RNA
sequence to generate a mixture of cDNA sequences, followed by
increasing the concentration of a desired segment of the
transcribed cDNA sequences in the mixture without cloning or
purification. Typically, RNA is reverse transcribed using a single
primer (e.g., an oligo-dT primer) prior to PCR amplification of the
desired segment of the transcribed DNA using two primers.
[0072] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e.., in
the presence of nucleotides and of an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0073] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that it
is detectable in any detection system, including, but not limited
to enzyme (e.g., ELISA, as well as enzyme-based histochemical
assays), fluorescent, radioactive, and luminescent systems. It is
not intended that the present invention be limited to any
particular detection system or label.
[0074] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
doubles single-stranded nucleic acid at or near a specific
nucleotide sequence.
[0075] As used herein, the term "an oligonucleotide having a
nucleotide sequence encoding a gene" means a nucleic acid sequence
comprising the coding region of a gene (i.e. the nucleic acid
sequence which encodes a gene product). The coding region may be
present in either a cDNA, genomic DNA or RNA form. When present in
a DNA form, the oligonucleotide may be single-stranded (i.e.., the
sense strand) or double-stranded. Suitable control elements (e.g.,
enhancers, promoters, splice junctions, polyadenylation signals,
etc.) may be placed in close proximity to the coding region of the
gene, if needed to permit proper initiation of transcription and/or
correct processing of the primary RNA transcript. Alternatively,
the coding region utilized in the expression vectors of the present
invention may contain endogenous enhancers, splice junctions,
intervening sequences, polyadenylation signals, or other sequences,
or a combination of both endogenous and exogenous control
elements.
[0076] Transcriptional control signals in eukaryotes comprise
"enhancer" elements. Enhancers consist of short arrays of DNA
sequences that interact specifically with cellular proteins
involved in transcription (Maniatis et al., Science 236:1237
[1987]). Enhancer elements have been isolated from a variety of
eukaryotic sources including genes in plant yeast, in sect and an
cells and viruses. The selection of a particular enhancer depends
on what cell type is to be used to express the protein of
interest.
[0077] The presence of "splicing signals" on an expression vector
often results in higher levels of expression of the recombinant
transcript. Splicing signals mediate the removal of introns from
the primary RNA transcript and consist of a splice donor and
acceptor site (Sambrook, et al, Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp.
16.7-16.8 [1989]). A commonly used splice donor and acceptor site
is the splice junction from the 16S RNA of SV40.
[0078] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires expression of signals directing the
efficient termination polyadenylation of the resulting transcript.
Transcription termination signals are generally found downstream of
the polyadenylation signal and are a few hundred nucleotides in
length. The term "poly A site" or "poly A sequence" as used herein
denotes a DNA sequence which directs both the termination and
polyadenylation of the nascent RNA transcript. Efficient
polyadenylation of the recombinant transcript is desirable as
transcripts lacking a poly A tail are unstable and are rapidly
degraded The poly A signal utilized in an expression vector may be
"heterologous" or "endogenous." An endogenous poly A signal is one
that is found naturally at the 3' end of the coding region of a
given gene in the genome. A heterologous poly A signal is one which
is isolated from one gene and placed 3' of another gene.
[0079] The terms "promoter," "promoter element," and "promoter
sequence" as used herein, refer to a DNA sequence which when placed
at the 5' end of (i.e., precedes) an oligonucleotide sequence is
capable of controlling the transcription of the oligonucleotide
sequence into mRNA. A promoter is typically located 5' (i.e.,
upstream) of an oligonucleotide sequence whose transcription into
mRNA it controls, and provides a site for specific binding by RNA
polymerase and for initiation of transcription.
[0080] The term "promoter activity" when made in reference to a
nucleic acid sequence refers to the ability of the nucleic acid
sequence to initiate transcription of an oligonucleotide sequence
into mRNA.
[0081] As used herein, the terms "nucleic acid molecule encoding,"
"nucleotide encoding," "DNA sequence encoding," and "DNA encoding"
refer to the order or sequence of deoxyribonucleotides along a
strand of deoxyribonucleic acid. The order of these
deoxyribonucleotides determines the order of amino acids along the
polypeptide (protein) chain. The DNA sequence thus codes for the
amino acid sequence.
[0082] The term "isolated" when used in relation to a nucleic acid
(e.g., "an isolated oligonucleotide") refers to a nucleic acid
sequence that is separated from at least one contaminant nucleic
acid with which it is ordinarily associated in its natural source.
Isolated nucleic acid is nucleic acid present in a form or setting
that is different from that in which it is found in nature. In
contrast non-isolated nucleic acids are nucleic acids such as DNA
and RNA which are found in the state they exist in nature. For
example, a given DNA sequence (e.g., a gene) is found on the host
cell chromosome in proximity to neighboring genes; RNA sequences,
such as a specific mRNA sequence encoding a specific protein, are
found in the cell as a mixture with numerous other mRNAs which
encode a multitude of proteins. However, isolated nucleic acid
encoding a polypeptide of interest includes, by way of example,
such nucleic acid in cells ordinarily expressing the polypeptide of
interest where the nucleic acid is in a chromosomal or
extrachromosomal location different from that of natural cells, or
is otherwise flanked by a different nucleic acid sequence than that
found in nature. The isolated nucleic acid or oligonucleotide may
be present in single-stranded or double-stranded form. Isolated
nucleic acid can be readily identified (if desired) by a variety of
techniques (e.g., hybridization, dot blotting, etc.). When an
isolated nucleic acid or oligonucleotide is to be utilized to
express a protein, the oligonucleotide will contain at a minimum
the sense or coding strand (i.e., the oligonucleotide may be
single-stranded). Alternatively, it may contain both the sense and
anti-sense strands (i.e., the oligonucleotide may be
double-stranded).
[0083] As used herein the term "coding region" when used in
reference to a structural gene refers to the nucleotide sequences
which encode the amino acids found in the nascent polypeptide as a
result of translation of a mRNA molecule. The coding region is
bounded, in eukaryotes, on the 5' side by the nucleotide triplet
"ATG" which encodes the initiator methionine and on the 3' side by
one of the three triplets which specify stop codons (i.e., TAA,
TAG, TGA).
[0084] As used herein, tie terms "structural gene" and "structural
nucleotide sequence" refer to a DNA sequence coding for RNA or a
protein which does not control the expression of other genes. In
contrast, a "regulatory gene" or "regulatory sequence" is a
structural gene which encodes products (e.g., transcription
factors) which control the expression of other genes.
[0085] As used herein, the term "regulatory element" refers to a
genetic element which controls some aspect of the expression of
nucleic acid sequences. For example, a promoter is a regulatory
element which facilitates the initiation of transcription of an
operably linked coding region. Other regulatory elements include
splicing signals, polyadenylation signals, termination signals,
etc.
[0086] As used herein, the term "gene" means the
deoxyribonucleotide sequences comprising the coding region of a
structural gene. A "gene" may also include non-translated sequences
located adjacent to the coding region on both the 5' and 3' ends
such that the gene corresponds to the length of the full-length
mRNA. The sequences which are located 5' of the coding region and
which are present on the m are referred to as 5' non-translated
sequences. The sequences which are located 3' or downstream of the
coding region and which are present on the mRNA are referred to as
3' non-translated sequences. The term "gene" encompasses both cDNA
and genomic forms of a gene. A genomic form or clone of a gene
contains the coding region interrupted with non-coding sequences
termed "introns" or "intervening regions"or "intervening
sequences." Introns are segments of a gene which are transcribed
into heterogenous nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRAN) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0087] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences which are present on the RNA transcript These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers which control
or influence the transcription of the gene. The 3' flanking region
may contain sequences which direct the termination of
transcription, post-transcriptional cleavage and
polyadenylation.
[0088] A "non-human animal" refers to any animal which is not a
human and includes vertebrates such as rodents, non-human primates,
ovines, bovines, ruminants, lagomorphs, porcines, caprines,
equines, canines, felines, aves, etc. Preferred non-human animals
are selected from the order Rodentia.
[0089] A "transformed cell" is a cell or cell line that has
acquired the ability to grow in cell culture for many multiple
generations, the ability to grow in soft agar and the ability to
not have cell growth inhibited by cell-to-cell contact. In this
regard, transformation refers to the introduction of foreign
genetic material into a cell or organism. Transformation may be
accomplished by any method known which permits the successful
introduction of nucleic acids into cells and which results in the
expression of the introduced nucleic acid "Transformation" includes
but is not limited to such methods as transfection, microinjection,
electroporation, and lipofection (liposome-mediated gene transfer).
Transformation may be accomplished through use of any expression
vector. For example, the use of baculovirus to introduce foreign
nucleic acid into insect cells is contemplated. The term
"transformation" also includes methods such as P-element mediated
germline transformation of whole insects. Additionally,
transformation refers to cells that have been transformed
naturally, usually through genetic mutation.
GENERAL DESCRIPTION OF INVENTION
[0090] The present invention provides methods and compositions for
the screening of compounds for agonistic or antagonistic activity
and means of activation of the NAG-1 gene promoter region.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to activate
this gene and activation of this gene by NSAIDs correlates with the
apoptotic elimination of certain cancers. The present invention
provides easy-to-use drug screens that allow for the identification
of compounds contemplated to have anti-cancer properties via the
activation of NAG-1. It is contemplated that the present invention
will facilitate the identification of compounds that are more
effective and efficient in the elimination of cancer than currently
used anti-cancer compounds.
[0091] Currently, NSAIDs are widely used in the treatment of
inflammatory disease (See egg., Vane and Botting, "Mechanism of
action of nonsteroidal antiinflammatory drugs" Am J Med 104:2S-8S,
1998). Although the present invention is not limited to any
particular mechanism, the anti-inflammatory effects of NSAIDs are
believed to result from their ability to inhibit the formation of
prostaglandins by prostaglandin H synthase (COX). As discussed
above, two isoforms of prostaglandin H synthase, COX-1 and COX-2
have been identified. COX-1 is constitutively expressed in many
tissues, while the expression of (OX-2 is regulated by mitogens,
tumor promoters, and growth factors (Herschman, "Prostaglandin
synthase 2" Biochim Biophys Acta 1299:125-140, 1996). High
expression of COX-2 has been reported in human colorectal tumors
and tumors from other tissues, suggesting a role for this enzyme in
regulating tumor growth. NSAIDs are effective in reducing human and
rodent colorectal, and possibly, breast and lung cancer (Castonguay
et al., "Inhibition of lung tumorigenesis by NSAIDS: a working
hypothesis" Exp Lung Res 24:605-615, 1998; Han et al., "Effects of
sulindac and its metabolites on growth and apoptosis in human
mammary epithelial and breast carcinoma cell lines" Breast Cancer
Res Treat 48:195-203, 1998; Taketo, "Cycrooxygenase-2 inhibitors in
tumerogenesis (Part I)" J Natl Cancer Inst 90:1529-1536, 1998;
Taketo, "Cyclooxygenase-2 inhibitors in tumerogenesis (Part 11)" J
Natl Cancer Inst 90:1609-1620, 1998). In addition, retrospective
and prospective studies link NSAID usage to a reduced risk for
colorectal cancer death. NSAIDs are effective in reducing the
number and size of polyps in animal models, and epidemiological
studies indicate that use of NSAIDs provide a 40-50% reduction in
mortality from colorectal cancer (Boolbol et al., "Cyclooxygenase-2
overexpression and tumor formation are blocked by sulindac in a
murine model of familial adenomatous polyposis" Cancer Res
56:2556-2560, 1996; Thun et al., "Aspirin use and risk of fatal
cancer [see comments]" Cancer des 53:1322-1327, 1993).
