U.S. patent application number 12/721881 was filed with the patent office on 2010-11-04 for glycine n-methyltransferase (gnmt) animal model and use thereof.
This patent application is currently assigned to NATIONAL YANG-MING UNIVERSITY. Invention is credited to Yi-Ming Chen, Shih-Ping Liu.
Application Number | 20100279897 12/721881 |
Document ID | / |
Family ID | 39967685 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279897 |
Kind Code |
A1 |
Chen; Yi-Ming ; et
al. |
November 4, 2010 |
Glycine N-methyltransferase (GNMT) Animal model and use thereof
Abstract
The present invention is a new type of Glycine
N-methyltransferase (GNMT) knockout mice model. This model can be
applied to screen drug, test of treatment and search for diagnostic
marker of hepatocellular carcinoma (HCC), glycogen storage disease,
liver dysplasia, fatty liver and other liver disease.
Inventors: |
Chen; Yi-Ming; (Taipei City,
TW) ; Liu; Shih-Ping; (Taipei City, TW) |
Correspondence
Address: |
WPAT, PC;INTELLECTUAL PROPERTY ATTORNEYS
2030 MAIN STREET, SUITE 1300
IRVINE
CA
92614
US
|
Assignee: |
NATIONAL YANG-MING
UNIVERSITY
TAIPEI CITY
TW
|
Family ID: |
39967685 |
Appl. No.: |
12/721881 |
Filed: |
March 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11832304 |
Aug 1, 2007 |
7759542 |
|
|
12721881 |
|
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Current U.S.
Class: |
506/17 ;
536/24.33 |
Current CPC
Class: |
A01K 2217/075 20130101;
A01K 2227/105 20130101; A61P 35/00 20180101; A01K 2267/0331
20130101; A01K 67/0276 20130101; A61P 39/02 20180101; C12N 15/8509
20130101; A61P 1/16 20180101; A61P 43/00 20180101; A61P 3/08
20180101; A01K 2267/03 20130101 |
Class at
Publication: |
506/17 ;
536/24.33 |
International
Class: |
C40B 40/08 20060101
C40B040/08; C07H 21/04 20060101 C07H021/04 |
Claims
1. A pair of prime, which is (i) SEQ ID Nos 1 and 2 or (ii) SEQ ID
Nos 3 and 4.
2. A database for regulatory genes in GNMT knock-out mouse
comprising up-regulatory genes as listed in Tables 3-1 and 3-2 and
down-regulatory genes as listed in Tables 3-1, 3-2 and 4.
3. A database for hepatocellular carcinoma signaling pathway genes
comprising (a) survival and proliferation: PTENPI3KAkt 1GSK3.beta.
or .beta.-catenin; (b) oncogenes: Cyclin D1C-myc or C-Jun; and (c)
tumor suppressor gene: Rb or p53.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 11/832,304 filed on Aug. 1, 2007, that is incorporated
herein by reference in its entirety.
[0002] Although incorporated by reference in its entirety, no
arguments or disclaimers made in the parent application apply to
this divisional application. Any disclaimer that may have occurred
during the prosecution of the above-referenced application(s) is
hereby expressly rescinded. Consequently, the Patent Office is
asked to review the new set of claims in view of the entire prior
art of record and any search that the Office deems appropriate.
FIELD OF THE INVENTION
[0003] The present invention relates to Glycine N-methyltransferase
(GNMT) animal model and use thereof. The present invention also
relates to the use of GNMT product in preventing or treating
cancer, especially liver cancer.
BACKGROUND OF THE INVENTION
[0004] One of the most common types of human diseases throughout
the world due to cell abnormalities is cancer, which is also the
leading cause of death nowadays. Cancers are fully developed
(malignant) tumors with a specific capacity to invade and destroy
the underlying mesenchyme, i.e., local invasion. In some cases,
invading tumor cells may further penetrate lymphatic vessels or
blood vessels newly formed in the tumor and then may be carried to
local lymph nodes or even to distant organs where they may produce
secondary tumors (metastases). Tumors are usually recognized by the
fact that the cells, which may arise from any tissue, are no longer
responsive to at least some normal growth controlling mechanisms
and hence show abnormal growth. Apart from the cancer, a tumor may
merely develop locally and never become malignant, i.e., a benign
tumor. Alternatively, cells of a tumor may merely have
morphological appearances of cancer cells but remain in their
place, i.e., an in situ tumor, although in this case the tumor may
sometimes precede a cancer in situ.
[0005] There are no absolute methods for diagnosing or assessing
the degree of malignancy of tumors. However, among the methods,
microscopic examination of tissue is still the most reliable method
for routine use. In a pathologic study, tumors can be graded by
making an approximate assessment of the degree of structural
dedifferentiation (anaplasia) based on histological and cytological
criteria by microscopically examining sections thereof. However, on
one hand, some cells may have lost their specific structural
characters but still retain differentiated biochemical features,
while others may still appear differentiated in structure but have
lost many normal function attributes. On the other hand, a tumor is
not homogeneous and may contain areas with more than one tumor
grade, therefore, a developed tumor may consist of a mixed
population of cells which may differ in structure, function, growth
potential, resistance to drugs or X-rays and ability to invade and
metastasize. The two limitations reduce the effectiveness of
histological examination of tumors. In another aspect, such an
examination by sampling specimens is not suitable for
investigations on a large scale.
[0006] Many attempts to find absolute markers of malignancy have
long been made. Other attempts to identify tumor-specific or
tumor-associated proteins, either by direct measurement or by
developing specific antibodies to these proteins, are still being
made at the moment. They seem to be promising approaches not only
in diagnosis but also in providing strategies of destroying cancer
cells. A variety of substances wherein the presence or
concentrations thereof in vivo may be indicative for certain
cancers have been reported, such as oncofetal antigens, e.g.,
alpha-fetoprotein; serum proteins, e.g., ferritin; enzymes;
polyamines; ectopic hormones; cell markers; receptors or
tumor-associated viral antigens. However, the most commonly used
method of diagnosis of cancers depends on histology rather than any
of the above substances. The lack of any absolute markers is a
major deficiency in studying cancer.
[0007] Recent observations provide some contemplation in searching
for the substances intimately associated with carcinogenesis.
Cancer is appreciated as a result of multiple gene aberrations
which cause both the activation of oncogenes and inactivation of
tumor suppressor genes. Further, the differential expression of
those critical genes associated with oncogenesis is able to be
reflected at the messenger RNA (mRNA) level in cells. For
effectively screening the altered ones of interest amongst a great
amount of mRNA, a powerful tool, i.e., differential display has
been established to identify and isolate a small subset of genes
which are differentially expressed between tumorous and normal
cells (Liang et al., Cancer Research 52, 6966-6968, 1992).
[0008] Human hepatocellular carcinoma (HCC), one of the world's
most common cancers, usually develops from chronic inflammatory
liver disease via viral infections that induce cirrhosis and
exposure to chemical carcinogens (Yu, M. W. et al., Crit. Rev.
Oncol. Hematol. 17, 71-91, 1994; Schafer, D. F. et al., Lancet 353,
1253-1257, 1999; Williams, J H et al., Am. J. Clin. Nutr. 80,
1106-1122, 2004). In some areas (e.g., China and Africa) HCC is
primarily caused by viral infections (HBV, HCV), food contaminated
by aflatoxin B1 (AFB1), and other forms of aflatoxin ingestion
(Williams, J. H. et al., Am. J. Clin. Nutr: 80, 1106-1122, 2004;
Chen, C. J., Hepatology 16, 1150-1155, 1992). Aflatoxin metabolites
are secondary products of Aspergillus flavus and Aspergillus
parasiticus fungi under hot and humid conditions. These ubiquitous
fungi affect such dietary staples as rice, corn, cassaya, nuts,
peanuts, chilies, and spices (McLean, M. & Dutton, M. F.,
Pharmacol. Ther: 65, 163-192, 1995). Chemicals or xenobiotics (such
as AFB1) that encounter biologic systems can be altered by
metabolic processes. In phase I of the detoxification pathway,
cytochrome P450 isoenzymes (induced by polycyclic aromatic
hydrocarbons and chlorinated hydrocarbons) add one atom of oxygen
to the substrate; bioactivation is an occasional sequela (Hsieh, D.
P. H., Elsevier Scientific Publishers, Amsterdam, 1986; Hsieh, D.
P. H., Academic, Cambridge, 1987; Aoyama, T. et al., Proc. Natl.
Acad. Sci. U.S.A 87, 4790-4793, 1990; Swenson, D. H. et al.,
Biochem. Biophys. Res. Commun. 60, 1036-1043, 1974). The reactive
intermediate aflatoxin B1 8,9-epoxide (produced by CYP isoenzymes,
such as cytochrome P450IA2 and P450IIIA4) is carcinogenic in many
animal species; its covalent binding to hepatic DNA has been shown
to be a critical step in hepatocarcinogenesis (Forrester, L. M., et
al., Proc. Natl. Acad. Sci. U.S. A 87, 8306-8310, 1990; Koser, P.
L. et al., J. Biol. Chem. 263, 12584-12595, 1988). Phase II enzymes
of primary importance belong to the GST group; these catalyze the
conjugation of potentially toxic electrophiles to the GSH
tripeptide, generally rendering them non-toxic (Degen, G H. &
Neumann, H. G, Chem. Biol. Interact. 22, 239-255, 1978; Hayes, J.
D. et al., Pharmacol. Thu: 50, 443-472, 1991). The reactive
aflatoxin B1 8,9-epoxide subsequently attacks and damages DNA. The
major AFB1-DNA adduct formed in vivo is AFB1-N-7-guanine (Croy, R.
G. et. al., Proc. Natl. Acad. Sci. U.S.A 75, 1745-1749, 1978;
Kensler, T. W. et al., Cancer Res. 46, 3924-3931, 1986). There are
at least two reports indicating that AFB1 binds covalently with DNA
and induces G:C to T:A transversions at the third base in codon 249
of p53-considered a hot spot for AFB1 mutagenesis (Bressac, B. et.
al., Nature 350, 429-431, 1991; Hsu, I. C. et al., Nature 350,
427-428).
[0009] GNMT is an intracellular enzyme which catalyzes the
synthesis of sarcosine from glycine. Through this enzyme, glycine
receives a methyl group from S-adenosylmethionine (SAM) and becomes
sarcosine, which can be subsequently oxidized to become glycine
again by sarcosine dehydrogenase. The latter reaction will generate
energy and release one carbon unit from SAM. GNMT thus plays a key
role in regulating the ratio of SAM to S-adenosylhomocysteine
(SAH). The properties of rat liver GNMT, such as its activity being
fluctuated and correlated with the level of methionine in the diet
and its inducibility with a methionine-rich diet, suggest that it
also plays a crucial role in regulating tissue concentration of SAM
and metabolism of methionine (Ogawa, H. et al., J. Biol. Chem.,
257:3447-3452, 1982). However, GNMT was found to be merely
responsible for the metabolism of 20% of total metabolized
methionine in vivo (Case et al., J. Nutr. 106: 1721-1736, 1976),
but this protein is abundant in liver of mature rats or mice,
almost 1% to 3% of the total soluble proteins in liver (Heady et
al., J. Biol. Chem., 248:69-72, 1973). Therefore, the GNMT protein
may exert other important physiological functions, one of which was
found to be identical to a folate-binding protein purified from rat
liver cytosol (Cook, R. J. et al., Proc. Natl. Acad. Sci. USA,
81:3631-3634, 1984). Recently, Raha et al. (J. Biol. Chem.,
269:5750-5756) proved that GNMT is the 4 S polycyclic aromatic
hydrocarbon-binding protein which interacts with 5'-flanking
regions of the cytochrome P4501A1 gene (CYP1A1).
[0010] Furthermore, as GNMT is the most abundant and efficient
methyltransferase in hepatocytes, the activity of GNMT may
influence other methyltransferases, e.g., the activity of tRNA
methyltransferase can be blocked by GNMT (Kerr et al., J. Biol.
Chem., 247:4248-4252, 1972). Results from various laboratories have
indicated that lipotropic compounds, such as SAM and its
precursors: methionine, choline and betaine, can prevent the
development of liver tumors induced by various carcinogens in a rat
or mouse model. Due to the findings that GNMT is tightly associated
with the SAM level in liver cells and its enzyme activity may be
activated by SAM, the GNMT may involve the chemopreventive pathway
way of liver cancer (Pascale et al., Anticancer Res., 13:1341-1356,
1993).
[0011] It has been reported that diminished GNMT expression levels
in both human hepatocellular carcinoma cell lines and tumor tissues
(Liu, H. H. et al, J. Biomed. Sci. 10, 87-97, 2003; Chen, Y. M. et
al., Int. J. Cancer 75, 787-793, 1998). Human GNMT gene is
localized to the 6p12 chromosomal region and characterized its
polymorphism (Chen, Y. M. et al., Genomics 66, 43-47, 2000).
Genotypic analyses of several human GNMT gene polymorphisms showed
a loss of heterozygosity in 36-47% of the genetic markers in
hepatocellular carcinoma tissues (Tseng, T. L. et al., Cancer Res.
63, 647-654, 2003). It also reported that GNMT were involved in the
benzo(a)pyrene (BaP) detoxification pathway and reduced BPDE-DNA
adducts that formed in GNMT-expressing cells (Chen, S. Y. et al.,
Cancer Res. 64, 3617-3623, 2004).
