Patent Application
20090269750
The present invention relates to a diagnostic cancer marker using variation in the gene expression of a granulocyte colony stimulating factor (G-CSF) and a method for preparing the same, and more specifically, relates to a method for diagnosing cancer and/or assessing the state of cancer progression using an oligonucleotide having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a G-CSF gene as a diagnostic cancer marker.
Cancer diagnosis is generally achieved by (1) morphological analysis using microscopes such as an optical microscope or electron microscope, (2) immunohistochemical assays which detect proteins specifically expressed in cancer tissues (Iran, Biomed. J., 3:99, 1999; Lancet, 2:483, 1986), or (3) molecular diagnosis which analyzes abnormal biomolecules found in cancer tissues, such as mutated genes. In comparison with the molecular diagnosis, the morphological and immunohistochemical diagnosis requires much longer time and higher cost. Since the molecular diagnosis has a relatively simple procedure and a short time to yield results, it has become a main subject in developing novel diagnostic methods for cancer. Recently, Health Digit Inc. developed a protein chip system for diagnosing various cancers, and gained on approval for clinical tests from the Chinese State Drug Administration (CSDA) for the first time in the world (www.health-digit.com). However, the protein chip system does not use only one biomarker to diagnose all kinds of cancer, but uses 10 or more proteins as biomarkers.
To effectively apply such diagnostic methods to cancer diagnosis, it is most important to select and use cancer diagnostic markers capable of more accurately and easily detecting cancer incidence. Several genes (Steve, M. et al, J. Clin. Oncology, 20:3165˜3175, 2002; Sridlhar, R. et al., J. Clin. Oncology, 20:1932˜1941, 2002) and proteins (Goessl, et al., Urology, 58:335˜338, 2001; Zhou, et al., Breast Cancer Res. Treat., 66:217˜224, 2001; Korea Pat. Publication No. 2001˜0061173) have been reported as diagnostic cancer markers, and some of them are being clinically used for diagnosis of cancer. Among conventional cancer biomarkers, CEA, BFP, TPA and IAP, which have low organ specificity, have low sensitivity, thus generating false positive data. Also, the biomarkers which have high organ specificity, such as AFP, PIVKA II, Esterase I, CA19-9, CA50, Span-1 antigen, CA15-3 and BCA 225, are useful only for target organs.
Many researchers have attempted to find genes having diagnostic applications, in developing diagnostic cancer marker candidates showing different results according to pathological and physiological condition using microarray technology (Liu, H. X. et al., Nat. Genet., 27:55˜58, 2001; Wilson, C. A. et al., Oncogene, 14:1˜16, 1997; Weissensteiner, T., Nucleic Acids Res., 26:687, 1998; Zolezzi, F. et al., Am. J. Med. Genet., 71:366˜370, 1997; Mottes J. R. and Iverson, L. E., Neuron, 14:613˜623, 1995; Crook, R. et al., Nat. Med., 4:452˜455, 1998; Jiang, Z. H. and Wu, J. Y., Proc. Soc. Exp. Biol. Med., 220:64˜72, 1999).
However, since diagnostic cancer marker candidates found by the above mentioned methods are mostly composed of expressed sequence tag (EST), they are just defined as a characteristic of data and thus it is difficult to select reliable specific candidates and to catch on the very genes from which they are originated. Specifically, the number of genes is known by human genome analysis and it is also known that many isoforms or variants are expressed there from to have biological function and its complexity. Therefore, it has become another big subject for the future to find out that, in which gene and condition variants throughout the whole genome are expressed and what their functions are. These various variants can be a good basis to figure out the correlation between the formation of abnormal variants among them and possibility of causing cancer (Cartegni, L. et al., Nat. Rev. Genet., 3:285˜298, 2002; Schweighoffer, F. et al., Pharmacogenomics, 1:187˜197, 2000; Blencowe, B. J., Treds Biochem. Sci., 25:106˜110, 2000; Cooper, T. A. and Mattox, W., Am. J. Hum. Genet., 61:259˜266, 1997).