[0092] As discussed above, our understanding of the mechanisms by
which NSAIDs exert their anti-tumor effect is not clear. One
possible mechanism is altered arachidonic acid (AA) metabolism,
since NSAIDs inhibit the formation of prostaglandins by COX-1 and
COX-2. However, an understanding of the mechanism(s) is not
necessary in order to use the present invention Recent data suggest
that inhibition of COX by NSAIDs may increase the cellular pool of
AA, resulting in the hydrolysis of sphingomyelin to ceramide, which
promotes apoptosis (Chan, "Mechanism underlying nonsteroidal
antiinflammatory drug-mediated apoptosis" Proc Natl Acad Sci
94:11514-11519, 1998). Some data link NSAID chemoprevention in
colorectal cancer cells to prevention of angiogenesis and induction
of apoptosis. COX appears to regulate angiogenesis induced by colon
cancer cells and NSAIDs appear to limit tumor growth (Tsujii et
al., "Cyclooxygenase regulates angiogenesis induced by colon cancer
cells [published erratum appears in Cell 1998 July 24:94(2):
following p271]" Cell 93:705-716, 1998). In epithelial cells of the
intestinal crypt, elevated COX-2 expression appears to attenuate
apoptosis (Tsujii and DuBois, "Alteration in cellular adhesion and
apoptosis in epithelial cells overexpressing prostaglandin
endoperoide synthase 2" Cell 83:493-501, 1995). Some evidence like
NSAID-induced apoptosis to inhibition of COX, but other data
suggest a prostaglandin-independent mechanism for the induction of
apoptosis (Hanif et al. "Effects of nonsteroidal anti-inflammatory
drugs on proliferation and on induction of apoptosis in colon
cancer cells by a prostaglandin-independent pathway" Biochem
Pharmacol 52:237-245, 1996; Murphy et al.,
"Cyclooxygenase-2-selective antagonists do not inhibit growth of
colorectal carcinoma cell lines" Cancer Lett 122:25-30, 1998).
PPAR.gamma. agonists also have anti-tumorigenic activity and
stimulate apoptosis. Ligand activation of PPAR.gamma. inhibits
proliferation of breast cancer cells (Mueller et al., "Terminal
differential of human breast cancer through PPAR gamma" Mol Cell
1:465-470, 1998). The addition of PPAR.gamma. ligands to cancer
cells induce apoptosis (Bishop-Bailey and Hla, "Endothelial cell
apoptosis induced by the peroxisome proliferator-activated receptor
(PPAR) ligand 15-deoxy-delta12, 14-prostaglandin J2" J Biol Chem
274:17042-17048, 1999; Chinetti et al., "Activation of
proliferative-activated receptors alpha and gamma induces apoptosis
of human monocyte-derived macrophages" J Biol Chem 273:25573-25580,
1998; Elstner et al, "Ligands for peroxisome proliferator-activated
receptor gamma and retinoic acid receptor inhibit growth and induce
apoptosis of human breast cancer cells in vitro and in BNX mice"
Natl Proc Acad Sci USA 95:8806-8811, 1998; Keelan; et al,
"15-deoxy-delta(12, 14)-prostaglandin J(2), a ligand for peroxisome
proliferator-activated receptor-gamma, induces apoptosis in JEG3
choriocarcinoma cells" Biochem Biophys Res Commun 262:579-585,
1999; Takahashi et al., "Activated of PPAR gamma inhibits cell
growth and induces apoptosis in human gastric cancer cells" FEBS
Lett 455:135-139, 1999). PPAR.gamma. agonists also inhibit the
growth of transplantable tumors in a nude mouse model (Clay et al.,
"Influence of J series prostaglandins on apoptosis and
tumorigenesis of breast cancer cells" Carcinogenesis 20:1905-1911,
1999).
[0093] Until the development of the present invention, the
stimulation of the expression of proteins that have both
anti-inflammatory and anti-tumorigenic activity by NSAIDS and/or
PPAR.gamma. ligands was not previously determined. As detailed in
the Experimental section below, NSAID-inducible genes were
identified by suppression subtractive hybridization (Diatchenko et
al., "Suppression subtractive hybridization: A method for
generating differentially regulated or tissue-specific cDNA probes
and libraries" Proc Natl Acad Sci USA 93:6025-6030, 1996) utilizing
the human colorectal adenocarcinoma cell line, HCT-116. NSAIDs and
PPAR.gamma. ligands were found to increase the expression of a
novel member of the transforming growth factor-b (TGF-b)
superfamily (NSAID Activated Gene, NAG-1). The expression of this
protein is strongly associated with NSAID-induced (or
PPAR.gamma.-induced) ligand-induced apoptosis in colorectal cells.
Furthermore, transplantable tumors derived from
over-expressing-NAG-1 HCT-116 cells were found to grow at a slower
rate in athymic nude mice than control cells, providing additional
evidence for the anti-tumorigenic activity of NAG-1.
[0094] As indicated above, the present invention provides methods
and compositions for identifying and testing agonists and
antagonists of NAG-1 promoter activation and inhibition.
Additionally, the invention provides methods and compositions
comprising drugs, drug therapies and gene therapies that allow for
the elimination or reduction of cancers.
[0095] A. Measurement Of Apoptosis--General Indicators
[0096] Although an understanding of the mechanism of the invention
is not necessary in order to use the present invention, it is
contemplated that one of the methods by which NAG-1 activation may
eliminate cancer cells is through the process of cell death called
apoptosis. Programmed cell death, or apoptosis, is the genetically
controlled, systematic dismantling of a cell. Apoptosis typically
happens during embryogenesis when much tissue remodeling is taking
place, but continues to happen throughout the life of an organism.
For example, the elimination of senescent cells, the involution of
tissues and the elimination of diseased cells occurs through
apoptosis. The hallmarks of the apoptotic process are morphological
changes consisting of chromatin condensation, membrane blebbing,
loss of membrane integrity and, ultimately, the disintegration of
the cell into apoptotic bodies that are engulfed by phagocytic
cells. On the molecular scale, DNA is cleaved into 180-200 kb
nucleosomal fragments resulting in a laddering appearance when run
on an agarose gel. Apoptosis prevents the release of cellular
constituents into the extracellular space thereby preventing an
inflammatory response and allows for the orderly remodeling of
tissues. In contrast, necrotic or accidental cell death is
exemplified by membrane rupture and the release of cellular
constituents into the extracellular space often resulting in an
inflammatory response by the body.
[0097] Traditionally, the measurement of apoptosis has involved
delineating the percentage of a cell population undergoing
apoptosis. However, determining the earliest detectable point in
which apoptosis could be accurately detected and determining the
kinetics of the apoptotic process have been problematic. The
changes in morphology and the DNA laddering discussed above,
although not overly quantitative, are the classic determinants of
apoptosis. Other measures of apoptosis include, but are not limited
to, terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP
nick end labeling (TUNEL) staining (indicative of early DNA strand
cutting by endonucleases), trypan blue staining (and various other
vital stains indicative of loss of membrane integrity), propidium
iodide (and various other DNA intercalating dyes indicative of loss
of DNA from the nucleus) and Annexin-V staining (indicative of
phosphatidyl serine exposure on the cell surface). These techniques
allow for better quantitative analysis of apoptosis on a population
level but do little to allow for the measurement of the effect of
agonists or antagonists on a specific apoptotic signaling
pathway.
[0098] B. Measurement of Apoptosis--Cell Pathway Specific
Techniques
[0099] As indicated above, an understanding of the mechanism is not
necessary in order to use the present invention. However, some
advances have been made into delineating pathway involvement in the
apoptotic process. In this regard, inhibitors have been made which
target some constituents of the apoptotic pathway. For example,
tetra-peptide inhibitors have been developed for several of the
caspases activated during apoptosis. Likewise, loss of function and
gain of function gene mutants have been made for several steps in
the apoptotic process. Additionally, reagents have been developed
which combine a fluorogenic substrate with caspase cleavage sites
allowing for the visualization of apoptosis-induced caspase
activation by flow cytometric methods. These previously developed
reagents, however, focus on the pro-apoptotic pathways and fail to
look at survival pathways.
[0100] C. Advances Conferred By The Present Invention
[0101] The present invention provides numerous advantages over the
presently available methods for identifying, characterizing, and
using drug and gene therapies for cancer treatment. For example, it
is contemplated that the present invention will find use in such
areas as the design and execution of screens to identity protein or
small molecules that interfere with or augment the activation or
inhibition of the NAG-1 promoter, as well as the development of
therapeutic protocols involving the use of (i) compounds that
regulate NAG-1 promoter activation or inhibition and (ii) the
establishment of drug and gene therapies for the treatment of
various cancers. Other uses of the present invention will be
apparent to those in the art.
[0102] D. Format of the Present Invention [0103] 1. Cell Based
Assays
[0104] One embodiment of the present invention provides
compositions and methods for the transfection of cell lines with
plasmids containing the NAG-1 promoter operationally linked to a
reporter gene or genes. These cells are then used in tests to
measure the effect of test compounds on apoptosis. Empty plasmids
are used as controls in some embodiments. In some embodiments, the
cells are transiently transfected, while in other embodiments, the
cells are stably transfected. However, it is contemplated that
stably transfected cell lines allow for more reliable enterozoa
comparisons. In preferred embodiments, the indication of promoter
activation by the reporter gene identifies compounds suitable for
further screening as potential anti-cancer agents (e.g., in
transgenic animal-based assays) as outlined below.
[0105] 2. Transgenic Animal Based Assays
[0106] The present invention also provides compositions and methods
for the generation of transgenic animals hat express reporter genes
activated by the NAG-1 promoter, thereby providing in vivo assay
systems for screening of potential drug candidates. As these
embodiments of the present invention allow for the testing of
agents in vivo, they facilitate determinations of potential side
effects of tested agents and the ability of the agents to eliminate
or reduce cancers. In preferred embodiments, the expression of the
reporter gene allows for the generation of data regarding the
mechanisms and locations of action of the test compound.