[0012] Previous results indicated that multiple proteins were
capable of binding aflatoxin B1 in rat liver cytosol (Taggart, P.
et al., Proc. Soc. Exp. Biol. Med. 182, 68-72, 1986). Cytosolic
proteins involved in AFB 1 binding may have the potential to
function in the transport, metabolism and even action of the
carcinogen (Dirr, H. W. & Schabort, J. C., Biochem. Int. 14,
297-302, 1987).
SUMMARY OF THE INVENTION
[0013] The present invention provides a knock-out mouse whose
genome is disrupted by recombination at Glycine N-methyltransferase
(GNMT) gene locus so as to produce a phenotype, relative to a
wild-type phenotype, comprising abnormal liver function of said
mouse, wherein the disruption occurs nucleotides 547-4875 of SEQ ID
No. 8.
[0014] The present invention also provides a method for screening a
candidate agent for preventing or treating liver disease or
disorder comprising: [0015] (a) providing the knock-out mouse of
the present invention; [0016] (b) administering to said the
knock-out mouse a candidate agent, and [0017] (c) comparing liver
function of the knock-out mouse to that of the knock-out mouse of
not administered said candidate agent; wherein the agent that
ameliorates liver function is selected as an agent that has
effectiveness against said liver disease or disorder.
[0018] The present invention further provides a pair of prime,
which is (i) SEQ ID Nos 1 and 2 or (ii) SEQ ID Nos 1 and 2.
[0019] The present invention further provides a database for
regulatory genes in GNMT knock-out mouse a database for regulatory
genes in GNMT knock-out mouse.
[0020] The present invention also provides a database for
hepatocellular carcinoma signaling pathway genes.
[0021] The present invention further provides a method for treating
or preventing disease caused by aflatoxin B1 (AFB1) in a patient
subject comprising administering the patient with an effective
amount of Glycine N-methyltransferase (GNMT) or plasmid including
GNMT.
[0022] The present invention also provides a composition for
treating or preventing disease caused by aflatoxin B1 comprising
Glycine N-methyltransferase (GNMT) and pharmaceutically or food
acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the strategy of constructing the targeting
vector.
[0024] FIG. 2 shows targeted modification of the Gnmt gene locus.
(A) Targeting vector was designed to replace Gnmt exons 1-4 and a
part of exon 5 with a neomycin resistance gene. Neomycin positive
selection marker is flanked by two homologous regions and followed
by a TK negative selection marker at the 3' end of the targeting
vector. (B) Southern blot analysis of embryonic stem cell clones.
BamHI (B)-BamHI DNA fragment size decreased from 7.9 kb (wild-type
allele) to 5.3 kb (recombinant allele). (C) Genotyping of Gnmt
knockout mice by PCR. The normal Gnmt allele yielded a 772 by
fragment and the disrupted allele a 409 bp fragment. +/+,
wild-type; +/-, Gnmt heterozygous and -/-, Gnmt-/- mice (D)
Expression of GNMT protein confirmed by western blot analysis. Each
lane contains 10 .mu.g hepatic lysate. GNMT molecular mass: 32 kDa.
GAPDH: internal control.
[0025] FIG. 3 shows the Real-time PCR analyses of mRNA expression
levels of the genes involved in one-carbon metabolism pathway. The
expression profiles of mRNA in WTM (wild-type male), KOM (Gnmt-/-
male) and KOF (Gnmt-/- female) liver tissue were normalized to the
WTF (wild-type female) mice. *, p<0.05. Ahcy,
S-adenosylhomocysteine hydrolase; Ms, methionine synthase; Cbs,
cystathionine beta-synthase; Mthfr, 5,10-methylenetetrahydrofolate
reductase; Mthfd1, methylenetetrahydrofolate dehydrogenase (NADP+
dependent); methenyltetrahydrofolate cyclohydrolase;
formyltetrahydrofolate synthase; AldhIII, aldehyde dehydrogenase 1
family; member L1; Atic, 5-aminoimidazole-4-carboxamide
ribonucleotide formyltranferase/IMP cyclohydrolase; Shmt2, serine
hydroxymethyl transferase 2; Mthfs, 5,10-methenyltetrahydrofolate
synthetase; Ftcd, formiminotransferase cyclodeaminase.
[0026] FIG. 4 depicts that gGnmt-/- mice had hepatomegaly and
significantly higher levels of serum ALT. (A) Ratio of liver weight
to body weight. (B) Comparison of serum ALT levels between
wild-type, Gnmt+/-, and Gnmt-/- mice. *, p<0.05; **, p<0.01,
both compared to wild-type mice.
[0027] FIG. 5 shows the pathological examination of wild-type and
Gnmt-/- mouse livers. Gross pathology of liver organs from male
wild-type (A), male Gnmt+/- (B), male Gnmt-/- (C), and female
Gnmt-/- mice (D). All the mice had been fasting for eight hours
before they were sacrificed. HE staining of liver tissue from
11-week-old male wild-type (E and I), male Gnmt+/- (F and J), male
Gnmt-/- (G and K), female Gnmt-/- (H and L), 9-month-old male
Gnmt-/- (Q), and 9-month-old female Gnmt-/- mice (S). PAS staining
of liver tissue from 11-week-old male wild-type (M), female
wild-type (N), male Gnmt-/- (O), female Gnmt null (P), 9-month-old
male Gnmt-/- (R), and 9-month-old female Gnmt-/- mice (T).
Magnification: 100.times. for E-H, 400.times. for I-T.
[0028] FIG. 6 shows Hematology and analysis of blood biochemical
parameters of wild-type and Gnmt-/- mice. (A) White blood cell,
neutrophil, lymphocyte, monocyte, eosinophil and basophil counts in
wild type mice (solid circles) and Gnmt-/- mice (open circles).
Horizontal bars indicate the mean counts. (B) Serum glucose,
cholesterol, and triglyceride levels of wild type (solid circles)
and Gnmt-/- mice (open circles). Horizontal bars indicate the mean
serum concentration. *, p<0.05; **, p<0.01, both compared to
wild-type mice.
[0029] FIG. 7 shows Real-time PCR analysis of mRNA expression
levels of genes linked with various types of GSD. The expression
profiles of mRNA were normalized to the wild type mice. (A) The
mice at 11 weeks of age; (B) The mice at 9 months of age. *,
p<0.05; **, p<0.01. Gys2, glycogen synthase 2; G6 Pase,
glucose-6-phosphatase; G6PT, glucose-6-phosphate transporter; Gaa,
alpha-glucosidase; Agl, amylo-1,6-glucosidase; Gbe1, branching
enzyme 1; Pygl, glycogen phosphorylase; Phka2, phosphorylase kinase
alpha 2; Fbp1, fructose 1,6-bisphosphatase; and PEPCK,
phosphoenolpyruvate carboxykinase.
[0030] FIG. 8 is the result of ultrasound, MRI, gross pathology, HE
stain and reticulin stain of male and male Gnmt-/- mice. Ultrasound
of liver organs from male Gnmt-/- (A), female Gnmt-/- (G). MRI and
MRI reconstruction of liver organs from male Gnmt-/- (B and C), and
female Gnmt-/- mice (H and I). Gross pathology of liver organs from
male Gnmt-/- (D), and female Gnmt-/- mice (J). HE staining of liver
tissue from male male Gnmt-/- (E), and female Gnmt-/- (K).
Reticulin staining of liver tissue from male male Gnmt-/- (F), and
female Gnmt-/- (1).
[0031] FIG. 9 shows Real-time PCR analysis of several early HCC
markers (glypican-3, LYVE1, survivin and alpha-fetoprotein) in the
wild-type and Gnmt-/- mice.
[0032] FIG. 10 shows (A-B) Nuclear translocation of glycine
N-methyltransferase (GNMT) following treatment with aflatoxin B1.
HA22T cells on coverslips were transfected with 5 .mu.g GNMT-Flag
and treated with DMSO solvent (A) or 40 .mu.M AFB1 (B) prior to
fixing and reaction with R4 (rabbit anti-GNMT) antisera. For
immunofluorescent staining we used FITC-conjugated goat antirabbit
antibodies. Nuclei were stained with Hoechst H33258. Bars: 20
.mu.M. (C-E) Model of benzo(a)pyrene (BaP) and aflatoxin B1 (AFB1)
docking with the tetrameric form of GNMT using the Lamarckian
genetic algorithm. (C) BaP (green) and AFB1 (red) molecules docked
with the S-adenosylhomocysteine-bound tetrameric form of rat GNMT
(cyan) (PDB code 1D2H). (B) A monomer showing docked models of BaP
(green) and AFB1 (red) molecules. GNMT amino acid residues (A1a64,
Va169, Leu136, Gly137 and Ser139) in close proximity to several
AFB1 carbon atoms are indicated according to the GNMT structure
(PDB code 1D2H) and the docking model of the GNMT-AFB1 complex. (E)
Structures of AFB1 (left) and BaP (right).
[0033] FIG. 11 shows GNMT antagonized the cytotoxicity effect of
AFB1. (A-C) AFB1-induced cytotoxicity is reduced by GNMT
overexpression. MTT assay was used to determine the survival
percentage of HuH-7 cells treated with AFB1. A. Survival curve of
HuH-7 cells treated with different amount of AFB1 at series time
points. The 50% inhibitory concentration is dependent on the
duration of treatment. The IC50 of AFB1 on HuH-7 cells be treated
for 72 hr is about 12 M. B. HuH-7 cells were infected with
Adenovirus carried GNMT gene or GFP control gene for 16 hr. After
72 hr of AFB 1 treatment, cells were subjected to MTT assay. The
survival rates of HuH-7 cells increased slightly by the dosage of
Ad-GNMT. At the group of HuH-7 cells treated with 8 M AFB1, the
survival rates of HuH-7 cells increased significantly by the dosage
of Ad-GNMT. C. Similar results were observed in another system in
which HuH-7 cells were transducted with the GNMT gene via a
lentiviral vector. *p<0.05, **p<0.01. (D-E) GNMT
overexpression reduced the formation of AFB1-DNA adducts. D,
SCG2-neg and SCG2-1-1 cells were treated with DMSO or indicated
concentration AFB1 prior to harvesting for DNA extraction. AFB1-DNA
adducts were measured with a competitive ELISA. White box and gray
box indicate SCG2-neg and SCG2-1-1, respectively. Data represent
the mean.+-.SD. *, p<0.01; **, p<0.001 by t-test. E, Ad-GFP-
and Ad-GNMT-infected HepG2 cells were used to perform this assay.
White box indicated Ad-GFP-infected HepG2 cells; gray box, 5 MOI
Ad-GNMT infected HepG2 cells; black box, 50 MOI Ad-GNMT-infected
HepG2 cells. *, by one way ANOVA.
[0034] FIG. 12 illustrates the expression profiles and enzyme
activity of GNMT in GNMT-TG and wild-type mice. A. GNMT protein
level in 1, wild type male (opened diamond); 2, transgenic male
(closed diamond); 3, wild type female (opened square) and 4,
transgenic female (closed square) were determined by Western blot
analysis (upper panel) and the quantitative data (lower panel). The
result showed that transgenic animals have more amount of GNMT
protein than wild type before 5 weeks old. B. Compared enzyme
activity of GNMT between 1, wild type male; 2, transgenic male; 3,
wild type female and 4, transgenic female.
[0035] FIG. 13 shows H&E and IHC staining for the four groups
of male mice livers. Photomicrographs of livers of
carcinogen-treated mice by H&E staining. (A) Wild type mice
treated with AFB 1. X 200, (B) GNMT transgenic mice treated with
AFB 1. X 200, Immunohistochemical analysis of the GNMT expression
in the paraffin-fixed tissue. (C) Wild type treated with AFB1. X
200, (D) GNMT transgenic treated with AFB 1. X 200. (E) Western
blot analysis of cell extracts from non-tumor tissue (N) and tumor
tissue (T). The result showed that the GNMT expression level in
tumor tissue was lower than non-tumor tissue in three groups of
mice.
[0036] FIG. 14 shows a construction of the pPEPCKex-flGNMT plasmid.
pPEPCKex (vector) and pSK-flGNMT (insert) digested with Not I and
Xho I and ligated to produced pPEPCKex-flGNMT. B. The expression of
the mouse endogenous and human GNMT mRNAs in various organs in the
transgenic or wild-type mouse was determined by Northern blot
analysis. 1) Kidney RNA of GNMT transgenic mice. 2) Liver RNA of
GNMT transgenic mice. 3) Brain RNA of wild-type mice. 4) Kidney RNA
of wild-type mice. 5) Liver RNA of wild-type mice. The result
showed that GNMT transgenic mice expressed human GNMT gene
(transgene) in liver and kidney.
DETAILED DESCRIPTION OF THE INVENTION
[0037] It is surprisingly found in the present invention that the
GNMT gene is differentially expressed between normal and tumorous
cells with a significant distinction. An objective of the present
invention is to provide a method of detecting abnormalities of
cells by determining the relative levels of gene expression of
GNMT. Furthermore, another objective of the present invention is to
provide a method of correcting the abnormalities of cells by
delivering GNMT into the abnormal cells.
[0038] Non-human transgenic animal models useful for screening
psychoactive drugs are provided. The animals have genetically
altered GNMT gene. Alterations to the gene include deletion or
other loss of function mutations, introduction of an exogenous gene
having a nucleotide sequence with targeted or random mutations,
introduction of an exogenous gene from another species, or a
combination thereof. The transgenic animals may be either
homozygous or heterozygous for the genetic alteration.