[0107] 3. Molecular Biological Based Assays
[0108] In a harder embodiment, the present invention provides
compositions and methods for the production of NAG-1 promoter cDNA
and RNA. This makes facilitates the performance of a wide range of
standard molecular biological assays including, but not limited to,
Northern and Southern blotting, PCR, cloning and various screening
assays for the detection of intraspecific and interspecific
homologs. Thus, the present invention provides many advantages for
the identification, development, and utilization of compounds as
anti-cancer agents.
DETAILED DESCRIPTION OF THE INVENTION
[0109] Generally, the nomenclature used herein, and the laboratory
procedures in cell culture, molecular genetics, and nucleic acid
chemistry and hybridization described below, are those well known
and commonly employed in the art. Standard techniques are used for
recombinant nucleic acid methods, polynucleotide synthesis, and
microbial culture and transformation (e.g.., electroporation,
lipofection). Generally enzymatic reactions and purification steps
are performed according to the manufacturer's specifications. The
techniques and procedures are generally performed according to
conventional methods in the art and various general references (See
generally, Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), and Current Protocols in Molecular Biology,
John Wiley and Sons, Inc., N.Y. (1996), both of which are
incorporated herein by reference).
[0110] Oligonucleotides can be synthesized by any means known in
the art including, but not limited to, use of an oligonucleotide
synthesizer (e.g., Applied BioSystems), according to specifications
provided by the manufacturer. Complementary oligonucleotides are
annealed by heating them to 90.degree. C. in a solution of 10 mM
Tris-HCl buffer (pH 8.0) containing NaCl (200 mM) and then allowing
them to cool slowly to room temperature.
[0111] Any suitable assays for detecting the ability of agents to
inhibit or enhance NAG-1 promoter region activity that provide for
facile high-throughput screening of agent banks (e.g., compound
libraries, peptide libraries, and the like) will find use with the
present invention to identify suitable antagonists or agonists. It
is contemplated that such NAG-1 promoter region activity
antagonists and agonists will be further developed as therapeutics
and diagnostic or prognostic tools for diverse types of cancers,
autoimmune diseases and hereditary diseases.
[0112] A. Screens to Identify Agonists of Antagonists of the NAG-1
Promoter
[0113] There are several different approaches provided by the
present invention to confirm the ability of small molecules to
specifically inhibit or enhance activation of the NAG-1 promoter.
One approach is to transfect expression constructs (i.e.,
constructs containing the NAG-1 promoter operationally liked to a
reporter gene) into cells and measure changes in the expression of
the reporter gene as compared to controls transfected with empty
constructs after the cells have been exposed to at least one
compound. In these screens, the cells are either transiently
transfected or stably transfected. Furthermore, transgenic animals
allow for in vivo assays to be conducted.
[0114] 1. In vitro Assays
[0115] Transfection assays allow for a great deal of flexibility in
assay development The wide range of commercially available
transfection vectors and reporter constructs permit the expression
of NAG-1 components in an extensive number of cell types. in one
particularly preferred embodiment, cells are transiently
transfected with an expression construct that includes the NAG-1
promoter and a luciferase gene allowing for the detection of NAG-1
promoter activation. In these embodiments, cells are exposed to the
agent suspected of modulating NAG-1 promoter activation, and
luciferase expression is turned on. Rates of luciferase expression
in cells expressing the construct are compared to cells transfected
with an empty expression vector. Rates of luciferase activation are
then quantitated by any of a number of ways reported in the
literature and known to those in the art.
[0116] In another preferred embodiment, stably transfected cells
lines are developed. The use of various reporter genes allows for
numerous types of assays depending on the organism used. Screening
assays for compounds suspected of modulating NAG-1 promoter
activity are conducted in the same manner as in the transient
transfection assays. Using stably transfected cell lines allows for
greater consistency between experiments and allow for
inter-experimental comparisons.
[0117] 2. In Vivo Assays
[0118] In another embodiment, transgenic animals are constructed
using standard protocols. The generation of transgenic animals
allows for the investigation of compounds that function by
activating the NAG-1 promoter in vivo.
[0119] B. Screens to Identify NAG-1 Promoter Homologs
[0120] In yet other embodiments, standard molecular biological
techniques are used to identify NAG-1 promoter homologs in humans
and other species. For example, preferred embodiments include, but
are not limited to, DNA-DNA hybridization techniques (e.g..
Southern blots) and DNA-RNA hybridization techniques (e.g. Northern
blots), as well as other suitable methods.
EXPERIMENTAL
[0121] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
constructed as limiting the scope thereof.
[0122] In the experimental disclosure which follows, the following
abbreviations apply: .degree. C. (degrees Centigrade); H.sub.2O
(water); aa (amino acid); bp (base pair); kb (kilobase pair); k-D
(kilodaltons); gm (grams); .mu.g (micrograms); mg (milligrams); ng
(nanograms); .mu.l (microliters); ml (milliliters); mm
(millimeters); nm (nanometers); .mu.m (micrometer); M (molar); mM
(millimolar); .mu.M (micromolar); nM (nanomolar); sec (seconds);
min(s) (minute/minutes); hr(s) and h (hour/hours); ab and Ab
(antibody); FITC (fluorescein isothiocyanate); PAGE (polyacrylamide
gel electrophoresis); PBS (phosphate buffered saline); SDS (sodium
dodecyl sulfate); Tris (tris(hydroxymethyl)aiinomethae); EDTA
(Ethylenediaminetetraacetic Acid); w/v (weight to volume); v/v
(volume to volume); RT (room temperature); S.D. (standard
deviation); S.E. (standard error); FACS (fluorescence activated
cell sorter); Millipore Millipore, Bedford, Mass.); Sigma (Sigma
Chemical Co., St. Louis, Mo.); Biofluids (Biofluids, Rockville,
Md.); Pharmacia (Pharmacia Biotech, Uppsala, Sweden); Dupont/NEN
(NEN Life Science Products, Boston, Mass.); Promega (Promega,
Madison, Wis.); Becton Dickinson (Becton Dickinson, San Jose,
Calif.); Cayman Chemical (Ann Arbor, Mich.); Clontech (Clontech,
Palo Alto, Calif.); and Life Technologies (Life Technologies,
Bethesda, Md.); ATCC (Manasssas, Va.); Cayman Chemical (Ann Arbor,
Mich); New England BioLabs (Beverly, Mass.); Ambion (Austin, Tex.);
Scion Corp. (Frederick, Md.); Schleicher & Schuell (Keene,
N.H.; Amersham Pharmacia Biotech (Piscataway, N.J.); Pfizer (N.Y.,
N.Y.); Parke-Davis (N.Y., N.Y.); Research Genetics (Huntsville,
Ala.); and Trevigen (Gaithersberg, Md.).
EXAMPLE 1
Identification of NSAID-Regulated Gene
[0123] Although an understanding of the mechanism is not necessary
to use the present invention, this Example describes experiments to
determine the possible molecular mechanisms for the
anti-tumorigenic activity of NSAIDs. These experiments involved the
determination of whether NSAIDs could stimulate the expression of
proteins which could participate in both the anti-inflammatory and
anti-tumorigenic activity of NSAIDs. Suppression subtractive
hybridization methods were used to identify NSAID-inducible
proteins. Cell lines used in these experiments were purchased from
ATCC (Manassas, Va.).
[0124] Human colorectal carcinoma cells, HCT-116 cells, were
maintained in McCoy's 5A medium supplemented with 10% fetal bovine
serum (FBS) and Gentamicin. A549 human lung epithelial carcinoma
cells, and U937 human monocytic cells were grown in RPMI 1640
medium supplemented with 10% FBS and Gentamicin. Breast cancer
cells, MCF-7 cells, were grown in EMEM medium with 10% PBS, and
PC-3 cells were grown in DMEM/F12 medium supplemented with 10% FBS
and Gentamicin. All NSAIDs in this study were purchased from Sigma.
Troglitazone (TGZ) was obtained from Parke-Davis, and was
reconstituted in DMSO. 15d-PGJ.sub.2 was purchased from Cayman
Chemical and dissolved in ethanol. All NSAIDs were dissolved in
DMSO (Sigma), except Sodium Salicylate was dissolved in PBS.
[0125] For this experiment, the human colorectal cell line, HCT-116
was selected, since anti-tumorigenic activity of NSAIDs is linked
strongly with human colorectal cancer. As confirmed by Western and
HPLC analysis, these cells do not express COX-1 and COX-2 (data not
shown). A clone, designated INDO29, was isolated from the
indomethacin (INDO)-induced library as follows. mRNAs were isolated
from INDO-treated (100 .mu.M) or vehicle-treated (0.2% DMSO)
HCT-116 cells using a poly(A) spin mRNA isolation kit (New England
BioLabs). The cDNA Subtraction kit was used to make the INDO(+) and
INDO(-) subtractive libraries according to the manufacturer's
protocol (Clontech). A gene containing 159 bp was isolated from the
INDO-induced library and designated INDO29. The full-length cDNA
containing the entire coding region was isolated by RT-PCR using
two primers from PTGFB sequence (GenBank # AF008393); sense
strand,
5'-ACCTGCACAGCCATGCCCGGGCA-3' (SEQ ID NO:4) and anti-sense strand,
5'-CAGTGGAAGGACCAGGACTGCTC-3' (SEQ ID NO:5), and designated as
NAG-1 (NSAID Associated Gene). Characterization of this 159 bp
fragment by sequence analysis indicated that INDO29 is identical to
the 3' region of a novel TGF-.beta. superfamily gene reported
recently by five different groups (FIG. 1A). Each clone is
represented as follows: Placenta Bone Morphogenic protein, PLAB
(GenBank # U88323); Placenta TGF-.beta., PTGFB (GenBank #
AF008303); Prostate Derived Factor, PDF (GenBank # AF003934);
Macrophage Inhibitory Cytokine-1, MIC-1 (GenBank # AF019770); novel
TGF-.beta. super family, HP00269 (GenBank # AB000584). The black
bar labeled INDO29 indicates 159 bp fragment first identified by
subtractive hybridization from HCT-116 cells, while "NAG-1"
indicates the PCR generated full length cDNA. Although these genes
are named differently, sequence analyses revealed that the five
genes are almost identical, and belong to the new TGF-.beta.
superfamily with unknown function. Specific PAR primers were used
to generate a full length clone from HCT-116 cells with sequence
identity to the five genes shown in FIG. 1A. As indicated above,
this NSAIDs activated-gene was designated "NAG-1".
[0126] The sequence of the NAG-1 gene encompasses a novel
5'-promoter region not disclosed with any of the other homologous
sequences isolated in these experiments. Briefly, recombinant
bacteriophage clones were isolated by the plaque hybridization
method, from human genomic chromosome-19 specific library (ATCC).
The library was screened using a DNA probe containing 966 bp of the
NAG-1 promoter, which was labeled by random priming (Ambion) in the
presence of [.alpha.-.sup.32P]dCTP (Du Pont-NEN, 3,000 Ci/mmol).
After three rounds of screening, large scale phage DNA was prepared
according to the procedures known in the art (See, Helms et al., "A
lambda DNA protocol based on purification of phage on
DEAE-cellulose" Methods Enzymol 153:69-82, 1987) using
DEAE-cellulose chromatography. Several .lamda. clones were isolated
by genomic library screening. One of the positive clones,
.lamda.NAG61, is in the schematic diagram, FIG. 5A. Exons are
indicated by .box-solid. and the number under the box is the exon
number. The bar indicates 3.5 kb SmaI fragment cloned into
luciferase vector (pNAG3421). The restriction sites are indicated
as follows: E, EcoRI; S, SmaI; X, XbaI. One of positive clones
(.lamda.NAG61) was purified and found to contain 9 kb NAG-1
promoter (SEQ ID NO:1) and the fall-length NAG-1 gene (SEQ ID
NO:2). The insert of .lamda.NAG61 clone was digested with EcoR1 and
subcloned into a plasmid vector pBlueScript II) for sequencing
analysis. In addition, the 3.5 kb SmaI fragment containing NAG-1
promoter was cloned into pGLBasic3 (Promega) luciferase reporter
vector and assayed for luciferase activity. The 3.5 kb sequence
(SEQ ID NO:3) were deposited to GenBank (AF231408).
EXAMPLE 2
NAG-1 Expression and Apoptosis Induced by Indomethacin is Dose- and
Time-Dependent
[0127] To confirm the increased expression of NAG-1 by INDO,
Northern and Western blot analyses were performed using HCT-116
cells treated with 100 .mu.M INDO for varying times, as follows.
Upon reaching 60-80% confluence in 10 cm plates, the cells were
treated at indicated concentrations and times with either different
NSAIDs or PPAR.gamma. ligands in the absence of serum. Total RNAs
were isolated using TRIzol reagent (Life Technologies) according to
the manufacturer's protocol. For Northern blot analysis, 10 mg of
total RNA was denatured at 55.degree. C. for 15 min and separated
in a 1.2% agarose gel containing 2.2 M formaldehyde, and
transferred to Hybond-N membrane (Amersham). After fixing the
membrane by UV, blots were prehybridized in hybridization solution
(Rapid-hyb buffer, Amersham) for 1 hr at 65.degree. C. followed by
hybridization with cDNA labeled with [.alpha.-.sup.32P]dCTP by
random primer extension (DECAprimeII kit, Ambion). The probes used
were either full-length NAG-1 fragment or PTGFB clone (generously
provided by Dr. Bento-Soar of the University of Iowa). After 4 hr
incubation at 65.degree. C., the blots were washed once with
2.times.SSC/0.1% SDS at RT and twice with 0.1.times.SSC(0.1% SDS at
65.degree. C. Messenger RNA abundance was estimated by intensities
of the hybridization bands of autoradiographs using Scion Image
(Scion). Equivalent loading of RNA samples was confirmed by
hybridizing the same blot with a .sup.32P-labeled b-actin probe
which recognizes RNA of approximately 2 kb.
[0128] The level of secreted NAG-1 was evaluated using Western blot
analysis with anti-human-NAG-1 antibody (generously provided by Dr.
Paralkar of Pfizer). HCT-116 cells were grown to 60-80% confluency
in 10 cm plates followed by 48 hr treatment in the absence of serum
with either NSAIDs or TGZ. The media were harvested and
concentrated approximately 15-fold using Centriprep 10
concentrators (Amicon). Proteins (30 .mu.g) were separated by 15%
SDS-PAGE and transferred for 1 hr onto nitrocellulose membrane
(Schleicher & Schuell). The blots were blocked for 1 hr with 5%
skim milk in TBS/Tween 0.05%, and probed with anti-NAG-1 antibody
(1 .mu.g/ml in TBS-0.2%-Tween20) at 4.degree. C. overnight. After
washing, the blots were treated with horseradish
peroxidase-conjugated secondary antibody for 1 hr and washed
several times. The signal was detected by enhanced
chemiluminescence system (Amersham) and autoradiography.
[0129] The NAG-1 MRNA expression increased with duration of
treatment, with a significant increase in expression observed at 24
hr and a further increase in expression observed at 48 hr of
treatment (See, FIG. 1B). The expression of NAG-1 protein also
increased with duration of INDO treatment, with a marked increase
in expression observed at 36 and 48 hr (See, FIG. 1B), indicating
time-dependent induction by INDO. HCT-116 cells were treated with
100 .mu.M INDO in the absence of serum for various times. In FIG.
1B, left panel, 10 .mu.g of total RNA was loaded in each lane.
Northern blotting was performed using the NAG-1 probe and re-probed
with .beta.-actin probe. The hybridization signals were quantitated
using Scion Image software (Scion). Levels of the 1.3 kb NAG-1
transcript were normalized to the levels of .beta.-actin
transcripts and represented relative to 0 hr treatment. For the
Western analysis, (FIG. 1B, right panel) HCT-116 cells were treated
with 100 .mu.M INDO for various times in the absence of serum. The
media were harvested and concentrated using the Centriprep column
(.about.700 .mu.l from 10 ml media). Then, 30 .mu.g of proteins
were subjected to 15% SDS-PAGE, transferred to nitrocellulose
membrane, and probed with anti-NAG-1 antibody (generously provided
by Dr. Paralkar of Pfizer). The signal was detected by ECL reagent
(Amersham).
[0130] To determine whether NAG-1 gene expression is associated
with NSAID-induced apoptosis or altered cell cycle kinetics, flow
cytometric analysis of the distribution of cells at various stages
of the cell cycle was performed. Apoptosis and cell cycle kinetics
of IADO-treated HCT-116 cells were measured at different time
points. HCT-116 cells were treated with INDO, stained with PI, and
analyzed by flow cytometry. Seven thousand five hundred cells were
examined by flow cytometry by gating on an area versus width dot
plot to exclude cell debris and cell aggregates. Apoptosis is
represented by the fold increase in sub-G1 population over 0 hr
treatment. All values represent mean.+-.S.D. A prolonged GI phase
and shortened S phase were observed, which was dependent on the
duration of INDO treatment (See, FIG. 1C). The changes in the cell
cycle are consistent with the previous report that NSAIDs affect
cell cycle progression in colon cancer cells. However, since G1
arrest begins to occur at an earlier time point than NAG-1 protein
expression, the cell cycle arrest and NAG-1 induction appear to be
independent events. In contrast, increases in NAG-1 protein
expression and induction of apoptosis exhibited similar time
courses, with a nearly 3-fold increase in apoptosis observed at 36
hr, and a 4.5 fold increase at 60 hr of exposure to INDO (See, FIG.
1C). Furthermore, both induction of apoptosis and the increase in
NAG-1 expression were dependent on the concentration of INDO.
HCT-116 cells were treated with several concentrations of INDO and
the cells harvested at 24 hr for mRNA and 48 hr for protein
expression. Apoptosis was measured after 72 hr of INDO exposure, as
described below.
[0131] The DNA content for NSAIDs, PPAR.gamma. ligand, and vehicle
treated HCT-116 cells was determined by FACS. Cells were plated at
a density of 4.times.10.sup.5 cells/well in 2 ml of medium in 6
well plates, incubated for 16 h, and then treated with NSAIDs or
TGZ in the presence of serum. After treatment, the cells were
harvested, washed with PBS, fixed by the slow addition of cold 70%
ethanol to a total of 1 ml, and stored overnight at 4.degree. C.
The fixed cells were pelleted, washed once with PBS, and stained in
1 ml of 20 .mu.g/ml of propidium iodide (PI), 1 mg/ml RNase in PBS
for 20 min. Seven thousand five hundred cells were examined by flow
cytometry using Becton Dickinson FACSort equipped with CellQuest
software, by gating on an area versus width dot plot to exclude
cell debris and cell aggregates. Each cell cycle distribution was
analyzed by the McCycle program using one cell cycle parameter.
Apoptosis was measured based on the level of sub-diploid DNA
contained in cells following treatment with NSAIDs or TGZ using
CellQuest software.
[0132] As a second method to detect apoptosis, TACS Annexin-V-FITC
kit (Trevigen) was used according to the manufacturer's protocol.
Annexin-V has specificity for phosphatidylserine, which becomes
asymmetrically distributed on the cell surface during early
apoptosis. Annexin-V positive/PI positive and Annexin-V positive/PI
negative cell populations were determined as apoptotic populations
from the total gated cells. As shown in FIG. 1D and FIG. 1E,
stimulation of apoptosis and the increase in NAG-1 mRNA and protein
expression were also dependent on the concentration of INDO.
HCT-116 cells were grown in varying concentrations of INDO for 24
hr or 48 hr. Results of Northern (left panel) and Western analysis
(right panel) for these experiments are shown in FIG. 1D. These
analyses were performed as described above. In this figure, "V"
indicates 0.2% DMSO. FIG. 1E shows the results of studies to
determine apoptosis and cell cycle kinetics of INDO-treated HCT-116
cells at different concentrations. Apoptosis analysis was performed
by FACS using HCT-116 cells treated with different concentrations
of INDO for 48 hr as described above. These results are supportive
of an association between NSAID-induced apoptosis and increased
expression of NAG-1 in HCT-116 cells.
EXAMPLE 3
Stimulation of NAG-1 Expression and Apoptosis by Other NSAIDs
[0133] To determine whether other NSAIDs increased apoptosis and
NAG-1 expression, tests were conducted to determine whether
variants of NSAIDs induced NAG-1 gene expression and NSAID-induced
apoptosis. Aspirin, ibuprofen, indomethacin, and sulindac sulfide
were selected since they are known COX inhibitors. Sulindac,
acetaminophen and sodium salicylate were also tested since these
drugs are weak COX inhibitors but are reported to induce apoptosis.
HCT-116 cells were treated with different NSAIDs (vehicle, 0.2%
DMSO; aspirin, 10 mM; ibuprofen, 1 mM; sodium salicylate, 5 mM;
sulindac sulfide, 50 .mu.M; sulindac, 40 .mu.M; acetaminophen, 100
.mu.M) for 24 hr in the absence of serum. Northern analysis was
performed using NAG-1 and .beta.-actin as probes. Total RNAs were
isolated from NSAID-treated HCT-116 cells after 24 hr treatment and
Northern blot was performed using NAG-1 EDNA as a probe (data not
shown). Each NSAID was tested at 3 different concentrations and
maximally effective doses were chosen. Each of the doses were as
follows: aspirin (1, 5, 10 mM); ibuprofen (100, 500, 1000 .mu.M);
salicylate (1, 2.5, 5 mM); sulidac sulfide (1, 10, 50 .mu.M);
sulindac (10, 20, 40 .mu.M); acetaminophen (10, 50, 100 .mu.M).
After 48 hr treatnent with NSAIDs in the presence of serum, cells
were harvested and assayed as described in FIG. 1C. Apoptosis
detected by PI staining was measured by FACS, as known in the art
(FIG. 2A; HCT-116 cells were treated with different NSAIDs
(vehicle, 0.2% DMSO; Aspirin, 10 mM; Ibuprofen, 1 mM; Sodium
Salicylate, 5 mM; Sulindac Sulfide, 50 .mu.M; Sulindac, 40 .mu.M;
Acetaminophen, 100 .mu.M) for 24 hr in the absence of serum.