[0039] GNMT undergoes nuclear translocation following AFB.sub.1
treatment. According to the results of tests of the present
invention, AFB.sub.1 binds with GNMT and competes with SAM for the
same binding site. Evidence was also found in support of the idea
that GNMT antagonizes AFB.sub.1-induced cytotoxicity by reducing
AFB.sub.1-DNA adduct formation and enhancing AFB.sub.1-treated cell
survival rate. Finally, results from GNMT transgenic mouse model
showed that overexpression of GNMT exhibited protective effect
against AFB.sub.1 induced hepatocellular carcinoma.
[0040] The present invention provides a method for treating or
preventing disease caused by aflatoxin B1 (AFB1) in a patient
subject comprising administering the patient with an effective
amount of Glycine N-methyltransferase (GNMT) or plasmid including
GNMT.
[0041] In a preferred embodiment, the disease is hepatocellular
carcinoma (HCC).
[0042] In the present method, the treatment or prevention is made
by blocking formation of AFB1-DNA adducts.
[0043] For gene therapy, the plasmid can be regarded as a plasmid
vaccine and could be directly administered to the body of the
patient by current technology for gene therapy.
[0044] The present invention provides a knock-out mouse whose
genome is disrupted by recombination at Glycine N-methyltransferase
(GNMT) gene locus so as to produce a phenotype, relative to a
wild-type phenotype, comprising abnormal liver function of said
mouse, wherein the disruption occurs nucleotides 547-4875 of SEQ ID
No. 8.
[0045] In particular, the nucleotides are GNMT exons 1-4 and a part
of exon 5. The phenotype of absence of Glycine N-methyltransferase
activity results from a diminished amount of mature Glycine
N-methyltransferase relative to the wild-type phenotype.
[0046] In the preparation of knock-out mouse, the Glycine
N-methyltransferase gene is disrupted by recombination with
heterologous nucleotide sequence (such as neomycin).
[0047] The term "abnormal liver function" herein is not limited but
includes elevation of S-adenosylmethionine (SAM), alanine
aminotransferase (ALT) or asparate aminotransferase (AST).
Transgenic Animals
[0048] The term "transgene" is used herein to describe genetic
material that has been or is about to be artificially inserted into
the genome of a mammalian cell, particularly a mammalian cell of a
living animal. The transgene is used to transform a cell, meaning
that a permanent or transient genetic change, preferably a
permanent genetic change, is induced in a cell following
incorporation of exogenous DNA. A permanent genetic change is
generally achieved by introduction of the DNA into the genome of
the cell. Vectors for stable integration include plasmids,
retroviruses and other animal viruses, YACs, and the like. Of
interest are transgenic mammals, e.g. cows, pigs, goats, horses,
etc., and particularly rodents, e.g. rats, mice, etc.
[0049] Transgenic animals comprise an exogenous nucleic acid
sequence present as an extrachromosomal element or stably
integrated in all or a portion of its cells, especially in germ
cells. Unless otherwise indicated, it will be assumed that a
transgenic animal comprises stable changes to the germline
sequence. During the initial construction of the animal, "chimeras"
or "chimeric animals" are generated, in which only a subset of
cells have the altered genome Chimeras are primarily used for
breeding purposes in order to generate the desired transgenic
animal Animals having a heterozygous alteration are generated by
breeding of chimeras. Male and female heterozygotes are typically
bred to generate homozygous animals.
[0050] Transgenic animals fall into two groups, colloquially termed
"knockouts" and "knockins". In the present invention, knockouts
have a partial or complete loss of function in one or both alleles
of the endogenous GNMT gene. Knockins have an introduced transgene
with altered genetic sequence and function from the endogenous
gene. The two may be combined, such that the naturally occurring
gene is disabled, and an altered form introduced.
[0051] In a knockout, preferably the target gene expression is
undetectable or insignificant. A knock-out of a GNMT gene means
that function of the GNMT gene has been substantially decreased so
that expression is not detectable or only present at insignificant
levels. This may be achieved by a variety of mechanisms, including
introduction of a disruption of the coding sequence, e.g. insertion
of one or more stop codons, insertion of a DNA fragment, etc.,
deletion of coding sequence, substitution of stop codons for coding
sequence, etc. In some cases the exogenous transgene sequences are
ultimately deleted from the genome, leaving a net change to the
native sequence. Different approaches may be used to achieve the
"knock-out". A chromosomal deletion of all or part of the native
gene may be induced, including deletions of the non-coding regions,
particularly the promoter region, 3' regulatory sequences,
enhancers, or deletions of gene that activate expression of GNMT
gene. A functional knock-out may also be achieved by the
introduction of an anti-sense construct that blocks expression of
the native genes. "Knock-outs" also include conditional knock-outs,
for example where alteration of the target gene occurs upon
exposure of the animal to a substance that promotes target gene
alteration, introduction of an enzyme that promotes recombination
at the target gene site (e.g. Cre in the Cre-lox system), or other
method for directing the target gene alteration postnatally.
[0052] A "knock-in" of a target gene means an alteration in a host
cell genome that results in altered expression or function of the
native GNMT gene. Increased (including ectopic) or decreased
expression may be achieved by introduction of an additional copy of
the target gene, or by operatively inserting a regulatory sequence
that provides for enhanced expression of an endogenous copy of the
target gene. These changes may be constitutive or conditional, i.e.
dependent on the presence of an activator or represser.
[0053] The exogenous gene is usually either from a different
species than the animal host, or is otherwise altered in its coding
or non-coding sequence. The introduced gene may be a wild-type
gene, naturally occurring polymorphism, or a genetically
manipulated sequence, for example having deletions, substitutions
or insertions in the coding or non-coding regions. The introduced
sequence may encode a GNMT polypeptide. Where the introduced gene
is a coding sequence, it is usually operably linked to a promoter,
which may be constitutive or inducible, and other regulatory
sequences required for expression in the host animal. By "operably
linked" is meant that a DNA sequence and a regulatory sequence(s)
are connected in such a way as to permit gene expression when the
appropriate molecules, e.g. transcriptional activator proteins, are
bound to the regulatory sequence(s).
[0054] Specific constructs of interest, but are not limited to,
include anti-sense GNMT gene, which will block native GNMT
expression, expression of dominant negative GNMT mutations, and
over-expression of a GNMT gene. A detectable marker, such as lac Z
may be introduced into the locus, where upregulation of expression
will result in an easily detected change in phenotype.
[0055] A series of small deletions and/or substitutions may be made
in the GNMT gene to determine the role of different exons in DNA
binding, transcriptional regulation, etc.
[0056] By providing expression of GNMT protein in cells in which it
is otherwise not normally produced, one can induce changes in cell
behavior.
[0057] DNA constructs for homologous recombination will comprise at
least a portion of the GNMT gene with the desired genetic
modification, and will include regions of homology to the target
locus. DNA constructs for random integration need not include
regions of homology to mediate recombination. Conveniently, markers
for positive and negative selection are included. Methods for
generating cells having targeted gene modifications through
homologous recombination are known in the art.
[0058] For embryonic stem (ES) cells, an ES cell line may be
employed, or embryonic cells may be obtained freshly from a host,
e.g. mouse, rat, guinea pig, etc. Such cells are grown on an
appropriate fibroblast-feeder layer or grown in the presence of
appropriate growth factors, such as leukemia inhibiting factor
(LIF). When ES cells have been transformed, they may be used to
produce transgenic animals. After transformation, the cells are
plated onto a feeder layer in an appropriate medium. Cells
containing the construct may be detected by employing a selective
medium. After sufficient time for colonies to grow, they are picked
and analyzed for the occurrence of homologous recombination or
integration of the construct. Those colonies that are positive may
then be used for embryo manipulation and blastocyst injection.
Blastocysts are obtained from 4 to 6 week old superovulated
females. The ES cells are trypsinized, and the modified cells are
injected into the blastocoel of the blastocyst. After injection,
the blastocysts are returned to each uterine horn of pseudopregnant
females. Females are then allowed to go to term and the resulting
litters screened for mutant cells having the construct. By
providing for a different phenotype of the blastocyst and the ES
cells, chimeric progeny can be readily detected.
[0059] Accordingly, the present invention also provides a cell or
cell line, which is prepared from the knock-out mouse of the
present invention. In a preferred embodiment, the cell or cell line
is an undifferentiated cell selected from the group consisting of:
a stem cell, embryonic stem cell oocyte and embryonic cell.
[0060] The chimeric animals are screened for the presence of the
modified gene and males and females having the modification are
mated to produce homozygous progeny. If the gene alterations cause
lethality at some point in development, tissues or organs can be
maintained as allogeneic or congenic grafts or transplants, or in
in vitro culture.
[0061] A genomic sequence of interest comprises the nucleic acid
present between the initiation codon and the stop codon, as defined
in the listed sequences, including all of the introns that are
normally present in a native chromosome. It may further include the
3' and 5' untranslated regions found in the mature mRNA. It may
further include specific transcriptional and translational
regulatory sequences, such as promoters, enhancers, etc., including
about 1 kb, but possibly more, of flanking genomic DNA at either
the 5' or 3' end of the transcribed region. The genomic DNA may be
isolated as a fragment of 100 kbp or smaller; and substantially
free of flanking chromosomal sequence.
Drug Screening Assays
[0062] The present invention further provides a method for
screening a candidate agent for preventing or treating liver
disease or disorder comprising: [0063] (i) providing the knock-out
mouse of the present invention; [0064] (ii) administering to said
the knock-out mouse a candidate agent, and [0065] (iii) comparing
liver function of the knock-out mouse to that of the knock-out
mouse of not administered said candidate agent; wherein the agent
that ameliorates liver function is selected as an agent that has
effectiveness against said liver disease or disorder.
[0066] Through use of the subject transgenic animals or cells
derived therefrom, one can identify ligands or substrates that bind
to, modulate, antagonize or agonize GNMT polypeptide. Screening to
determine drugs that lack effect on these polypeptides is also of
interest. Of particular interest are screening assays for agents
that have a low toxicity for human cells.
[0067] A wide variety of assays may be used for this purpose,
including in vivo behavioral studies, determination of the
localization of drugs after administration, labeled in vitro
protein-protein binding assays, protein-DNA binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, and the like. Depending on the particular assay, whole
animals may be used, or cell derived therefrom. Cells may be
freshly isolated from an animal, or may be immortalized in culture.
Cell of particular interest include neural and brain tissue.
[0068] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of affecting the
biological action of GNMT polypeptide. Generally a plurality of
assay mixtures are run in parallel with different agent
concentrations to obtain a differential response to the various
concentrations. Typically, one of these concentrations serves as a
negative control, i.e. at zero concentration or below the level of
detection.
[0069] In a preferred embodiment, the agent is for preventing or
treating hepatocellular carcinoma (HCC), glycogen storage disease,
liver dysplasia or fatty liver.
[0070] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including, but not limited to: peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0071] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0072] Screening may be directed to known pharmacologically active
compounds and chemical analogs thereof. known anti liver cancer or
disease drugs include the Drugs with a significant hepatoprotective
effect are of particular interest.
[0073] To prepare the knock-out mouse, the present invention also
provides a pair of prime, which is (i) SEQ ID Nos 1 and 2 or (ii)
SEQ ID Nos 3 and 4.
[0074] The present invention further provides a database for
regulatory genes in GNMT knock-out mouse to study up-regulatory and
down-regulatory genes.
[0075] The present invention further provides a database for
hepatocellular carcinoma signaling pathway genes comprising [0076]
(a) survival and proliferation: PTENPI3KAkt 1GSK3.beta. or
.beta.-catenin [0077] (b) oncogenes: Cyclin D1C-myc or C-Jun; and
[0078] (c) tumor suppressor gene: Rb or p53.
[0079] The present invention also provides a composition for
treating or preventing disease caused by aflatoxin B1 comprising
Glycine N-methyltransferase (GNMT) and pharmaceutically or food
acceptable carrier. In a preferred embodiment, the GNMT is in
dimeric or tetrameric forms.
[0080] The composition of the present invention could be applied as
a supplementary additive for a dietary staple (such as rice, corn,
cassaya, nut, peanut, chilies or spices).
[0081] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, constructs, and reagents described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0082] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise.
[0083] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0084] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for
example, the cell lines, constructs, and methodologies that are
described in the publications which might be used in connection
with the presently described invention. The publications discussed
above and throughout the text are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention.
[0085] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
Example 1
[0086] To construct a targeting vector, DNA fragments digested from
lambda phage clones 3-2 and 5-3 were inserted into a
plasmid-pBluescrip II KS. Left arm was digested from the phage
clone 5-3 by using Pst I and inserted into the pNeo vector. Right
arm was digested from the phage clone 3-2 by using Hinc II and
inserted into the TK vector. The fragment containing right arm and
TK gene was digested by using Not I and inserted into the pNeo
vector containing left arm to generate the targeting vector (FIG.
1).
[0087] The neomycin gene (to replace exons 1-4 and part of exon 5
of the mouse Gnmt gene) was framed with two DNA fragments (3.1 kb
and 3.7 kb) in the targeting vector. The thymidine kinase gene was
used as a negative selection marker (FIG. 2A). The 40 .mu.g
targeting vector was linearized using AscI and introduced into
embryonic stem cells (129/Sv-derived) by electroporation. After
screening 278 clones using southern blot analysis (FIG. 2B), a
recombinant clone was isolated and used for microinjection into
blastocytes. Four male chimeric mice were obtained and used to
breed female C57BL/6 mice. Agouti F.sub.1 offspring were subjected
to PCR to detect the germline transmission of the disrupted allele.