Northern analysis was performed using NAG-1 and .beta.-actin as
probes. The open bars represent the relative expression of NAG-1
mRNA over the control (vehicle). The hatched bars represent the
relative apoptosis ratio over vehicle treatment by PI staining
methods. N=3.+-.S.D.).
[0134] FAGS Ann -V-FITC was also used to detect apoptosis according
to the manufacturer's protocol. Annexin-V positive cell populations
were determined as early/late apoptotic populations from the total
gated cells. The hatch bars in FIG. 2A represent the relative
apoptosis ratio over vehicle treatment by Annexin-V assay. Aspirin,
ibuprofen, salicylate and sulindac sulfide treatment increased
NAG-1 gene expression by 3-4 fold at the highest concentrations
tested, while acetaminophen and sulindac did not induce NAG-1
expression at any concentration (data not shown). Treatment with
different NSAIDs increased NAG-1 expression in a
concentration-dependent manner in HCT-116 cells. Therefore, the
concentrations giving the highest fold increase in NAG-1 expression
were chosen to stimulate apoptosis. As shown in FIG. 2B, each NSAID
tested that induced NAG-1 also induced apoptosis, suggesting a
correlation between apoptosis and NAG-1 expression. The
NSAID-induced apoptosis was confirmed by annexin V assay (FIG. 2A,
hatched bar), which can detect early apoptotic and late
apoptotic/necrotic cell populations. Except for sulindac sulfide, a
strong correlation was found between PI staining and Annexin-V
assay in the detection of apoptosis. The association between
NSAID-induced apoptosis and the increase in NAG-1 expression is
more clearly illustrated in FIG. 2B. A strong correlation
(r.sup.2=0.859) was found to exist between NSAIDs which induce
NAG-1 expression and NSAIDs which induce apoptosis in HCT-116
cells. Similar data are presented in Table 1, below.
[0135] In the Table below, HCT-116 cells were treated with
different NSAIDs (vehicle, 0.2% DMSO) for 24 hr in the absence of
serum. Northern analysis was performed using NAG-1 and .beta.-actin
as probes. The data represent relative expression of NAG-1 mRNA
over the control (1.0). Each NSAID was tested at 3 different
concentrations and maximally effective concentration used for
apoptosis determination. Apoptosis ratio by PI staining was
measured by FACS. After 48 hr treatment with NSAIDs in the presence
of serum, cells were, harvested and assayed. The data represent
relative apoptosis ratio over vehicle treatment by PI staining
methods. TACS Annexin V-FITC kit was also used to confirm
apoptosis.
TABLE-US-00001 TABLE 1 NAG-1 Induction and NSAID-Induced Apoptosis
NAG-1 Concen- Induction tration (mRNA) Apoptosis (.mu.M) Used Fold
(PI Staining) Conventional NSAIDs Indomethacin 10-100 2.2 2.9 .+-.
0.121 Sulindac 10-40 1.5 1.1 .+-. 0.173 Sulindac Sulfone 100-400
1.3 1.6 .+-. 0.043 Sulindac Sulfide 1-50 4.6 4.3 .+-. 0.737
Piroxicam 200-1000 2.5 1.9 .+-. 0.145 Diclofenac 50-200 3.7 4.0
.+-. 0.315 Aspirin 1,000-10,000 3.5 3.5 .+-. 0.451 Ibuprofen
100-1,000 4.5 4.3 .+-. 0.666 Sodium Salicylate 1,000-5,000 3.0 1.8
.+-. 0.252 Acetaminophen 10-100 0.9 0.9 .+-. 0.153 COX-2 Specific
Inhibitors NS-398 10-100 0.9 1.029 .+-. 0.109 DFU 10-100 1.0 0.54
.+-. 0.091 Celecoxib* 0.01-0.1 1.0 0.722 .+-. 0.098 LM-4101 10-100
3.0 4.057 .+-. 0.119 LM-4108 10-100 1.0 1.7 .+-. 0.229 LM-4115
10-100 1.0 ND.sup.# *This compound was toxic to the HCT-116 cells
at the .mu.M concentration. .sup.#ND: Not Determined
EXAMPLE 4
NAG-1 Induction by NSAIDs in Lung, Breast, Leukemia, and Prostate
Cells
[0136] NSAID-induced apoptosis is not restricted to colorectal
cells, as NSAIDs can induce apoptosis in breast, lung, leukemia,
and prostate cell lines. To test for the induction of NAG-1 by
NSAIDs in different cell lines, A549 lung epithelial cells, MCF-7
mammary cells, PC-3 prostate cancer cells, and U937 leukemia cell
lines were treated with several concentrations of INDO and aspirin.
Then, as described above, Northern blot analysis was performed. As
shown in FIG. 3, NAG-1 was induced in different cell lines, A549
lung carcinoma cells (A), MCF-7 breast carcinoma cells (B), U937
leukemia cells (C), and PC-3 prostate cells (D). Quiescent cells
were grown for 24 hr in the presence of indicated NSAIDs. Total
RNAs were isolated and subjected to Northern analysis using NAG-1
and .beta.-actin as probes. The blots contained 10 .mu.g of total
RNA from INDO- or Aspirin (Asp)-treated cells with indicated doses.
"V" indicates total RNAs from vehicle (0.2% DMSO) treated cells.
The results shown are representative of those obtained in two
experiments.
[0137] As shown in FIG. 3, NAG-1 gene expression was induced by
INDO or aspirin in all of the cell lines tested. Interestingly, in
U937 cells, which did not express basal NAG-1, INDO treatment
resulted in only a modest increase in NAG-1 expression, while
aspirin (5 mM) significantly increased NAG-1 expression. All of the
cell lines tested in these experiments showed high NAG-1 induction
by 10 mM Aspirin. Thus, the ability of NSAIDs to increase the
expression of NAG-1 is not specific for colorectal cells but is
also observed in other cells.
EXAMPLE 5
NAG-1 Induction and Apoptosis by PPAR.gamma. Ligands
[0138] The absence of COX expression in HCT-116 cells suggested
that the induction of NAG-1 by NSAIDs is not liked to inhibition of
prostaglandin formation Although an understanding of the mechanism
is not necessary in order to use the present invention, one
possible mechanism of NAG-1 induction is through the activation of
the peroxisome proliferator-activated receptors, PPAR.gamma. and
PPAR.gamma.. INDO, ibuprofen and other NSAIDs bind to and activate
PPAR.gamma. and PPAR.gamma. (Lehmann, et al., 1997; Ricore, et al.,
1998) at similar concentrations used in this investigation.
Furthermore, PPAR.gamma. is expressed in human colon tumors and
several colon cancer cell lines, including HCT-116 cells (DuBois,
et al., 1998), and the activation of PPAR.gamma. results in growth
arrest in colon cancer cells (Brockman, et al., 1998). The
observation that NSAIDs activate PPAR.gamma., and PPAR.gamma.
ligands induce apoptosis and growth arrest in vitro, led to
experiments to determine whether conventional PPAR.gamma. and
PPAR.gamma. ligands could increase NAG-1 expression in HCT-116
cells. While it was determined that PPAR.gamma. ligands were not
effective (data not shown), as shown in FIG. 4A,
15-deoxy-.DELTA..sup.12,14-prostaglandin J.sub.2 (15d-PGJ.sub.2)
and Troglitazone (TGZ), PPAR.gamma. ligands, induced NAG-1 gene
expression in a concentration-dependent manner (3-fold at 1 .mu.M
and 5 .mu.M, respectively). Interestingly, TGZ-induced NAG-1
expression was observed at earlier times than were observed after
INDO treatment. An increase in NAG-1 mRNA was also observed as
early as 3 hr after treatment, and an increase in NAG-1 protein was
also observed at 6 hr after treatment with 5 .mu.m TGZ (See, FIG.
4B). Cells were grown in the absence of serum for 16 hr and treated
with 5 .mu.M TGZ for varying times. Total RNAs were subjected to
Northern analysis and concentrated medium was used for Western
analysis as described in Example 1. In FIG. 4C, (black bars)
apoptosis is represented by the fold induction of sub G1 population
over 0 hr treatment. All values represent mean.+-.S.D. To determine
whether TGZ also induces apoptosis in HCT-116 cells, FACS analysis
was performed (See, FIG. 4C). Cell cycle and apoptosis analyses
were performed as described below. HCT-116 cells were plated in
6-well plates at a density of 4.times.10.sup.5 cells/well in 2 ml
of medium, incubated for 16 hr, and treated with TGZ (5 .mu.M) in
the presence of serum over different time points. G1 arrest was
seen at the same time point as INDO treatment (12 hr). However,
apoptosis was observed as early as 12 hr after treatment.
Interestingly, the apoptosis induced by TGZ was observed at earlier
times compared to apoptosis induced by INDO treatment (12 hr and 36
hr, respectively). The stimulation of apoptosis by TGZ is
consistent with previous publications reporting that PPAR.gamma.
ligands cause the induction of apoptosis in a number of colon cell
line including HCT-116, in breast cancer cells (MCF7), and in
monocyte-derived macrophage cells (U937). Taken together, these
results support an association between NAG-1 expression and
apoptosis.
EXAMPLE 6
Transcriptional Regulation of NAG-1 by PPAR.gamma. Ligands and
NSAIDs
[0139] To investigate transcriptional activation of NAG-1, the
NAG-1 promoter was cloned and examined. A 966 bp P fragment
corresponding to the 5' end of the human PTGFB was used as a probe
to isolate .gamma.NAG61, a 17 kb human NAG-1 genomic clone.
Southern blot analysis of .gamma.NAG61, using this 966 bp fragment
as a probe, indicated that the .gamma.NAG61 clone contains
full-length NAG-1 containing two exons and a 9 kb 5' region of
NAG-1 promoter (See, FIG. 5A). In this Figure, exons are indicated
by .box-solid. and the number under the box is the exon number. The
bar indicates a 3.5 kb SmaI fragment cloned into luciferase vector
(pNAG3421). The restriction sites are indicated as follows: E,
EcoRI; S, SmaI; X, XbaI.
[0140] In other experiments, a BAC clone (BAC182K4) containing
NAG-1 gene was purchased from Research Genetics and characterized
by Southern analysis. The 3.5 kb SmaI fragment was characterized
and found to contain the NAG-1 promoter in both the .lamda.NAG61
and the BAC clone. To characterize the human NAG-1 5' flanking
region and identify the PPAR.gamma. ligand and/or NSAID response
element in the NAG-1 promoter, a 3.5 kb fragment containing NAG-1
promoter was cloned into luciferase reporter vector, and was
transiently transfected into HCT-116 cells.
[0141] FIG. 5B shows deletion analysis of the NAG-1 promoter. The
pNAG3421 clone was deleted by ExoIII nuclease to generate several
deletion clones. The clones were transiently transfected into
HCT-116 cells, treated either with TGZ (10 .mu.M), or INDO (100
.mu.M), and the luciferase activity was measured after 24 hr
treatment. The number next to the clone names in FIG. 5B indicates
the length (bp) of the promoter. The data presented in this Figure
represent the mean.+-.S.D. of the relative luciferase activity of
the test compared with that obtained with the vehicle
treated-pNAG474 clone, which was taken as 1.0. The pGLB3
promoterless vector was used as a negative control, and the pRL-TK
vector was used as internal control.
[0142] HCT-116 cells were plated in 6-well plates at
2.times.10.sup.5 cells/well in McCoy's 5A media supplemented with
10% fetal bovine serum. After growth for 16 hr, plasmid mixtures
containing 1 .mu.g of NAG-1 promoter linked to luciferase and 0.1
.mu.g of pRL-TK (Promega) were transfected by lipofectamine (Life
Technologies) according to the manufacturer's protocol. After 24
hr, the media were changed to serum-free media and NSAIDs or TGZ
were added. Cells were harvested in 1.times. luciferase lysis
buffer after 24 hr of growth, and luciferase activity was
determined and normalized to the pRL-TK luciferase activity using
the Dual Luciferase Assay Kit (Promega).
[0143] The 3.5 kb NAG-1 promoter sequences were deposited in
GenBank (accession number AF241308) and some restriction enzyme
maps are shown in FIG. 5A. Several deletion clones were also
generated by ExoIII nuclease from the 3.5 kb SmaI fragments. The
deletion clones, pNAG2092, pNAG2021, pNAG1086, pNAG474, and pGL3
negative control vector, were transiently transfected into HCT-116
cells. As shown in FIG. 5B, the TGZ and/or INDO response elements
are located between -1086 bp to -2021 bp of NAG-1 promoter. These
data also suggest that INDO and TGZ might act by different
mechanisms, since the TGZ showed greater induction of luciferase
activity than INDO. Since several NSAIDs induce NAG-1 transcripts,
the pNAG2021 clone containing the TGZ and/or INDO response element
was used to determine if this region also responses to treatment
with different NSAIDs. The treatment with TGZ or with various
NSAIDs was found to induce reporter activity by 3-fold or 2- to
4-fold, respectively. Figure SC shows luciferase activity of 2021
bp NAG-1 promoter in the presence of TGZ or several NSAIDs. In
these experiments, HCT-116 cells were transiently transfected with
pNAG2021, treated with vehicle (0.2% DMSO), TGZ (10 .mu.M), INDO
(100 .mu.M), aspirin (10 mM), ibuprofen (ibu, 1 mM), salicylate
(SSA, 5 mM, sulindac sulfide (50 .mu.M), sulindac (40 .mu.M), or
acetaminophen (acet, 100 .mu.M) for 24 hr. The internal control
vector (pRL-TK) was used to normalize the results to determine the
transfection efficiency. The data presented here represent the
mean.+-.S.D. from 3 different experiments. Sulindac and
acetaminophen did not increase luciferase activity (See, FIG. 5C).
These data are consistent with the Northern data using NAG-1 as a
probe (See, FIG. 2A). Interestingly, there is no obvious
PPAR.gamma. response element (PPRE) with a direct repeat (DR-1) in
this position, but there are 15 putative PPREs with a 4 bp mismatch
in -1086 to -2021 position, suggesting that NAG-1 induction by
NSAIDs and/or TGZ may be complex. However, the NAG-1 promoter
between -1086 bp to -2021 bp does respond to the PPAR.gamma.
ligands and/or NSAIDs.
EXAMPLE 7
Ectopic Expression of NAGS1 Induces Apoptosis
[0144] HCT-116 cells were stably transfected with an expression
vector containing the full-length NAG-1 coding region in the sense
and antisense orientations. The sense cells expressed NAG-1 protein
at a level that was 2.0-2.5-fold greater than the vector
transfected cells, while the anti-sense cells expressed slightly
lower NAG-1 levels compared to vector transfected cells. FIG. 6A
shows a Western analysis of ectopic expression of NAG-1 in HCT-116
cells. The fill-length NAG-1 (sense strand and antisense stand) was
cloned into the pCDNA3.1 expression vector and transfected into
HCT-116 cells. Each cell was grown under G418 (500 .mu.g/ml) for 2
weeks and treated with either vehicle or INDO (100 .mu.M). After
growing for 48 hr, the media was harvested, concentrated, and
subjected to Western analysis. Thirty .mu.g of total protein was
loaded in each lane, and the arrow indicates the NAG-1 protein
band.
[0145] Despite repeated attempts, construction of NAG-1 highly
expressing cells which may be linked to high rates of apoptosis
were not generated (data not shown). The sense-HCT-116 cells
exhibited a slower growth rate compared to vector transfected cells
or antisense-HCT-116 cells (data not shown). This could be due to
effects on cell proliferation and/or an increase in cell death. A
significant percentage of the sense-HCT-116 cells were found to
undergo spontaneous apoptosis, as compared to the vector
transfected HCT-116 cells. In contrast the antisense-HCT-116 cells
demonstrated lower spontaneous apoptosis at 48 hr (See, FIG. 6B).
The stably transfected cells were then incubated with 100 .mu.M
indomethacin for 48 hr and the percentage of cells in apoptosis
determined by FACS analysis as described in Example 1. The data
shown in FIG. 6 represent mean.+-.S.D. Indomethacin enhanced the
percentage of apoptotic cells by approximately 2 fold in the
vector-HCT-116 and sense cells. In contrast, indomethacin did not
stimulate apoptosis or increase the expression of NAG-1 in
antisense HCT-116 cells. These results support the conclusion that
NAG-1 expression is responsible, in part, for the INDO-induced
apoptosis in HCT-116 cells.
EXAMPLE 8
NAG-1 Expression Reduces Transplantable Tumor Growth Rate
[0146] Since in vitro experiments showed that NAG-1 is strongly
associated with apoptosis, anti-tumorigenic activity of NAG-1 in
vivo was evaluated using NAG-1 stably transfected HCT-116 cells
growing in athymic nude mice as follows. Twenty male nude mice
(athymic NCr-nu) were purchased at 5 weeks of age from NCI/Taconic
and maintained in pathogen-free conditions. Exponentially growing
cells (1.times.10.sup.6 cells) vector and NAG-1 transfected HCT-116
cells were inoculated subcutaneously behind the anterior forelimb
into bilateral sides of each mouse. Ten mice were injected at two
sites for each cell line. Tumors were measured externally on the
indicated days in two dimensions using calipers. Tumor measurement
began when the size was more than 3 mm. in diameter (around 10 days
after injection). Tumor volume was determined by the equation
V=(L.times.W.sup.2).times.0.5, where L=length and W=width of the
tumor. Values are the mean (S.E. of 9 xenograftst See, FIG. 7B).
"Vector" represents vector transfected HCT-116 cells, whereas
"NAG-1" represents NAG-1 transfected HCT-116 cells. FIG. 7A
provides a Western blot of transfected HCT-116 cells probed with
anti-NAG-1 antibody.
[0147] As shown in FIG. 7C, the vector transfected HCT-116 cells
rapidly developed visible tumors and dramatic growth was observed
throughout the time course. In contrast the NAG-1 transfected
HCT-116 cells grew at a slower rate in the nude mice and suppressed
tumor growth. Thus, data from in vitro and in vitro studies,
indicate that NAG-1 has anti-tumorigenic activity which may be
mediated by an increase in apoptosis.
[0148] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the art of
molecular biology, oncology, and/or related fields are intended to
be within the scope of the following claims.
Sequence CWU 1
1
518902DNAHomo sapiens 1gaattcgagc tcggtacccg gggatcgatc ctctagagtc
gacctgcagg catgcaagct 60tgcggccgcc ctaggactag tctcgaggct agcccatggg
gcgccgggcc cggatcacct 120gaggtccgga gttcgagatc agcctgggca
acacggtgaa acccccgtct ctactaaaaa 180ttcaaaaatt agctgggcat