Heterozygous F.sub.1 male mice were backcross with female wild-type
C57B/6 mice to generate C57BL/6 genome background mice. PCR was
developed to differentiate wild-type (+/+), Gnmt heterozygous
(+/-), and Gnmt-/- mice. The primers used for PCR were shown as the
following: GNMT-F (5'-GCGGCGGCCGCATGCTGGTGGAAGAGGGC) (SEQ ID NO: 1)
and GNMT-R (5'-TTGCAGTCTGGCAAGTGAGC) (SEQ ID NO: 2) for GNMT;
neomycin-F (5'-GTTCCTTGCGCAGCTGTGCT) (SEQ ID NO: 3) and neomycin-R
(5'-CGGCCACAGTCGATGAATCC) (SEQ ID NO: 4) for neomycin. The normal
Gnmt allele yielded a 772 bp fragment by Gnmt primers and the
disrupted allele yielded a 409 bp fragment by neomycin primers
(FIG. 2C). The expression of GNMT protein in liver was analyzed
using western blot; the results show that compared to the
wild-type, GNMT expression decreased approximately 50% in the
livers of Gnmt+/- mice and GNMT was undetectable in the livers of
Gnmt-/- mice (FIG. 2D).
[0088] At 11 weeks of age, male and female wild-type, Gnmt+/- and
Gnmt-/- mice 6 mice per group) were sacrificed for phenotypic
analysis. SAM and SAH concentrations were detected by using HPLC.
Compared to wild-type mice of the same gender, hepatic
concentrations of SAM in Gnmt-/- mice significantly increased in
both male and female mice (p<0.05). In contrast, the hepatic
concentration of SAM in Gnmt+/- mice was 2.8 fold lower than in
wild-type mice (Table 1), and hepatic concentrations of SAH in male
and female Gnmt-/- mice were similar to those in the wild-type
mice. Accordingly, the SAM/SAH ratio increased 42- and 67-fold in
the male and female Gnmt-/- mice, respectively (Table 1).
Homocysteine levels remained unchanged across the different mouse
groups. Methionine levels in the Gnmt-/- mice were 1.9 to 2.4-fold
greater than in the wild-type mice (Table 1).
TABLE-US-00001 TABLE 1 Concentrations of hepatic SAM and SAH and
levels of serum homocysteine and methionine from wild-type and Gnmt
KO mice. SAM (nmol/g liver) SAH (nmol/g liver) SAM/SAH Total
Homocysteine (.mu.M) Methionine (mg/dl) Wild-type Male 45.0 .+-.
23.4 71.9 .+-. 26.2 0.63 5.7 .+-. 0.03 0.74 .+-. 0.03 Female 52.4
.+-. 29.3 64.2 .+-. 16.8 0.82 5.7 .+-. 0.04 0.74 .+-. 0.01 Total
48.0 .+-. 24.6 68.8 .+-. 22.2 0.69 5.6 .+-. 0.13 0.75 .+-. 0.02
Gnmt+/- Male 18.0 .+-. 4.6 77.1 .+-. 8.9 0.23 5.9 .+-. 0.10 ND
Female 16.2 .+-. 10.2 70.4 .+-. 11.9 0.23 5.9 .+-. 0.20 ND Total
17.1 .+-. 7.1 73.7 .+-. 10.1 0.23 5.9 .+-. 0.17 ND Gnmt-/- Male
3085.4 .+-. 1276.9 73.3 .+-. 33.6 42.09 5.8 .+-. 0.10 2.04 .+-.
0.64 Female 3882.0 .+-. 1978.8 57.7 .+-. 6.2 67.28 5.8 .+-. 0.30
2.47 .+-. 0.89 Total 3453.1 .+-. 1617.9 66.5 .+-. 25.4 51.93 5.8
.+-. 0.19 2.23 .+-. 0.76
[0089] The phenotypes of Gnmt-/- mice have been followed up
biweekly using magnetic resonance imaging (MRI) and ultrasound
since they were 13 months of age. Among 6 GNMT-/- mice sacrificed,
1 male and 1 female mice had an early HCC nodule and at age of 18
and 14 months, 1 female mouse had an early HCC nodule and liver
fatty nodules at age of 14 months, 1 female and 1 male mice had
dysplastic nodules with markedly fatty changes in liver, and 1 male
mouse had liver necrosis at age of 14 months (Table 2).
TABLE-US-00002 TABLE 2 The liver tumor formation of the Gnmt-/-
mice at the age of 13-21 months liver/body No. of nodules Gender
Age (m) weight nodules >0.5 cm <0.5 cm Pathology data GNMT-/-
M 13 7.23% 0 0 0 Fatty change, Necrosis M 13 7.58% 0 0 0 Necrosis M
18 9.48% 1 1 0 Dysplastic nodules, Early HCC GNMT-/- F 14 10.62% 7
3 4 Fatty change, dysplastic nodule, Early HCC F 15 6.23% 2 0 2
Fatty change F 21 5.94% 2 2 0 Fatty change, Carvernous
hemangioma
[0090] Microarray analysis was applied to observe the difference
metabolism between GNMT knockout and wild-type mice. Significantly
increase of mRNA levels in both female (GNMT KO compared to wild
type) and male (GNMT KO compared to wild type) mice were shown for
1896 and 2429 genes, respectively. Among these genes, 543 and 843
genes with more than twofold changes were selected to further
functional analysis. We used CrossPath program
(http://ibs.sinica.edu.tw/crosspath/) to classify these genes by
their functions based on KEGG pathway databases. Tables 3 and 4
showed the functional pathways in which increase two or more
differentially expressed genes have been categorized in the given
pathways. The major pathways increase in female and male GNMT KO
mice are PPAR signaling pathway and cell cycle. In addition,
cytokine-cytokine receptor interaction and MAPK signaling pathway
were decreased in female and male GNMT KO mice. Table 5 shown the
ratio of mRNA expression levels of genes belonging to different
pathways in various tissues from Gnmt-/- mice vs. that expressed in
liver tissues from age-matched wild type mice measured using
real-time PCR. The results shown several survival and proliferation
genes were significantly down-regulated in Gnmt-/- mice and three
oncogenes were up-regulated in Gnmt-/- mice.
TABLE-US-00003 TABLE 3-1 Functional classification of regulatory
genes in female and male GNMT KO mice at 11 weeks of ages by KEGG
pathway database. Pathways that up-regulated in female Pathways
that up-regulated in male GNMT-/- mice No.* GNMT-/- mice No.* PPAR
signaling pathway 17 Cell cycle 16 Biosynthesis of steroids 11 Gap
junction 13 MAPK signaling pathway 10 PPAR signaling pathway 12
Glutathione metabolism 9 Pyrimidine metabolism 12 Pyruvate
metabolism 7 Purine metabolism 11 Xenobiotics by cytochrome P450 7
Focal adhesion 10 Adipocytokine signaling pathway 7 Oxidative
phosphorylation 9 Insulin signaling pathway 6 Tight junction 8
Jak-STAT signaling pathway 6 Glutathione metabolism 7 Colorectal
cancer 6 Cytokine-cytokine receptor interaction 7 Pathways that
down-regulated in female Pathways that down-regulated in male
GNMT-/- mice No.* GNMT-/- mice No.* Cytokine-cytokine receptor
interaction 21 MAPK signaling pathway 24 Cell adhesion molecules 17
Cytokine-cytokine receptor interaction 16 Focal adhesion 16 Focal
adhesion 15 MAPK signaling pathway 15 Regulation of actin
cytoskeleton 13 Regulation of actin cytoskeleton 15 Complement and
coagulation cascades 12 Neuroactivate ligand-receptor interaction
12 Wnt signaling pathway 12 Calcium signaling pathway 11
Xenobiotics by cytochrome P450 12 Wnt signaling pathway 11 Insulin
signaling pathway 12 Hematopoietic cell lineage 11 Calcium
signaling pathway 12 Insulin signaling pathway 10 Linoleic acid
metabolism 11 Jak-STAT signaling pathway 10 Arachidonic acid
metabolism 10 *Number of genes that exhibited at least twofold
expression difference between GNMT KO and wild type mice
TABLE-US-00004 TABLE 3-2 Functional classification of regulatory
genes in female and male Gnmt-/- mice tumorous and tumor adjacent
tissues at 14-18 months of ages by KEGG pathway database. Pathways
that up-regulated in female Pathways that up-regulated in male
GNMT-/- mice No.* GNMT-/- mice No.* PPAR signaling pathway 17 Cell
cycle 16 Biosynthesis of steroids 11 Gap junction 13 MAPK signaling
pathway 10 PPAR signaling pathway 12 Glutathione metabolism 9
Pyrimidine metabolism 12 Pyruvate metabolism 7 Purine metabolism 11
Xenobiotics by cytochrome P450 7 Focal adhesion 10 Adipocytokine
signaling pathway 7 Oxidative phosphorylation 9 Insulin signaling
pathway 6 Tight junction 8 Jak-STAT signaling pathway 6 Glutathione
metabolism 7 Colorectal cancer 6 Cytokine-cytokine receptor
interaction 7 Pathways that down-regulated in female Pathways that
down-regulated in GNMT-/- mice No.* female GNMT-/- mice No.*
Cytokine-cytokine receptor interaction 21 MAPK signaling pathway 24
Cell adhesion molecules 17 Cytokine-cytokine receptor interaction
16 Focal adhesion 16 Focal adhesion 15 MAPK signaling pathway 15
Regulation of actin cytoskeleton 13 Regulation of actin
cytoskeleton 15 Complement and coagulation 12 cascades
Neuroactivate ligand-receptor interaction 12 Wnt signaling pathway
12 Calcium signaling pathway 11 Xenobiotics by cytochrome P450 12
Wnt signaling pathway 11 Insulin signaling pathway 12 Hematopoietic
cell lineage 11 Calcium signaling pathway 12 Insulin signaling
pathway 10 Linoleic acid metabolism 11 Jak-STAT signaling pathway
10 Arachidonic acid metabolism 10 *Number of genes that exhibited
at least twofold expression difference between tumorous and tumor
adjacent tissues from Gnmt-/- mice
TABLE-US-00005 TABLE 4 Functional classification of down-regulatory
genes in female and male GNMT KO mice by KEGG pathway database.
Pathways that down-regulated in female Pathways that down-regulated
in male GNMT-/- mice No.* GNMT-/- mice No.* Cytokine-cytokine
receptor interaction 21 MAPK signaling pathway 24 Cell adhesion
molecules 17 Cytokine-cytokine receptor interaction 16 Focal
adhesion 16 Focal adhesion 15 MAPK signaling pathway 15 Regulation
of actin cytoskeleton 13 Regulation of actin cytoskeleton 15
Complement and coagulation cascades 12 Neuroactivate
ligand-receptor interaction 12 Wnt signaling pathway 12 Calcium
signaling pathway 11 Xenobiotics by cytochrome P450 12 Wnt
signaling pathway 11 Insulin signaling pathway 12 Hematopoietic
cell lineage 11 Calcium signaling pathway 12 Insulin signaling
pathway 10 Linoleic acid metabolism 11 Jak-STAT signaling pathway
10 Arachidonic acid metabolism 10 *Number of genes that exhibited
at least twofold expression difference between GNMT KO and wild
type mice
TABLE-US-00006 TABLE 5 The ratio of mRNA expression levels of genes
belonging to different pathways in various tissues from Gnmt-/-
mice vs. that expressed in liver tissues from age-mached wild type
mice measured using real-time PCR. 11-w Gnmt-/- mice 18-m Gnmt-/-
14-m Gnmt-/- Male Female Male Female Gene category KO/WT.sup.a
KO/WT.sup.a TA/WT.sup.b T/WT.sup.c TA/WT.sup.b T/WT.sup.c Survival
and proliferation PTEN 1.32 1.07 0.60 0.37 0.56 0.08 PI3K 0.60 0.47
0.26 0.36 0.40 0.69 Akt 1 1.40 1.28 0.95 0.74 0.64 0.48 GSK3.beta.
1.20 0.71 0.75 1.14 1.20 0.70 .beta.-catenin 1.58 1.07 0.65 0.65
0.78 0.47 Oncogenes Cyclin D1 6.55 5.57 3.35 2.41 1.73 1.50 C-myc
1.97 1.56 1.88 1.42 0.78 0.65 C-Jun 2.55 2.47 2.54 3.97 1.41 1.47
Tumor suppressor gene Rb 3.20 0.75 0.64 1.00 1.57 0.73 p53 1.96
1.16 1.40 1.62 1.07 0.83 PTEN, phosphatase and tensinhomolog; PI3K,
phosphatidylinositol 3-kinase; GSK3.beta., glycogen synthase
kinase; Rb, retinoblastoma; T, tumorous tissues; TA, tumor adjacent
tissues; KO, Gnmt-/- mice; WT, wild-type mice .sup.aratio of the
gene expression profiles between Gnmt-/- and wild-type mice at the
11 weeks of age. The raw data was normalized to GAPD internal
control. .sup.bratio of the gene expression profiles between tumor
adjacent tissues and wild-type mice liver tissues. The raw data was
normalized to GAPD internal control. .sup.cratio of the gene
expression profiles between tumorous tissues and wild-type mice
liver tissues. The raw data was normalized to GAPD internal
control.
[0091] To demonstrate that the Gnmt-/- mouse model is more
susceptible to carcinogens, aflatoxin B1 (AFB1) was used to
challenge the mice. The AFB1 was administrated intra-peritoneally
twice with the following dosages: 10 .mu.g per body weight in gram
on the 7.sup.th day of age and 40 ug at the 9.sup.th weeks of age.
The results showed that liver nodules were detected in all (5/5)
female and 57.1% (4/7) male Gnmt-/- mice treated with AFB1, while
neither the wild-type mice treated with AFB1 nor the other groups
of mice treated with solvent (tricaprylin) have developed liver
tumors at the age of 13-14 months old (Table 6)
TABLE-US-00007 TABLE 6 The liver tumor formation of the 2 genotypes
mice treated with solvent or AFB1 at the age of 13 months old.
Nodules No. mice with Gender No. No. mice with nodules >0.5 cm
<0.5 cm nodules >0.5 cm ALT (U/L) AST (U/L) AFB1 wild type M
8 0/8 (0%) 0 0 0/8 (0%) 27.5 .+-. 0.71 72.00 .+-. 58.92 F 8 0/8
(0%) 0 0 0/8 (0%) 22.83 .+-. 3.92 42.50 .+-. 8.22 Gnmt-/- M 7 4/7
(57.1%) 5 6 3/7 (43.9%) 89.67 .+-. 63.13 106.00 .+-. 54.21 F 5 5/5
(100%) 4 3 4/5 (80%) 182.33 .+-. 95.26 266.33 .+-. 249.52 Solvent
wild type M 6 0/6 (0%) 0 0 0/6 (0%) 20.00 .+-. 0.00 74.00 .+-.
21.21 F 8 0/8 (0%) 0 0 0/8 (0%) 20.17 .+-. 4.45 47.67 .+-. 6.56
Gnmt-/- M 4 0/4 (0%) 0 0 0/4 (0%) 124.00 .+-. 127.28 155.50 .+-.
144.96 F 4 0/ 4(0%) 0 0 0/4 (0%) 76.00 .+-. 26.92 98.50 .+-.
18.43
[0092] HE staining of histological mouse liver sections revealed no
abnormalities in the wild-type mice treated with AFB1. However, we
observed Dysplastic nodule, early HCC and fatty nodules in the male
Gnmt-/- mice treated with AFB1 (FIGS. 9A and B). Sclenosing HCC,
Dysplastic nodule with focal fatty change were observed in the
female Gnmt-/- mice (FIGS. 9C and D).
Example 2
[0093] Materials and Methods
Cell Culture and Treatment.
[0094] Hepatocellular carcinoma cell line [HA22T/VGH] was prepared
according to Waxman, D. J. & O'Connor, C. Growth Hormone
Regulation of Sex-Dependent Liver Gene Expression. Molecular
Endocrinology 20, 2613 (2006), and the stable expression clones
from human hepatoblastoma cell line-HepG2 was prepared based on
Mode, A. & Gustafsson, J. A. Sex and the Liver--A Journey
Through Five Decades. Drug Metabolism Reviews 38, 197-207 (2006),
[SCG2] as stated in the paper Chen, S. Y. et al. Glycine
N-methyltransferase tumor susceptibility gene in the
benzo(a)pyrene-detoxification pathway. Cancer Res. 64, 3617-3623
(2004). were used in this example. Cells were maintained in
Dulbecco's Modified Eagle Medium (DMEM) (GIBCO BRL, Grand Island,
N.Y.) containing 10% fetal bovine serum (Hyclone). AFB.sub.1 was
solved in DMSO and treatment was performed in culture medium.
Immunofluorescent Staining and Confocal Microscopy.
[0095] Cultured HA22T/VGH cells were placed on coverslips and
treated with 20 .mu.M AFB.sub.1 or 0.1% DMSO for 3 hr, fixed with
4% paraformaldehyde in PBS pH 7.4 (solution I) at room-temperature
for 20 min After the cells were incubated in permeabilization
solution (fixing solution I plus 0.5% Triton X-100) at room
temperature for 5 min, the coverslips were incubated with blocking
buffer (5% BSA in PBS) at room temperature for 1 hr and then with
rabbit anti-GNMT antiserum (1:200) for another 1 hr. The
FITC-conjugated goat anti-rabbit IgG (Chemicon, Temecula, Calif.,
USA) were used as secondary antibodies. Nuclei were counterstained
with Hoechst H33258 (Sigma-Aldrich). Confocal microscopy was
performed using an Olympus IX70 inverted fluorescence microscope
outfitted with the Olympus Fluoview Argon/Krypton scanning laser
system and Fluoview image analysis software (Olympus, Melville,
N.Y.).
LGA Dockings.
[0096] LGA was used to elucidate interaction sites between
AFB.sub.1 and various forms of GNMT. Autodock 3.0 software was used
to identify the most favorable ligand binding interactions. As
mentioned earlier, X-ray crystallography data from rat GNMT was
used for docking purposes because it has a 91% amino acid sequence
homology with human GNMT (Pakhomova, S. et al., Proteins Structure
Function and Bioinformatics 57, 331-337, 2004). Parameters included
10 runs, a population size of 50, and a run termination criterion
of 27,000 generations or 2.5.times.10.sup.5 energy evaluations
(whichever came first). A root mean square deviation conformational
clustering tolerance of 0.5 .ANG. was calculated from the ligand's
crystallographic coordinates. Procedural details are available in
previous report (Morris G M et al. J Comput Chem 19, 1639-1662,
1998).
Cytotoxicity Assay.
[0097] MTT assay was used to determine the cytotoxicity effect of
AFB.sub.1. In brief, cells were seeded on 96 well plate. At the
time of assay, culture medium was replaced by 100 .mu.L fresh
medium containing 10 .mu.L of 5 mg/mL MTT stock solution for each
well. After 4 hr of labeling cells with MTT, medium was removed
then 100 .mu.L DMSO was added to each well for 10 min at 37.degree.
C. Samples were mixed and read absorbance at 540 nm For half lethal
concentration (LC50) determination, seven thousand HuH-7 cells were
seeded on 96 well plate for 18 hr. Cells were treated with
different concentration of AFB.sub.1 and MTT assay was performed at
series time points, triplicate for each set. The survival
percentage is calculated by dividing the OD value of treatment
group by solvent control group. For cytotoxicity assay, five
thousand HuH-7 cells were infected with different amount of
adenovirus or lentivirus carrying GNMT cDNA for 8 hr then refreshed
medium for another 10 hr. After 72 hr of AFB.sub.1 treatment, MTT
assay was performed to determine the survival percentage.
Competitive ELISA for Quantifying AFB.sub.1-DNA Adducts.
[0098] To evaluate the protective effect of GNMT, stable SCG2-1-1
cell clones or GNMT recombinant adenovirus (Ad-GNMT) infected HepG2
cells were cultured overnight in a 10-cm petri dish, treated with
AFB.sub.1 or 0.1% DMSO for 16 hrs, then harvested for DNA
extraction. All samples were treated with 15 mM Na.sub.2CO.sub.3
and 30 mM NaHCO.sub.3 (pH 9.6) at 37.degree. C. for 2 hrs to ensure
the neutralization of all adducts in ring-opened form.
AFB.sub.1-DNA adduct levels were measured with a competitive ELISA
using 6A10 antibody, as previously described as Hsieh, L. L. et
al., Immunological detection of aflatoxin B1-DNA adducts formed in
vivo. Cancer Res. 48, 6328-6331 (1988). Each ELISA assay was
performed in triplicate. Absorption levels were read at 490 nm.
The pPEPCKex-flGNMT Transgenic Construct.
[0099] We used a liver and kidney-specific transgenic vector,
pPEPCKex, which contains the mouse phosphoenolpyruvate
carboxykinase (PEPCK; Valera, A. et al., Proc. Natl. Acad. Sci.
U.S. A 91, 9151-9154, 1994) promoter, a 0.3-kb synthetic intron and
a 0.6-kb human growth hormone poly (A) signal. The pPEPCKex-flGNMT
transgenic plasmid was constructed by placing the 1.2-kb human GNMT
cDNA (from the 9-1-2 plasmid; Chen, Y. M. et al., Int. J. Cancer
75, 787-793, 1998) into the NotI and XhoI sites of the pPEPCKex
vector. The pPEPCKex-flGNMT plasmid was digested with AscI and the
linear 4.3-kb fragment was used for the microinjection.
Generation of GNMT TG Mice.
[0100] The GNMT TG mice were generated by pronucleus microinjection
of FVB fertilized eggs. Mice were bred in a specific pathogen free
facility. The tails of individual mice were cut on weaning at 3
weeks of age. Genomic DNA was isolated by proteinase K/SDS
digestion (Promaga) and phenol/chloroform extraction method. The
genotype of TG mice was determined by PCR. To detect the GNMT-TG,
the 668-bp human GNMT specific DNA fragment was amplified by PCR
with cycling conditions of 94.degree. C. for 30 sec, 60.degree. C.
for 30 sec, 72.degree. C. for 1 min for 30 cycles using primers
GNMT-F 5'-GCGGCGGCCGCATGCTGGTGGAAGAGGGC-3' (SEQ ID NO: 1) and
GNMT-R 5'-GCGCTCGAGTCAGTCTGTCCTCTTGAGCAC-3' (SEQ ID NO: 5).
AFB.sub.1 Challenge.
[0101] AFB.sub.1 (Sigma Co, St Louis, Mo.) was dissolved in
tricaprylin (Sigma) at a concentration of 0.2 mg/mL. At 7 days of
age, more than 6 mice in each group were injected with aflatoxin
B.sub.1 (10 mg AFB.sub.1/kg of body weight) intraperitoneally, and
boosted the same dose at 2 months. The protocol has been modified
by Ghebranious and Sell previously (Ghebranious, N. & Sell, S.
Hepatology 27, 383-391,1998). At 9 months after the injection, the
mice were sacrificed for pathological examination. Two-fifth of the
organs (including liver, lung and kidney) were fixed with 10%
formalin for histopathological exam; the remaining parts of the
organs were stored at -80.degree. C. for in situ hybridization,
DNA, RNA, and protein analyses.
RNA Analysis.
[0102] Total RNA was extracted from frozen tissue using TRIzol
reagents (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. The amount for all RNA samples was
quantified by spectrophotometer. Northern blot hybridization was
performed as described previously. The cDNA probes generated from
human GNMT gene of plasmid 9-1-2. Complementary DNA was produced
from hepatic RNA (2 .mu.g) using a SuperScript II RNase H-Reverse
Transcriptase Kit (Invitrogen). The sequences of primers for GNMT
were F1: GCGGCGGCCGCATGCTGGTGGAAGAGGGC (SEQ ID NO: 1) and R1:
GCGCTCGAGTCAGTCTGTCCTCTTGAGCAC (SEQ ID NO: 5); and for .beta.-actin
were F2: GTGGGGCGCCCCAGGCACCA (SEQ ID NO: 6) and R2:
CTCCTTAATGTCACGCACGATTTC (SEQ ID NO: 7). The PCR conditions were as
follows: pre-denaturation at 94.degree. C. for 5 min followed by 30
cycles of amplification at 94.degree. C. for 30 secs, 60.degree. C.
for 30 secs, and 72.degree. C. for 1 min, followed by one 10 min
extension at 72.degree. C.
Western Blot Assay.
[0103] To perform western blot analysis, 10 .mu.g of whole liver
protein extract was separated by 10% SDS-PAGE and transferred to
polyvinylidene difluoride membranes (PVDF; Amersham Pharmacia
Biotech, Piscataway, N.J.). Procedures have been described in
Waxman, D. J. & O'Connor, C. Growth Hormone Regulation of
Sex-Dependent Liver Gene Expression. Molecular Endocrinology 20,
2613 (2006). In this test, mouse anti-GNMT monoclonal antibody
(mAB) 14-1 was used to detect GNMT.sup.20.
GNMT enzyme activity assay.
[0104] This method is modified from procedures reported at Cook, R.
J. & Wagner, C. Glycine N-methyltransferase is a folate binding
protein of rat liver cytosol. Proc. Natl. Acad. Sci. U.S.A 81,
3631-3634 (1984). It was used to measure the GNMT enzyme activity
of liver tissues from GNMT-TG mice. Portions of liver was
homogenized with three volumes of ice-cold phosphate buffer (10 mM,
pH 7.0) containing 0.25 M sucrose, 1 mM EDTA, 1 mM sodium azide,
and 0.1 mM phenymethylsulfonylflouride. After centrifugation at
20,000.times.g for 30 min, the resulting supernatant was removed
and 2-mercaptoethanol is added to a final concentration of 10 mM.
The concentration of the protein was measured and 250 ug protein
was added to a 100 ul reaction mixture containing of 100 mM Tris
buffer (pH 7.4), 50 mM glycine, 0.23 mM SAM, and 2.16 uM
S-adenosyl-.sub.L-[methyl-3H]-methionine (76.4 Ci/mmol). Following
incubation at 37.degree. C. for 30 min, reaction is terminated by
the addition of a 50 ul mixture of 10% trichloroacetic acid and 5%
activated charcoal. Each reaction is performed in triplicate.
Immunohistochemical Staining.
[0105] The immunohistochemical detection of the GNMT protein was
performed using a monoclonal antibody (mAB) 14-1 at 1:200 dilution.
The paraffin-embedded liver sections (4 um) were incubated with the
GNMT antibody and detected with the DAB kit (DakoCytomation)
according to the manufacturer's instructions.
Example 3
GNMT Nuclear Translocation is Induced by AFB.sub.1
[0106] GNMT cDNA transfected HA22T/VGH cells (FIGS. 10A and B) with
AFB.sub.1 or DMSO (solvent control) for 16 hours. As shown in FIG.
10, GNMT distribution was initially restricted to the cytoplasm
(FIG. 10A), but was partly translocated to cell nuclei following
AFB.sub.1 treatment (FIG. 10B). These results showed that
AFB.sub.1, as well as BaP, induces the nuclear translocation of
GNMT.