ggtggtgcat gcctgtaatc ccagctactc gggaggctaa 240ggcaggagaa
tcgtttgaac ccaggaggtg gagactgcag tgagtcgaga tggcgccatt
300gcactccagc ctgggcaaca agagtaaatc tccatctcac aaaaaaaaaa
aaaaaaagca 360aggcacagtg gcatcaggct ggggtgggaa gatcacttga
gcccagccat tcgagaccaa 420cctgtgcaac acaggaaaac cccattctac
aaaatagtaa aaaattatca gggcatggtg 480gcacatgcct atagtcccag
ctactcggga gagtgatggg ggaggatcac ttgagcccag 540gaggtcgagg
ctgcggtgac ctatgattgt gccactgcac tccagcctgg gctacacagc
600cagaccctgt ctaaaaagaa acaaaacagg caggacgcgg tggctcacac
ctgtaatccc 660agcactttgg gaggccgagg tgggcggatc acctgacatc
aggagttcga gaccagcctg 720tccaacatcg tgaaatcccc ctctctacta
aaatacaaac attaaccggg catggtagtg 780ggtgcctgta atcctaccta
ctcgggaggc tgaggcagga aaattgcttg aacctgggag 840atggaggttg
caatgagccg agatcgcgcc actgcacgac agcctaggcg acagaacaag
900actccatctc aaaaaaaaaa gaaaaagaaa aaaagaagaa actctcattg
atgggtgttc 960attgatggag cactttttgg gaatgggttg acatgctcac
agtgttttta acctcacctc 1020gtctctgaag aggtgcagat aaaagtcacc
tggtgaggaa aatgcctttt taaaaacaat 1080ttttagaggg ccagcacggt
gactcacgac tgtaatctga gcaatttggg aggccgaggt 1140gggcggatca
cttgaggtca ggagttcaaa accagtctgg ccaacatggt gaaatcctgt
1200ctctactaaa aatacaaaaa ttagttgggc atggtggcac atgcctgtaa
tcccagctac 1260tcgggaagct gaggcaggag aatcacttta acccaggagg
tggaagttgc agtgagccaa 1320gatcgcgcca gtgcactcca gcctgggcaa
cagagcgaga ctctgtctca aaagaaaaaa 1380aaaagagatg gggtcttggg
gtctcactgt gttgcccagg ctggtctcca actcctggcc 1440tcaagcaatc
ctcctgcttt ggcctcccaa agtgctagga ttacatgcct gagccactgt
1500gttcaaccag ggtgtccctt ttctgagact gcattcttcc caccttgccc
aagtattgca 1560ggagaaacac aagccccatg ctgtgctggc gaaatctcat
ctcatgctgg cctgttaggg 1620tggccatatc ctctcatttc aagaggctct
gagggagatg gaacaggctc agcggctggc 1680tgcttggggc tctaacccca
gccctgctgt cttctggctg tgtggccgtg acgagcccac 1740ctctctggcc
tcagtttgtc atctgtgaaa tggacataat ctatccacca catcaggctg
1800tataaagatt aggccaggtg tggtgcctca cacctgtaat cccagcgctt
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ctggccaaaa tggtgaaacc 1920ctgtctctaa taaaaataca aaaattagct
gggcatggtg gcatgtacct gtaatcccag 1980ctacccagga ggctgaggca
ggagaatcgc tggaacccgg gaggcagagg ctgcagtgag 2040ccaagattgc
accactgcac tccagcctga gtgacagagc gaaactccgt ctcagagaaa
2100aaaaaaaaaa aaaaagatat gtccaactcc taacgctcag tatctgtgac
cttatttgga 2160aagaaggtct ttgcaaatgt aattaaacta aagatcttgt
actgagatca ttctgaatta 2220aaaatggacc cgtggctggg cacagtggct
cacacctgta atcccagcac tctgggaggc 2280tgaggtgggc aaatcacctg
aaatcaggag ttcaaaacca gcctggccaa catggcgaaa 2340ccccgtctct
actaaaaata caaaaaaaaa ttagctgggc gttgtggtgc acacctgtaa
2400ttccagctac tcgggaggct gaggcaggag aatcacttga acctgggagg
ctgcagtgag 2460ccgagattgc gccactgcat tccagcctag gcaacaagag
tgaaactctg tctcgaaaat 2520aaaaacataa gaaagagtgg acccatggcc
aggcacagtg gctcatgcct ataatcccaa 2580cactttggga ggccaagatg
gaaagatcgt ttgaactcag ccgtttgaaa ccagcctggg 2640caacatagtg
agaccctgtg tctaaataca aacacaaaca gaaaacaaaa agaacccggg
2700ctggctgcgg aaccccctag atggcacagg cacatccggg ctttcgagga
cagtcacgag 2760catgagacag catttcttct tgtcctcggg gcctgcagga
agtttttaag taatctgctt 2820ttgtgcctct tccaaatctc ctcctatgac
cctgtttacc ttacatcaca agaaacaatc 2880aataaaagtg gtttgtgttc
gtgagtgccg tgtgtttgac aaaggcgttg ataactgaga 2940gctggtagtg
gaggggggag attttgggag attggggccc tgccaggtgg acggactggc
3000ctgacctccg gcccctgcgc tctgtttact tccatcccag ctccacctct
ggggacccat 3060ggaagctgtc ggtgaggaac acaggatccg acctgctgag
ctcatctctg gccacctgcc 3120tgcatccgcc ccatctctcc tctcagcttg
gccatgaggc gggctatgag gtcattggtt 3180aaagatttgg ggaagcagag
ccaggcgagt ggccagatgt ggtggctcat gccatcacag 3240gatccagcac
tgtgagagga caaggaagga ggatccttga cctcaggagt tcaaaaccag
3300cctgtacaac atagcaagac ccccgtctct acaaaatata aaattaacca
gttgtggagg 3360cacgcgcctc tattcccaac tactcgggag gttgaggtga
caggatccct tgacccagga 3420ggtcgaggct gcattgagtg ggtaacagag
ccaaaccctg tctctaaaac aaacaaaaca 3480gaaacaaaaa gtacactact
tactgtcctg cttctcacca ttccagcctg ggtagtcctt 3540tttttttttt
ttgagacagg gtcttgctca ttgctcaggc tggagtgcag aagcgtaatc
3600acagctcact gcagccttga tctccaaaaa gactaccagc ttggctgggt
gtggggactc 3660atgcctataa ccccagcatt ttgggagcca gaggcaagag
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cctatctcta aaattttttt ttttgagatg 3780gaatttagct ctcattgccc
aggctagagt gcaatggtgc aatctcagct caccacaatc 3840tccacctccc
aggttcaagc aaaattctcc tgcctcagcc tcccgagtag ctgggattac
3900aggcatgcgc caccatgccc ggctaatttt gttttttaaa gagacggggt
tctccatgtt 3960ggtcaggctg gtctcaaact cccaacctca ggtgatctgc
ctgccttggc ctccctatct 4020cctcattggg ggccatagga ttaaatagct
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cagtggcaca atcacggctc acggcaacct ccgcctcctg 4140cctcagccta
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4200tgtttgagta atctcggtat atatccattt tgtctatgaa gctcgttaat
ttgggcccca 4260aagcacccgc tggcaatggc ctttaagtgc gaaaatcctg
gcattaggcg tcccccacct 4320tttttttttt tttttgttgg gacggatgta
ggtgtgtcgc ccaggctgga gtgcagtggc 4380gcaatctcgg ctcactgcaa
gctccgcctt ccgggttcac gcctttctcc cacctcagcc 4440tcccgagtag
ctgagactac aggcacccac caccatgcct ggctaatttt tttgtatttt
4500tagtagagag ggggtttcac cgtgttagcc aggatggtct tgatctcctg
acctcgtgat 4560ccgcctgacc tcggccttcc aaagtgctgg gattacaggc
aagagccacc gcgcccggcc 4620tggtgtcctt atttcttggg aaaggataca
gagggtcaga gaggcgcccc agcttgtgca 4680ggaatgcatt cctgagatct
ggcctggccg tggtcactgg agtgtttact ctgcttacag 4740ggtctggtgt
agtgggggat ggctgggtag gaacgtgcgg gctataaagc cagaccccag
4800gtgtcagtgc ggttcagaaa gcaactagca cagccatgcc caggccagga
cccgtgacac 4860agcgatgctc tcagatgctg ctgatgttac agatgttgtt
gggggcaccc tgactctgca 4920ggtgagacct tctgggttcc taggaccctt
ggacaagcac tttgatccgg atgactccag 4980gtcccaggag ttgcagaaac
gccaccagga ctttcagagc cccagttaga caaggagagc 5040ccaggaggag
ttggccccca ccctcatccc aaaagcacag gtgagctttg agacctccca
5100cccccaggac agcccccatg tctattttct ttttctttaa agatgttctt
atttgggggc 5160ccaggcgtgg tggctcatgc ctgtaatccc agcactttgg
gaggccgagg caggcggatc 5220acctgagatc aggagttcga gaccagactg
gccaacatga cagggtgaga ccccatcttt 5280acaacaaata caaaaattac
ccaggcatgg tggcacacgc ctgtaatccc agctactagg 5340gaggatgaag
caggagaatt gcttgaaccc gggaggcgga gattgtagtg agttgagatc
5400gcacaactgc actccagcct gggcaacaga gggagactcc aatccaaaaa
aaagaaaaaa 5460atcccctagg acagggctgt ggctgagacc ctgagggctg
gaggcttggc tggccttgca 5520cagcagcggg tgcatgctgg ggtggggaga
ggcctggaga ccctgtgact ccactggggg 5580ccttgctgtg tgacccccta
gtgagtcctt gtgtctctta cccacgcatg cctgtcacat 5640gcagacaccc
acacacaccc agtatctgcc ggacagggca gcccttcctc tccgcagcca
5700ggaagctgga cataggcaca agggctgacg cctggggcca ggaatcctgc
ctgagcatta 5760ggataaggtc tgggaacccc agggggaagg gcactcctgg
ggcatcccct gcccctctac 5820catctggtgg gcttggactc ttacaccaag
cctgccctgc tctaaaaccc cactctcaat 5880tctgtgccac ctcctctctg
ggcccagaca agagcagatt catccctgcc ccaaaggaac 5940caccagtctt
gggtcagcag agctgggcac agacacttcc agtgccgtgg ggctgtaact
6000gtgataggtc tgttgctcca tgcacttggc aagtcaacag ctgagaaaac
aggttgcaac 6060atggaaagag ttttaatccg accggcgtgg tggctcatgc
ctgtaatccc agcactttgg 6120gaggctgagg cgggtggatc atctgaggcc
agcagttcaa gatcagcctg gccaaccagg 6180tgaaacctca tctctactaa
aaatacaaaa attagccagg cgtgatggtg cgcacctgta 6240atcccagcta
cttgggaggc tgaggcagga gaatcaattg aacctgggag gcggaggttg
6300cagtgagcca agatcatacc actgcactcc agcctgggca acagagcgag
agactgtctc 6360ggaagaaaaa aaaaaaaaaa aggctggggg cagtggctca
tgcctgtaat ccccacactt 6420taaaggcaga ggccgacgga tgacttgagg
tcaagagttc gagacctgcc tggcaacatg 6480gtgaatcacc gtctctacta
aaaacacaaa attagccagg tgtggtggcg catgactgta 6540atcccagcta
ctcagtaggc tgaggcagga ggcggaggtt gcagtgagcc gagattgtgc
6600cactgcactc caacctgggg gacagagaga aactccgtct caaaaaaaaa
aaaaaaaaga 6660gatttaatcc tagggccacc caatgaggag ataggaggga
acctcaaatc catttccagg 6720aggaggagtt tggggccata taaatgtata
tatacaaata tatatatatt tttaagatgg 6780agtcagccgg ccgtggtggc
tcacgcctct aatcccaata ctttgggagg ccgaggccgg 6840cggatcacaa
ggtcaggaga ttgagaccat cttagctaac acggtgaaac cccatctcta
6900ctaaaaatac aaaaaaaaat tagccaggcg tggtgggggg tgatcatagt
cccagctact 6960caggaggttg aggcaggaga atagcatgaa cctgggagct
ggagcttgca ggttggagtg 7020caatggcatg atctcagctc aatgcacctc