[0107] It demonstrated that GNMT exhibited nuclear translocation in
AFB.sub.1 treated cells (FIG. 10). It also showed that GNMT can
reduce the formation of AFB.sub.1-DNA adducts and increase the
survival rate of AFB.sub.1-treated cells. AFB.sub.1-DNA adducts
formation have been implicated in liver carcinogenesis (Bressac, B.
et al., Nature 350, 429-431, 1991; Hsu, I. C. et al., Nature 350,
427-428, 1991). It also proofed that the depletion of GNMT in
hepatocyte raised the sensitivity of liver to this carcinogen.
Given the choice, GNMT is involved in a cellular defense mechanism
against these environmental carcinogens.
Example 4
Modeling GNMT-AFB.sub.1 Interaction
[0108] A combination of a Lamarckian genetic algorithm (LGA) and
X-ray crystallography data was used to predict physical
interactions between GNMT and AFB.sub.1. Due to its 91% amino-acid
sequence homology with the human GNMT protein, we relied on X-ray
crystallography of the rat GNMT protein for our AFB.sub.1 docking
experiments. According to the data presented in Table 7, AFB.sub.1
bound to both dimeric (Protein Data Bank code 1D2C) and tetrameric
(1D2G) forms of GNMT at low binding energy levels (-9.41 and -10.06
kcal/mol, respectively).
TABLE-US-00008 TABLE 7 Lamarckian Genetic Algorithm Dockings
Between GNMT Protein and AFB.sub.1 Molecules.sup.a PDB Small
Cluster Cluster Mean Energy Number of Code.sup.b Molecule Number
Population (kcal/mol) Evaluations Protein Details 1D2C.sup.c
AFB.sub.1 5 5 -9.41 2.5 .times. 10.sup.5 GNMT dimmer 1D2H.sup.c
AFB.sub.1 2 9 -9.83 2.5 .times. 10.sup.5 GNMT R175K mutant tetramer
binding with SAM 1XVA.sup.d SAM 2 5 -9.85 2.5 .times. 10.sup.5 GNMT
dimer without SAM 1XVA.sup.e AFB.sub.1 4 4 +53.25 2.5 .times.
10.sup.5 GNMT dimer binding with SAM 1D2G.sup.c AFB.sub.1 1 10
-10.06 2.5 .times. 10.sup.5 GNMT R175K mutant tetramer
[0109] Results from a comparison of the binding energy of AFB.sub.1
with GNMT dimer (1XVA) without SAM (-9.85 kcal/mol) and AFB.sub.1
with GNMT dimer already bound with SAM (53.25 kcal/mol) suggest
that AFB.sub.1 competes with SAM to bind with GNMT. With slight
differences in molecular orientation, the AFB.sub.1 molecule, as
well as BaP, is located at the same position inside the molecular
basket in both the GNMT dimer and tetramer (FIG. 11A). GNMT amino
acid residues in close proximity to AFB.sub.1 (A1a64, Va169,
Leu136, Gly137, and Ser139) are shown in FIG. 11B.
[0110] The example demonstrated (a) an AFB.sub.1-binding domain at
the substrate
[0111] (SAM)-binding site of GNMT, and (b) that AFB.sub.1 binds
with both dimeric and tetrameric forms of GNMT. The R175K mutant
form of the GNMT tetramer (1D2G) was used to demonstrate that R/K
residue in close vicinity (.about.5 .ANG.) of the binding site
exerts practically zero effect on GNMT-AFB.sub.1 cluster formation
(Table 7). This result corresponds with the argument that GNMT is
an example molecular basket. This unique structure feature might be
consistent with the fact that GNMT can not only bind to SAM but
also the polycyclic aromatic hydrocarbon (PAH) molecules such as
benzo[a]pyrene. According to the crystal structure of GNMT, there
are many tyrosine residues (33, 44, 177, 194, 220, 242, 283)
located at the inner surface of the active site. This and other
residues may provide an interacting environment to carcinogens. It
is plausible that GNMT can also bind to AFB.sub.1.
Example 5
AFB.sub.1-Induced Cytotoxicity Antagonized by GNMT
[0112] MTT assay was used to determine the percentage of survived
cells. To optimize the condition of cytotoxicity assay, HuH-7 cells
were treated with different concentrations of AFB.sub.1 for series
time course. As shown in FIG. 11A, the half lethal concentration
(LC50) of AFB.sub.1 was dependent on the duration of treatment. The
cytotoxicity effect of treatment up to 16 .mu.M AFB.sub.1 was
obscure within 24 hr. However, the survival rate of HuH-7 cells
dropped significantly after 48 hr of treatment, even in the group
treated with 4 .mu.M AFB.sub.1. The 72 hr LC50 of AFB.sub.1 was
about 12 .mu.M. To determine the effect of GNMT on AFB.sub.1
treated cells, we expressed GNMT in HuH-7 cells by infecting
adenovirus carrying GNMT cDNA. Compared to HuH-7 cells infected
with Ad-GFP control virus, the survival percentage of AFB.sub.1
treated HuH-7 cells increased slightly but significantly by the
dose of Ad-GNMT (FIG. 11B). Similar results were observed in
another system in which GNMT gene was transducted via lentiviral
vector (FIG. 11C). These results demonstrated that GNMT can
antagonize the cytotoxic effect induced by AFB.sub.1 treatment.
Example 6
Inhibitory Effect of GNMT on AFB.sub.1-DNA Adduct Formation
[0113] To determine the effects of GNMT on AFB.sub.1-DNA adducts
formation, we conducted a competitive enzyme immunoassay (EIA) was
conducted to measure AFB.sub.1-DNA adduct formation with antibody
6A10, utilizing a pair of stable clones from HepG2 cell
line-SCG2-1-1 and SCG2-neg and GNMT recombinant adenovirus-infected
HepG2 cells.
[0114] Cells were treated with DMSO and various concentrations of
AFB.sub.1 for 16 hr prior to DNA extraction. There is no obvious
cytotoxicity effect during the treatment. The quantities of
AFB.sub.1-DNA adducts in SCG2-1-1 cells were reduced to
approximately 50% of those in SCG2-neg cells (FIG. 11D).
Furthermore, GNMT over-expression by GNMT recombinant adenovirus
(Ad-GNMT) infection also reduced AFB.sub.1-DNA adducts formation in
a dose-dependent manner (FIG. 11E). Compared to Ad-GFP-infected
cells, HepG2 cells infected with 5 MOI of Ad-GNMT resulted in a
greater than 40% reduction of AFB.sub.1-DNA adduct formation in
both AFB.sub.1 concentrations. It observed that a decrease of
approximately 70% of AFB.sub.1-DNA adduct formation in HepG2 cells
infected with 50 MOI Ad-GNMT. Results were calculated by
AFB.sub.1-DNA formation quantity based on inhibition percentage. It
indicated from the test data that compared to the control cells
(SCG2-neg cells and HepG2 cells infected with Ad-GFP), the number
of AFB.sub.1-DNA adducts formed in GNMT-expressing cells was
significantly reduced. It demonstrated that GNMT have a protective
role in AFB.sub.1-treated cells by reducing AFB.sub.1-DNA adduct
formation.
Example 7
Generation of GNMT-TG Mice
[0115] In order to determine the effect of GNMT on AFB.sub.1
induced carcinogenesis in vivo, a human GNMT transgenic mice (TG)
model was established. The plasmid used to generate the GNMT-TG
mice was shown in FIG. 14a. The pPEPCKex-flGNMT plasmid was
constructed with human GNMT expression driven from the mouse PEPCK
promoter (Valera, A. et al., Proc. Natl. Acad. Sci. U.S. A 91,
9151-9154, 1994). The GNMT-TG mice were generated by pronucleus
microinjection of FVB fertilized eggs. Northern blot analysis
demonstrated that the human GNMT was specifically expressed in the
mouse liver and kidney (FIG. 14B), as expected.
[0116] The expression profiles of GNMT in GNMT-TG and wild-type
mice were determined by RT-PCR and western blot analysis. As shown
in FIG. 12A, the mRNA expression level of GNMT increased by age in
wild type mice, and came to the plateau at the age of seven
week-old. The gene expression levels of GNMT mRNA in GNMT-TG mice
were higher than the wild-type mice, specifically at 1 and 3
week-old. In addition, western blot analysis showed that the
protein level of GNMT in male wild-type mice at 1-3 week-old was
under the detection limit, while that was detectable in female
wild-type mice at 1 week-old despite in a low expression level. By
contrast, the protein amount was higher in both male and female
GNMT-TG mice at the age of 1 week-old (FIG. 12A). These results
indicated that the expression of GNMT in GNMT-TG mice is higher
than in wild-type mice at 1-3 week-old. Moreover, we detected GNMT
enzyme activity in GNMT-TG and wild-type mice liver lysate were
detected. GNMT enzyme activity in GNMT-TG mice was significantly
higher than wild type mice liver at 9 and 11 week of age
(p<0.05) with one exception: male at 9 week-old (FIG. 12B).
[0117] In this test, GNMT-TG mice were not placed on the specific
diet. The gene expression levels of GNMT in GNMT-TG mice liver were
higher than in wild-type mice, specifically at 1 and 3 week-old
(FIG. 12A), while it came to the plateau at the seven week-old.
Example 8
AFB.sub.1 Induced Liver Tumor Formation Blocked in GNMT-TG Mice
[0118] The GNMT-TG and wild-type mice were charged with AFB.sub.1
intraperitoneally. Mice were sacrificed at 11 month-old. The
overall incidences of pathologically confirmed hepatic tumors for
male and female mice are showed in Table 8.
TABLE-US-00009 TABLE 8 The liver tumor formation of the 2 genotypes
mice treat with solvent or AFB1. No. liver/body mice with Gender
No. weight % nodules ALT AFB1 wild M 6 4.7 .+-. 0.3 4/6 (67%) 134
.+-. 112 type F 21 4.0 .+-. 0.7 0/21 (0%) 109 .+-. 97 GNMT M 7 4.5
.+-. 0.9 0/7 (0%) 61 .+-. 37 F 10 3.7 .+-. 0.4 0/10 (0%) 100 .+-.
73 Solvent wild M 6 4.1 .+-. 0.4 0/6 (0%) 55 .+-. 23 type F 7 3.8
.+-. 0.2 0/7 (0%) 54 .+-. 16 GNMT M 7 4.7 .+-. 1.0 0/7 (0%) 48 .+-.
12 F 6 3.8 .+-. 0.5 0/6 (0%) 40 .+-. 60
[0119] There was no liver tumor formed in both male and female
GNMT-TG mice treated with AFB.sub.1, while 4 of 6 (67%) male
wild-type mice treated with AFB.sub.1 developed hepatic tumors. No
tumors were observed in mice treated with solvent (tricaprylin).
Serum alanine aminotransferase (ALT) levels were measured in order
to monitor liver function of GNMT-TG and wild-type mice at 11
month-old. The mean levels of serum ALT in the male wild-type mice
were higher than male GNMT-TG mice in AFB.sub.1 treated group, but
there was no difference between the female GNMT-TG and wild-type
mice. Pathological examination revealed the presence of dysplasia
and HCC in the male wild-type mice treated with AFB.sub.1 (FIG.
13A), while a normal pattern was observed in AFB.sub.1 treated male
GNMT-TG mice (FIG. 13B). Immunohistochemical staining demonstrated
that the GNMT was expressed abundantly in the cytosol of the normal
liver cells (FIG. 13D), while it was diminished in the tumor cells
(FIG. 13C). This phenomenon is confirmed by western blot analysis
(FIG. 13E) which is also observed in human HCC.
[0120] In the AFB.sub.1 challenge experiment, the mice were
injected with AFB.sub.1 at the time that the expression of GNMT in
GNMT-TG mice was higher than wild-type mice. The results showed
that there were no liver tumor formation in both male and female
GNMT-TG mice, but 4 of 6 (67%) male wild-type mice had liver tumor
formation treated with AFB.sub.1 (Table 8). In the example, the
percentage of liver tumor formation in male wild-type mice was
increase from 10% to 67%. At the same challenge procedure, the male
GNMT-TG mice were no liver tumor formation. Moreover, though the
level is relative low, GNMT protein is detectable by western blot
analysis as early as 1 week-old in female mice liver. Those GNMT
protein exerted a protective effect and prevented AFB1 induced
liver tumor formation in female wild-type mice (none of AFB1
challenged female mice developed liver tumor). The test also
demonstrated that GNMT can provide a protection effect against
AFB.sub.1 induced liver tumors formation in the male GNMT-TG
mice.
[0121] Further, pathological examination confirmed that the liver
tumor formed in the wild-type mice treated with AFB.sub.1 is HCC
(FIG. 13A). The results from immunohistochemistry staining and
western blot showed that the expression levels of GNMT in tumor
tissue were decreased (FIGS. 13C and E). This result was consistent
to our previous findings in the human HCC. It also suggested that
the regulation of GNMT during carcinogenesis in mice is very
similar to those in human. Therefore, GNMT is helpful for the
prevention of carcinogenesis.
Sequence CWU 1
1
8129DNAArtificialprimer of PCR 1gcggcggccg catgctggtg gaagagggc
29220DNAArtificialprimer of PCR 2ttgcagtctg gcaagtgagc
20320DNAArtificialPrimer of PCR 3gttccttgcg cagctgtgct
20420DNAArtificialPrimer of PCR 4cggccacagt cgatgaatcc
20530DNAArtificialprimer of PCR 5gcgctcgagt cagtctgtcc tcttgagcac
30620DNAArtificialprimer of PCR 6gtggggcgcc ccaggcacca
20724DNAArtificialprimer of PCR 7ctccttaatg tcacgcacga tttc
24814777DNALambda phagemisc_feature(5953)..(5953)n is a, c, g, or t
8tctctgtgag ttcaaggcca gccaagacta tacagagaaa ccttgtctca aaaaacaaac
60aaacaaacaa acaaaatgca ccaccatgcc tggttcccca gttttaaaaa ccctaagtaa
120ataaaagata ccacttaaga tgctgagatg agatttgaaa aaagtctagg
attgataggt 180ttacccccca aatgctttga tatgtggcat tggatttact
tgaaattccc aaatacagtt 240tctcctctct gctttgtctc cacagggact
gatgtagccc agatttgcct tgaagtctat 300gtaccctaga atatctttga
actccagctt ttctgttctc agctgccaag tacaggactt 360acaggcacac
accatcacac tcagcctcga gaatctttct gagcatagtg tcagaggcct
420gtgtgtaggg acattgagtc cccaagactg agcctggtca gaaccatttg
agattggcgc 480tgtgctaaca aaagtttaaa aatgtgggct ggagagatag
gtcagccatt aaaagcccta 540tctgcagcta gggtcgtagc gcacactttt
agccctagcc cttgggaggc agaggcagtt 600ggatctctga gttcgaggcc
agcctggttt acagagtgaa ttctaggaca gccagagaaa 660ccctttctca
caaaaaccaa accaaaccaa acaaaccact tggtcctctg tggggttcag
720tgtcctcagc accacccagt ggctccccac tgtctacaac tgcaggttca
gggtatctga 780agctctcgat tctgggctcc atgggcgtca gacacatact
gacatctatg aagtcaaaac 840agccataccc actataagaa ggttaagtca
cacaactgtg ggcctccttc catctccagg 900gttcaccctg cattgggaag
gcgaagggcg ggcgggcggg cgggatgctc cctgtcttct 960ctcttctttc
cacgtaacga gtgttcacag agttggaagt gtgtgggcag gcggggcagg
1020cggggcaggc ttctcgttgc tcacacgcat cttccagagc cgactttagc
ccggctggga 1080tcttgtcctt ggtggggtaa aaagggtgag cgggtaacac
gaaacggacc ctcaggttat 1140acagtgacgc tagcccgagc gggcgccatg
agcaccttcc ctgccgaccc ccaactttcc 1200tctctgaagg tgctcccatg
gaaagcagat ggaaggcttg ctatgtctcc cacattgttc 1260actaattcat
tgagcactaa taatcctcct gtctcacctt actacaccca gcctttaaga
1320gattgcagag tataaagccc atggattgga aggcccaggc taccttagga
ggctcctggc 1380tggtatgggg gaggggaaga gagacacgcc tatctccagg
tgtactcttg tacttggatg 1440agcatctttc tcaaagactg ttcataatgt
cccgacacag tggctctctt tagcggagtg 1500ttccctccac taaatgataa
gcagtgttgt gtagacactg tgctgggctg ggcatgtaat 1560aattctgttt
tgggtggatt tcaggcttct tccccttcct ccttgggtag gggtggagtg
1620atctctcatt gctttacttt gtgagttcct cccggccacc ctggcgagtc
ctatctgacc 1680tgtcttacta agccaggtcc aggattgctg tgctgagcca
tcctcaaggc agaggcagca 1740agcctggctc tgggcccagg gcccagcagg
ggcgtgtccc tgctctcacc tgccattggc 1800caggtgggac tgccttgcca
gaggattata agtgcggatc gcgtggcctg agagccaggc 1860gccggtcagg
atggtggaca gcgtgtaccg tacccgctcc ctgggggtgg cggccgaagg
1920gctcccggac cagtatgcag atggggaggc cgcacgtgtg tggcagctgt
acatcgggga 1980cacccgcagc cgtaccgcag agtacaaggc gtggttgctt
gggctgttgc gccagcacgg 2040gtgccacagg gtgctggacg tagcctgtgg
cacagggtga gtccaaacgg gccggcctgc 2100ttaggccagt ctgggcagcc
tctgcgggct ggagcctggc aggcagtact agggggtcaa 2160gagcccttgc
tcatcagggt agtatgggca tgggagagca tctgccgaga aacagtgcgg
2220gaaggggcca gggtgtgcgc agagaagtat tcatgggaca gagcaaaatc
cgagctttgg 2280gtcaaggtgg atccttggct ccggcgctgt tcctcctgcg
attggcctct ggcagtgtga 2340acagaacatt tgtagcacgt ttgcacagcc
aagtgctgtg ccaggagttt gggtacgtgt 2400gttgcaagct gtaacaaaac
cccacgaggt gagcagcgct tttctccaac cagtagtact 2460aaagaaaaag
tcatttccaa gaggtaaaag gcccccattt ggtggagagg atacagacca
2520ggatagtgtc tggcccacac tcccgactca gcccagaact cacggacacc
tctgattgag 2580ttatttccag gagcaccagt atgccaggtt ctcaagtgca
cttttgtaga atgcctgact 2640atgatttaaa aacaaaaaca acaacaaaaa
gccattgagc cggtgagatg gtccgactac 2700taagagcccc ttaggctctg
gcagaagaca ggctctcagt gcctagcatc tgcatcaggt 2760ggctcataat
cgcctggaat tccgttccag ggggaggtta gcgctccctt ctggccgtgg
2820agggcactgt actcacatgt actacacata cagacataat taaaaataaa
aaaatgtgtt 2880gatgtcagga gtagtggcac atatatttaa tcccagcact
cagggactcc taaataggtg 2940gctctctgtg agttccaggc cctgcagggc
tacattagga gaccctgttt ctaataagag 3000gcagctgggg agatggcaca
tgttcttaca gaggacccaa atttggtgcc cagcaccctc 3060tctggggtct
gcagacacaa ggtatgcaca tgatgcacat attcaggcac gacacatgaa
3120attaaatttt gaaaaaaaat ttttttgaaa cagaatctct ctttgtagcc
ccagctgtct 3180tggaactcac tgtttaaacc aggctaaaaa aaaaaaaaaa
aaaacaaaaa ctctcaaaaa 3240aattttttgc tacctgataa ggaaaattct
tttttttttt taaagattta tttattatta 3300tatgtaagta cactgtagct
gtcttcagac acaccagaag agggagtcag atcttgttac 3360ggatggttgt
gagccaccat gtggttgctg ggatttgaac tctggacctt cagaagagca
3420gtcgggtgct cttacccact gagccatctc accagccccc aggaaaattc
ttaatgggtc 3480acttgagacc agtagagatg gcctgggaca ggaagcagag
gaacggactc tggaatgtta 3540tgtctgggga aatgtcacac acatttggcc
tcccaggctc ctctgactgg tcctctagtc 3600ccccgtttct gacctcatcc
agagtggact ccatcatgct ggtggaagag ggcttcagcg 3660tgatgagcgt
ggacgccagc gacaagatgc tgaaatatgc gcttaaggag cgctggaacc
3720ggaggaaaga gccatccttt gacaattggg gtaaatctgt ctgtccaggc
cccccagaca 3780tgcacctcca gggtcactct ctgccctggc tctctcttgc
tgtggagctg aatggttttt 3840cctcagtgca cggtacagtc cctgtcaact
tcctaaaaag gaaggctttg gcatcagagt 3900aggattccgg ctggtaagag
caagtttgtt gtatttatgg tgctaggcat tgaacttggg 3960catgccaagt
agacccttga tcactgggct gcattaccag cggtttttat gcttttcccg
4020tgttgcctgg cttggctaag ctagatttaa gtgatgctcc tgtctaacag
agtctagagg 4080tgtacgtcaa gtcactgggc cctacagagc tacttttaac
tgtcttatca catcctgtct 4140ctcctcccta caaaagcccc taagtggagg
gctggctttg ccaactgtgg ctctctccat 4200ccctaatgag tccccatgtg
tctctgggag gaggtggttg cttctgtgct gtgctataat 4260tctcgactcc
ccccccccaa cccctgccct ggcctccagt cattgaagaa gccaactggt
4320tgacgctgga caaagatgtg ctttcaggag atggctttga tgctgtcatc
tgtcttggga 4380acagttttgc tcacttgcca gactgcaaag gtaagctggt
ggccttggct gtggctcttg 4440ccttgagctt tcccacgtcc gaagatggct
ggcctgctgc cctgataagg cagacctgca 4500ttttgcctgt cttctttagt
gctggcactt gggagggaga caggggcggg aggtggtgct 4560ggaggcctaa
gcagatggtg tctgtggccc tgccgggaac ccaggtgacc agagcgagca
4620ccggctggca ctaaagaaca ttgcaagcat ggtgcggccc gggggcctgc
tggtgatcga 4680ccaccgcaac tacgactata tcctcagcac aggctgtgcg
cccccgggga agaacatcta 4740ctataaggtt gggctgcccc ccaatgggaa
gcgggagctg gagggccaga agccccgtgg 4800tggctcaggg aatgacgcaa
cccatgccct cagagtgacc tgaccaagga cattacgacg 4860tcagtactga
cagtcaacaa caaagcccac atggtaaccc tggactacac agtgcaggtg
4920ccaggcactg gcagagatgg ctctcctggc ttcaggtatg acgtggtttg
gcaggagaga 4980ggtggtggtg gtggggagac ctgaggccag cagccctcat
atctggctgg gggccctgtt 5040gcagtaagtt ccggctctct tactacccac
actgtttggc gtctttcacg gagttggtgc 5100gagcagcctt tgggggcagg
tgccagcaca gcgtcctggg tgacttcaag ccctacaagc 5160ctggccaggc
ctacgttccc tgctacttca tccatgtgct caagaagaca gactgagttt
5220ctccggctcc cagaagccca tgctcaggca atggccccta ccctaagacc
atcccctaat 5280gcagatattg catttgggtg cagatgtggg ggtcgggcaa
acggagtaaa caatacagtc 5340tgcattctcc aagcctgtgc ctggtgtttc
ttcagaagta acgtgttttt atggtgtccc 5400ccccccccaa tcccccctca
agacctcagc ctcccataac cttgcctttt gcaccgcaac 5460ctctcacaag
accagtagga ggcagtatac actttatttt ctactccaga actagtgtct
5520ccgcacaggt cacaggctgc caagccccta gctgccagcc ctgcctctcc
tcctgagcac 5580taggggcccc cactagggct cacaagggat ctctgggagg
aggcagcctt caggcgacag 5640gcgggaggta ccttcagtca gggggcatag
gcagacggca ggcaggcagg caggcaggca 5700gctcctagca ggctgcaaac
ttgcgttgga tgcgcttgta tcgcaacagc tcctgctcac 5760tgactgaggg
ctgcagccgg gctgcggcct gcaacaggtc ctccatggtg agcagcagtg
5820ctgagctccg cagctctagc cctaaggggt ggaaaggaag actaaggcca
gagccccagc 5880cctagctgaa gtacagaggc tgcagtctat gcctaggtag
gagagccact ccgggggggg 5940gggggggggg acngcacggg acgacgacga
acgacacccc ctgctcagcc attcctctgg 6000aattcagggc caggactgtc
tccctagctt tatggttccc atccagggct ggcaggtggg 6060ctcaccttcc
tctaggtctc gaaccctgcg tttgagggca gtcatcatgg cgtcagagca
6120gagagaatag agatctgcac cagtcagctg tgggtgggca gcaatccagc
acgtttgcca 6180ggctcacaga gggctccagc ttgaacctat cacacccaca
ccagggatgt taaagctggc 6240ctcacctcct ccctcagaag tcatgatgac
tcgcacacat cccctaactg tctaagcccc 6300caccccagct ctctgactac
gcatacttcc gtgtgatggc gctcagcaca cgcagctggg 6360aggcccggtc
ctcactcgcc cctacaaaca ccagcttgtc aaatcttcag agagacagag
6420acaggaagtg ttagggctgc caaccatgag aggtgaagga tgcccaggat
agaggttaag 6480gcgcaaagga gccttgtgga ggggagaggt ccagggtccc
tacctgccag gccgcagaag 6540ggcagggtcc aggaggtctg gtctgttggt
ggctccgatc acaaacacat cctgggtgct 6600gtggagccca tccagctcgg
ctaggagctg agacacaact ctgtacggga ggaaggcggc 6660aaaggttgca
gagtccttgc tgtgccctca ggccagctgc gcttctcttg aagactgctt
6720cagcaaggtc ccttgctaag ctctgcccca tgtccctgcc acactggtga
gcctcctcaa 6780agttccccac tcctttttag cttcccttct agctctgaac
tagaagggca ggaacctctg 6840tcttagaggc ctgggctgga gactgatgtg
actcaagata tatacacaga catatataca 6900tatacgcata tatacatatg
tatatgtgtg tgcatataca cacacacaca catacacaca 6960cacacatatc
tcacatccct ccccagactc cagaacccct gacctgtcca tcacccctcc
7020agaatctcca ctccgtccgc ggcttggagc taaggaatcc agttcatcaa
agaagatgat 7080acagggagca gcagccctgg ccctggcaaa cactgagaaa
gaggctcgca ggagagtggt 7140gtcaaggagc ttcaagtcag gggagaagga
tggcacatgt gcccgacagt tcctccccgg 7200ccattgccca ctgagctgac