cgcctcctgg ttcagcgatt tctcctgcct 7080caggctcccg agcagctggg
attacaggca tgtgcccacc acgaccggtt aatttctgaa 7140ttttttagta
gagacaggtt tcatcatgtt gtcaggctgg tctcgaactc ctgacctcag
7200gtgatccgcc cgccttggcc tcccaaagtg ctgggattac aggcgtgagc
cactgcacct 7260ggccaggggc tagagtttta atttttaagg attttggagt
gggctgaagt gtggaggtca 7320ttgcttggtg aaagagtgca ggaggtgaag
tcacagacag taagaaactg tattctcatg 7380cagattccgt tcctctgtgg
ggtcttcaca ctggtgggtg tcattggtta aggatttcaa 7440aaacatctta
agaaattctt ctaaaaagtc ttatgattct aaggtcggaa atcacatcta
7500tagcaaatgg tcggtatcag gtgctacaag caacttgcgg tcacaaggaa
gtgggtcaaa 7560gtgcagcctg attagtgctt aattataact aagtttctgt
ccagaattct ttttttttga 7620gacagagttt tgctcttgtt gatcaggcgg
aagtgcaatg gtgaaaactt ggctcactgc 7680aacctccgcc ctctgggttc
aagcgattct cttgcttcag cctctcgaat agctgggatt 7740acaggcatgt
aatcccacca ccaagcccag ctaattttgt atatttagta gagacagggt
7800ttctccatgt tggtcaggct agtctagaac tcttgacgtc agatgatcca
cgtgcctcgg 7860cctcccaaag tgctgggatt acaggcgaga gccaccgtgc
ccggcgcaga attctttttt 7920ttagagatga ggtattgcca tcttgcccag
acttgtctcg aactcctggg ctcaaacaat 7980ccacccacct cggcctccca
aagtgctgag attactgaca taagccacca tgcctggccc 8040ccagaattat
gaatcctgtg aggatggctt caaggtgagc gctgagccag acaaaaggat
8100ggggtttggg agcaccctgc ttagactgga aagataatgt tggagaagac
ttcctggaag 8160aggggctttt tgcgtagagt tttgaagaat gagtaggagt
tctccagagg aggatgagta 8220actgcaataa cacccagttt atcaagtgcc
tcctatgtgt ctggccctgt gctttacccc 8280tcatttgacc acctctccag
tgagagtctc agtccttttt ttcctggtga ggaaacaggc 8340atggcagaga
ggcatgacac atcaaggttg cccttcctgg ctccatctag cccgttctcc
8400tctgcttcct ttgtttttca ccatctttag cctttgaccc caaccaaaaa
gagaagagag 8460gaaatcccat gggcatagac agccacctct taaactcttg
tctggaattt ttcacatagt 8520aacaatgtct ttttttcctc caaaaagact
cccaggctgg aatggtgtcc tcatatcgag 8580gaagaggata ctgaggccca
gaaatgtgcc ctagctttac taggagcgcc cccacctaaa 8640gatcctcccc
ctaaatacac ccccagaccc cgcccagctg tggtcattgg agtgtttact
8700ctgcaggcag ggggaggagg gcgggactga gcaggcggag akggacaaag
tccggggact 8760ataaaggccg gtccggcagc atctggtcag tcccagctca
gagccgcaac ctgcacagcc 8820atgcccgggc aagaactcag gacggtgaat
ggctctcaga tgctcctggt gttgctggtg 8880ctctcgtggc tgccgcatgg gg
890221202DNAHomo sapiens 2gcggccgctg cacagccatg cccgggcaag
aactcaggac gctgaatggc tctcagatgc 60tcctggtgtt gctggtgctc tcgtggctgc
cgcatggggg cgccctgtct ctggccgagg 120cgagccgcgc aagtttcccg
ggaccctcag agttgcacac cgaagactcc agattccgag 180agttgcggaa
acgctacgag gacctgctaa ccaggctgcg ggccaaccag agctgggaag
240attcgaacac cgacctcgtc ccggcccctg cagtccggat actcacgcca
gaagtgcggc 300tgggatccgg cggccacctg cacctgcgta tctctcgggc
cgcccttccc gaggggctcc 360ccgaggcctc ccgccttcac cgggctctgt
tccggctgtc cccgacggcg tcaaggtcgt 420gggacgtgac acgacctctg
cggcgtcagc tcagccttgc aagaccccag gcgcccgcgc 480tgcacctgcg
actgtcgccg ccgccgtcgc agtcggacca actgctggca gaatcttcgt
540ccgcacggcc ccagctggag ttgcacttgc ggccgcaagc cgccaggggg
cgccgcagag 600cgcgtgcgcg caacggggac cactgtccgc tcgggcccgg
gcgttgctgc cgtctgcaca 660cggtccgcgc gtcgctggaa gacctgggct
gggccgattg ggtgctgtcg ccacgggagg 720tgcaagtgac catgtgcatc
ggcgcgtgcc cgagccagtt ccgggcggca aacatgcacg 780cgcagatcaa
gacgagcctg caccgcctga agcccgacac ggtgccagcg ccctgctgcg
840tgcccgccag ctacaatccc atggtgctca ttcaaaagac cgacaccggg
gtgtcgctcc 900agacctatga tgacttgtta gccaaagact gccactgcat
atgagcagtc ctggtccttc 960cactgtgcac ctgcgcgggg gaggcgacct
cagttgtcct gccctgtgga atgggctcaa 1020ggttcctgag acacccgatt
cctgcccaaa cagctgtatt tatataagtc tgttatttat 1080tattaattta
ttggggtgac cttcttgggg actcgggggc tggtctgatg gaactgtgta
1140tttatttaaa actctggtga taaaaataaa gctgtctgaa ctgttaaaaa
aaaaaaaaaa 1200aa 120233535DNAHomo sapiensmisc_feature(65)..(65)"n"
= any nucleic acid 3cccgggaggc ggagattgta gtgagttgag atcgcacaac
tgcactccag cctgggcaac 60agagngagac tccaatccaa aaaaaagaaa aaaatcccct
aggacagggc tgtggctgag 120accctgaggg ctggaggctt ggctggcctt
gcacagcagc gggtgcatgc tggggtgggg 180agaggcctgg agaccctgtg
actccactgg gggccttgct gtgtgacccc ctagtgagtc 240cttgtgtctc
ttacccacgc atgcctgtca catgcagaca cccacacaca cccagtatct
300gccggacagg gcagcccttc ctctccgcag ccaggaagct ggacataggc
acaagggctg 360acgcctgggg ccaggaatcc tgcctgagca ttaggataag
gtctgggaac cccaggggga 420agggcactcc tggggcatcc cctgcccctc
taccatctgg tgggcttgga ctcttacacc 480aagcctgccc tgctctaaaa
ccccactctc aattctgtgc cacctcctct ctgggcccag 540acaagagcag
attcatccct gccccaaagg aaccaccagt cttgggtcag cagagctggg
600cacagacact tccagtgccg tggggctgta actgtgatag gtctgttgct
ccatgcactt 660ggcaagtcaa cagctgagaa aacaggttgc aacatggaaa
gagttttaat ccgaccggcg 720tggtggctca tgcctgtaat cccagcactt
tgggaggctg aggcgggtgg atcatctgag 780gccagcagtt caagatcagc
ctggccaacc aggtgaaacc tcatctctac taaaaataca 840aaaattagcc
aggcgtgatg gtgcgcacct gtaatcccag ctacttggga ggctgaggca
900ggagaatcaa ttgaacctgg gaggcggagg ttgcagtgag ccaagatcat
accactgcac 960tccagcctgg gcaacagagc gagagactgt ctcggaagaa
aaaaaaaaaa aaaaggctgg 1020gggcagtggc tcatgcctgt aatccccaca
ctttaaangc agangccgac ngatgacttg 1080aggtcaagag ttcgagacct
gcctggcaac atggtgaatc accgtctcta ctaaaaacac 1140aaaattagcc
aggtgtggtg gcgcatgact gtaatcccag ctactcagta ggctgaggca
1200ggaggcggag gttgcagtga gccgagattg tgccactgca ctccaacctg
ggggacagag 1260agaaactccg tctcaaaaaa aaaaaaaaaa agagatttaa
tcctagggcc acccaatgag 1320gagataggag ggaacctcaa atccatttcc
aggaggagga gtttggggcc atataaatgt 1380atatatacaa atatatatat
atttttaaga tggagtcagc cggccgtggt ggctcacgcc 1440tctaatccca
atactttggg aggccgaggc cggcggatca caaggtcagg agattgagac
1500catcttagct aacacggtga aaccccatct ctactaaaaa tacaaaaaaa
aattagccag 1560gcgtggtggg gggtgatcat agtcccagct actcaggagg
ttgaggcagg agaatagcat 1620gaacctggga gctggagctt gcaggttgga
gtgcaatggc dtgatctcag ctcaatgcac 1680ctccgcctcc tggttcagcg
atttctcctg cctcaggctc ccgagcagct gggattacag 1740gcatgtgccc
accacgaccg gttaatttct gaatttttta gtagagacag gtttcatcat
1800gttgtcaggc tggtctcgaa ctcctgacct caggtgatcc gcccgccttg
gcctcccaaa 1860gtgctgggat tacaggcgtg agccactgca cctggccagg
ggctagagtt ttaattttta 1920aggattttgg agtgggctga agtgtggagg
tcattgcttg gtgaaagagt gcaggaggtg 1980aagtcacaga cagtaagaaa
ctgtattctc atgcagattc cgttcctctg tggggtcttc 2040acactggtgg
gtgtcattgg ttaaggattt caaaaacatc ttaagaaatt cttctaaaaa
2100gtcttatgat tctaaggtcg gaaatcacat ctatagcaaa tggtcggtat
caggtgctac 2160aagcaacttg cggtcacaag gaagtgggtc aaagtgcagc
ctgattagtg cttaattata 2220actaagtttc tgtccagaat tctttttttt
tgagacagag ttttgctctt gttgatcagg 2280cggaagtgca atggtgaaaa
cttggctcac tgcaacctcc gccctctggg ttcaagcgat 2340tctcttgctt
cagcctctcg aatagctggg attacaggca tgtaatccca ccaccaagcc
2400cagctaattt tgtatattta gtagagacag ggtttctcca tgttggtcag
gctagtctag 2460aactcttgac gtcagatgat ccacgtgcct cggcctccca
aagtgctggg attacaggcg 2520agagccaccg tgcccggcgc agaattcttt
tttttagaga tgaggtattg ccatcttgcc 2580cagacttgtc tcgaactcct
gggctcaaac aatccaccca cctcggcctc ccaaagtgct 2640gagattactg
acataagcca ccatgcctgg cccccagaat tatgaatcct gtgaggatgg
2700cttcaaggtg agcgctgagc cagacaaaag gatggggttt gggagcaccc
tgcttagact 2760ggaaagataa tgttggagaa gacttcctgg aagaggggct
ttttgcgtag agttttgaag 2820aatgagtagg agttctccag aggaggatga
gtaactgcaa taacacccag tttatcaagt 2880gcctcctatg tgtctggccc
tgtgctttac ccctcatttg accacctctc cagtgagagt 2940ctcagtcctt
tttttcctgg tgaggaaaca ggcatggcag agaggcatga cacatcaagg
3000ttgcccttcc tggctccatc tagcccgttc tcctctgctt cctttgtttt
tcaccatctt 3060tagcctttga ccccaaccaa aaagagaaga gaggaaatcc
catgggcata gacagccacc 3120tcttaaactc ttgtctggaa tttttcacat
agtaacaatg tctttttttc ctccaaaaag 3180actcccaggc tggaatggtg
tcctcatatc gaggaagagg atactgaggc ccagaaatgt 3240gccctagctt
tactaggagc gcccccacct aaagatcctc cccctaaata cacccccaga
3300ccccgcccag ctgtggtcat tggagtgttt actctgcagg cagggggagg
agggcgggac 3360tgagcaggcg gagacggaca aagtccgggg actataaagg
ccggtccggc agcatctggt 3420cagtcccagc tcagagccgc aacctgcaca
gccatgcccg ggcaagaact caggacggtg 3480aatggctctc agatgctcct
ggtgttgctg gtgctctcgt ggctgccgca tgggg 3535423DNAHomo sapiens
4acctgcacag ccatgcccgg gca 23523DNAHomo sapiens 5cagtggaagg
accaggactg ctc 23
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