caggagcagc cactggggaa gatggtgttg gggaagggcc 7260cgagactcgc
tcgctctccc cgctcactca ctcaccttcc cggacattct cctcgctttg
7320gcccacatac atgttgatga gctccggtcc ctttacactg agcagtggga
tggacggaca 7380aatgagaccc atcagacgtg ctgtcctccc ctcccctccc
accacctgcg acaactcccc 7440tccccctttc tgcaccggct acagctccca
gcctcccaca tttctcccca gccctgccaa 7500cctgaggaag gtgaggctgc
actcagtggc tacggccttg gccagcaggg tcttgccagt 7560gcctgggggc
ccatggagca gaaggcctga tcgtcttagg cccaggctga gcagctcagg
7620gtgttccaga ggcagctgga tggtttctag gatctccttc ttcacatcct
gcagcccgcc 7680cacgtcgtgc caggacactg aagggatcta gaagacggac
cagtggtcag ggacgtgtca 7740gcagagagag cccttgcccc cccccccccc
cacggaccag gattctcacc ctgggtgctc 7800ccacagcttg ggagtgagct
gtctgcagtt gatctaatgc ctgcccgaag tcctcagcca 7860gcaaaggaaa
gccagccaca cacaggtccc cctcatcctc ctcactcaaa ccacctgccg
7920agctgcaaag aaacacgatg ggggggaggg ggtaggctta cagctgtacc
ctcagcatct 7980gggaccagca ctcatggggc agaggcaggt gatctctatg
agtccaagac tagcttggtc 8040tccacagtga gtccaggcca gccaaagact
acatagtgag tccctgcctt aaggataaaa 8100aaggaccggg ccaatcaggg
tatggattaa ggctctaaca aacgagagac agagagagag 8160agagagagag
agagagagag agagagacag agacagagag agagagacag agacagagag
8220agagagagag agagagaccc cctctctagc agggtcccat tgccttccca
gccacctggg 8280atgagtctgt cttaccccga ggctctgatc ctggtgcagg
ctgcccggca ggtatgggtc 8340agaagggcat agaggtcccc caccacaaag
ccctagggag ttacacaaaa ggacacatgg 8400gtagggcagg gcacaggagg
cagaggccaa gtctggcttc cagctggagc agaggctttc 8460gtggtttcca
gtctcacggg gcagcagaaa cgaggccctg tgtgacaggg accctgtgac
8520ggcatagtgt caaggcccct gtgcagacac tcaccgcaca gcgccgtgcc
agctggggca 8580ggttaacctc ctggcctaag ggaaggtggg cagtgagggc
ctgcaggata ctgagccgtt 8640gagcctcaga cagcactggc acctctagct
catgaggaaa tgccgtctgc acatcggtgg 8700gcaggtcctg gacacggctt
gtggtagcca ccaccatgag aggagggcac ctgcagacag 8760ggacgtgggc
aaggtctggc atggctggca gctcctgcct ttggtgtgca ggaatgggga
8820gggttggtgt caggactggg tagggatggg catcatccag agtccattat
cccctgatgg 8880tggggacacc atgaagggag accctgcttc acacgggggg
ggggggtgct cactggggac 8940aaaagcttca accttgtgtt cccttcaaaa
tgttcaacct tgtgttccct tcaaaatgct 9000agaccgcacc ctcctacctc
tggacactgt aaattggccc caaggacagg taggtgagag 9060ttctgagaag
ctagcagggt gtgaggaatt gagggctgaa taggggggtt cccttcataa
9120cctgcctgct ctatacatac ctgctgagag cgtcctcatc aaggaggaga
tgacggagtg 9180tggccgcaac acgggcatcc tcacccagtc cgtctcggtc
ccggcccagg aggtccacag 9240ctgtcaagag gaggactgca ggcctgcagc
ggtgggcccg ggagaaggtg gcctgcagct 9300ttgtctccac ggccctactg
ctgtctgcac agaggctgga gcagggcacc tggggaagag 9360accagatggg
tcagcatgaa gggagaaggg tgactaggcc atctttgtga gtagggacag
9420gtgaaggagg gaccctggcc aggtgggagc tttggttcac cctatccttt
gcctggagtg 9480tgcagccttg aaacccctcc ttcctgagag gagctctgag
tagctgagca tcaaggggct 9540gtatactggc ctgaccactg ggcgagctct
gagtagctgg gcatcaaggg actgtacact 9600ggcctaacta ctgggctgaa
gcattagggc tgccaaatgg taatgaggga acccaggaga 9660agagagagca
aggcagagtc tgagctcagt ggaagcggct ggggctcgtg ccagaggcct
9720tcaccttcag caaatggagc ccaaggcggc tgcatgcagc cgtgacggct
gtggtcttcc 9780cgctgcctgg gggaccctgc agaagcacac agctggttcc
tgtgagcaat gttcctctgc 9840aattagagaa tagatattgt ctcccatttt
tctgcccaaa tgttaagacc ctagtaagac 9900agggacccct ggatttcata
agtgtcccat ctctccttgt gcctccctaa cccccatcca 9960ggcagtgtcc
acagtccgag gccttgtgtc atgccatctc tggtgcccgg gcagaagcac
10020tgagaacatt ttgcagccgc agaccagtta gccttcagtg aagcccagct
ggataggtct 10080ggatctagac ttgggccact ggagtggggc actctgctct
ttgggagatg tagtgggaaa 10140ggccaggacg gatctcgggc tccccaagca
gaaaagcagc tttaggatca gctgctgggg 10200agtctgcttg tgtaagtggg
tagcctgtgg cctggactgg gggcctggct tgtggatccc 10260cgacttgggg
tagactactg gaagtaattt ccccggatgc tgctctacag agatgctctg
10320agcaggcccc tctcacggcc cgaattcaca cctcccggcg tggcctcact
cacccgggct 10380gcaggtgagg cttcaggata gcacacagct catttaccaa
ggcctccagg cctggaggag 10440acaagctgtc ccagggtggg gaccttcctg
aaggcagcga tggcacatga ctcagggcgg 10500tgccagcctg tgaaagcagg
aagcccagca aggttctaca gggtccctac ccctttgacg 10560ctctcctccc
aggaacccac ggccttacca ggtataaaga ggtgtgggtg gtatcagcca
10620gaaaggcact ggctggcccc tctggggctt ctccaaccgt tttcttcact
ttgaaaaaca 10680tctcccgcca cctgtaagaa aggagggtca gggaaccagg
accgagcgtc cacatgtgca 10740cctcttgggg agtgggtagc tttctcacag
aggaccaact taggagccct gagactgatg 10800aggcaaggcg gggcccccag
agggctgggc agggaggaag tgggggatac agaagggcag 10860ccccctcatt
acagccatca aatagcccag gaagggcatc tctctggcca tatagcctag
10920ccacctactc taaaaggtga agccaccatc attacacaca gcaacctggg
gccgtttccc 10980tcagtttctg aacagtctct ccctcctaca gatcttcacc
caccagggtc ccagatctca 11040gacacaagtg tcccttcatt tttattcaac
aattcaacta attctaacta aagttagagt 11100ctttaatcat ggtgtctaac
tctagtcctg gggagttgag gcaggcggga aggtcttaag 11160acagaccagg
ctgggctacg aagtgagagt gattctctgt tccagaatta cctgtgaagc
11220ttgcttaatt aattaattaa ttgtatacga gtcttcagac acactagaag
agggtaccag 11280acctcattac agatggttgt gagccaccat gtggttgctg
agaattattg aactcaggac 11340ctctggaaga gcagtcaggt attttgtttt
tgttttccgt ttttttgaga cagggcttct 11400ctgtatagcc ctggctgtcc
tggaactcac tctgtagact aggctggcct cgaactcaga 11460aatccacctg
cctctgcttc ccaagtgctg ggattaaagg tgtgtgccac ccccgcccgg
11520cttgagcagt cagtgcttaa ccactgagcc atttctccag ctcccttgtg
aaactttaaa 11580aataagtata tttcatgcat tctgtgagtc cactcaacag
gctggtgagt gccaggatca 11640ggggacaagg caagaatgag ggagtgactg
ttagtgccca tggtttgtgt ggctaggaga 11700gctaaagaac cagagctatc
tgcaccctcc tgtacccacg gcaaaccctc tgagccgtgc 11760cactcctgta
cccacggcaa accctctgag ccgtgccact cctgtaccca tggcaaaccc
11820tctgagccgt gccactcctg tacccatggc aaaccctctg agccgtgcgt
acacttgttc 11880attctggaag atgtacactt gttcattata ggagacattt
aatgtctcct agaatagact 11940caaactcata acctcaaaag tttagaagag
gctcaagtgg tggtgtaaac cctgaatctc 12000aacactctgg aggcagaggc
aggaggatct ccgtgaatcc gaggccagcc tggtttacac 12060agcaagttcc
aggaaagcaa ggactacata gaaagaccct gtctcagccg ggcagtggtg
12120gcgcacgcct ttaatcctag cacttgggag gcagaggcag gtggatttct
gagtttgagg 12180ccagcctggt ctacagagtg agttctagga cagccagggc
tacacagaga aaccctgtct 12240cagaaaaaag aaaaaggaaa gaaaaaagaa
aaaagaaaag aaagaaaaaa gaaagacctt 12300atctcaaatt ctcccacccc
caaaaaagtc taagaggctg aatcatcatc acacacacac 12360acacacacac
acacacacac acacacacac acacttactt caaacattaa tgctggggcc
12420acccagatgg ctcagtaggt aaaggcactc gctagcaggc ctgactacct
gagttcagtc 12480ctgagaattc atagggtagg agcgaaccga cgcttacagc
tcgccttctg acctctgtaa 12540gctgctgata cacatttttt tcattcaaga
taggtatttc tctgtgtagc cctggctgtc 12600ctaatacttg ctttgtagac
cagactggct tcaaattcat agacatccac ctgcctgtac 12660ctccagagtg
ttggaattaa aggtgtgtgc caccatgcct gtcccaatac acattttata
12720aattaaatgc aatttttagc aaaataagct ggatggtagt ggcatatgcc
ttttcccccc 12780acattaactt atttattcac tttacattct gataacaggc
caccttctcc acccagtccc 12840ctctcataca gcttctccac tcaaccccct
ccccttctcc tctgaaaagg aggaggtccc 12900ccttgggttc caatccaccc
tgacatatca agtcactggg tggcgcacac ctttaatcac 12960agcactcagg
aggcagaggc aggtgaatct ctgagttcaa gactagattg gtctgcagag
13020tgagttccag gacagctagg gctacacaag caaaccctgt cccaaaataa
aaaaatagaa 13080tataagtaat aataataata atgatgataa tattaaatac
tacatcatcc tttgaaagga 13140aaagatggca caagccctag tttcatctcg
attctatcta actggtgtgg ggtgtgactc 13200tgtattacat gcagtctgga
agcacagcca gctgagcggt tattaaccag cagcacggag 13260actcagagat
atttacgcta ctcactcgga agacggtaag tgcatgtagc agccagtaac
13320ttaatctcct agagacatca ctagatagag ctcctttaac tcctcggcag
ggagacacca 13380tggaacagag cacactgtag gcctctctaa gacccaccag
gccgggcagc acgcctttaa 13440ttccagcact tgggaggcag aggcaggtgg
atttctgagt tcgagtccag cctggtctat 13500agagtgagtt ctaggtcagc
cagggttata gagaggaacc ttgtttggga naccccaaaa 13560aaaaaaaaaa
aaaaaaaaaa aaaagaccca ccaggatgca aactctttta cttaaaatgt
13620tttagttgcc attggcagca acagtggctt caaagtagac aaaggttttt
gttacaaaat 13680tcttgtaagt tgggctgggg agatggctca gctggtgaag
tactacccaa acaagtatga 13740agagcagtgt tcagcatcct agaaccacag
aaaagatgcc tgtatgacag cctgcccatc 13800atcccagcaa gcacggaagg
agacagggaa tgaccagggc aagctgtcca cagagtgaga 13860gaccctgctt
caaaatgcag tcagagtgca gttgaaggca ctgatatcca ccaccatggg
13920gcctccaggt gtgcatctgc acacccatgc actcacgctc acaaacccaa
aatggatttc 13980ctctgagtta ttccagttgg cagtggctgt ggttaaaaac
taaaatgtaa ggagaaagat 14040gcacagtgca tgaagcctag agcttggtgc
gcaacagggg aaagatgcct gtgagctgtg 14100ctctagctca gaaaaccacc
tcattgtggc ttaggtaggg tacagcctcg tgcctctatc 14160aagcatcctg
tcggaacatt ctggaagcac tgcataggga agcaatgagg cgcagctctg
14220agggaagtta cgaaggaacc accttcttca gtaggggatt ggtgctccac
ttcagagagc 14280agaaaggcca gtaatctaga ccctaccaca gccctgctga
gggagcaggg gccagagccc 14340ggtggggcac acagaagagt
tgtgactgtc tgcaaagtag ctttggtttg tgaagcagag 14400tacagaggac
gctgaagagg gtccaaccat gatgaaactc ctttctaggg aatataagat
14460ctctgggttt cccttatcaa gtctgagtgg actcagcggt ttacggtacc
gagcctgcta 14520ggcacccaga ggtgggcagg tttcaggagc tgggtcagca
gcagtgctga aaatggcctg 14580ctgtgtctca gaatgcctga tcacctgttt
ttctttgtgg gcactaagtg tgggaggtct 14640gcaggtcttc tacagaaaac
aaagtgaggg actgttggta gaatgactgc ccagctccta 14700aaagtcttag
gactcagatg gtaggtagaa gcaggaaggt cagaagttca aggccatcac
14760tcacacagat tttgagt 14777
* * * * *
References