The present inventors have also conducted studies for a long time to develop a new diagnostic cancer marker which can diagnose various kinds of cancers, consequently, confirmed that deletion of exon 3 region was specifically shown in tumor cells or tumor tissues during transcription of G-CSF gene, thereby filing an application regarding a method for diagnosing cancer using G-CSF mRNA, cDNA variants fragment or protein as a diagnostic cancer marker (WO 2003/027288 A1). In microarray which uses G-CSF gene fragment as a diagnostic cancer marker of the above application patent, any one or more fragments among exons 1, 2, 4 and 5 DNA fragments of G-CSF gene together with exon 3 DNA fragment of G-CSF gene are used as nucleic acid probes to detect G-CSF gene fragment having deleted exon 3 region among biological samples. This inventive method for diagnosing cancer, by detecting deletion of exon 3 region of G-CSF gene expression is one of the technologies which diagnose cancer using characteristics of gene variants, and is considered to be a useful diagnostic cancer marker candidate, since the variants appear in most cancer.
Meanwhile, most genes including G-CSF gene generally express many isoforms and variants, so, probe fragments fixed on a microarray must have high sensitivity in detecting the deletion of exon 3 region of G-CSF gene. Also, the expression of normal G-CSF or their fragments can exist together with that of mutated G-CSF isoforms in tumor cells or tumor tissues, thus diagnosis for cancer only by detecting the presence of exon 3 region of G-CSF in its gene expression can lead to loss of credibility or low sensitivity and, moreover, it has a problem in assessing the state of cancer progression.
Accordingly, the present inventors have made extensive efforts to develop a new nucleic acid probe for detecting G-CSF gene fragment not having exon 3 region which can satisfy the above requirement or solve the above problem, and as a result, found that it has remarkably increased high sensitivity in cancer diagnosis compared with other probes, when an oligonucleotide containing a nucleic acid sequence having the 3′-terminal end of exon 2 region of G-CSF gene linked to the 5′-terminal end of exon 4 region of G-CSF gene is used as a diagnostic cancer marker, and confirmed that the state of cancer progression can be accurately diagnosed by using an oligonucleotide containing nucleic acid sequence having 3′-terminal end of exon 2 region of G-CSF gene linked to the 5′-terminal end of exon 4 region of G-CSF gene together with an oligonucleotide having sequences of a part or the entire region of exon 3 region of G-CSF gene as a diagnostic cancer marker, thereby completing the present invention.
Therefore, the main object of the present invention is to provide an oligonucleotide for diagnosing cancer, essentially containing a nucleic acid sequence of a splice junction site having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a granulocyte colony stimulating factor gene.
Another object of the present invention is to provide a diagnostic kit for cancer diagnosis containing the oligonucleotide and a method for diagnosing cancer using the oligonucleotide.
To achieve the above objects, the present invention provides an oligonucleotide for a diagnostic cancer marker, essentially containing a nucleic acid sequence of a splice junction site having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a G-CSF gene.
Preferably, the oligonucleotide according to the present invention essentially contains nucleic acid sequences of SEQ ID NOs: 1 or 2.
The present invention also provides a diagnostic kit for cancer diagnosis containing the oligonucleotide.
In the present invention, the diagnostic kit for cancer diagnosis is preferably a diagnostic kit for assessing the state of cancer progression which additionally contains an oligonucleotide essentially containing sequences of a part or the entire region of the exon 3 region of G-CSF gene.
The present invention also provides a method for diagnosing cancer, the method comprising the steps of: (a) obtaining a G-CSF nucleic acid sample from a mammal biological sample; (b) amplifying the obtained G-CSF nucleic acid sample; and (c) detecting oligonucleotide containing a nucleic acid sequence of a splice junction site having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a G-CSF gene, in the amplified sample.
In the inventive method, the step (c) preferably contains the step in which simultaneously detects an oligonucleotide containing sequences of a part or the entire region of the exon 3 region together with an oligonucleotide containing a nucleic acid sequence of splice junction site having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a G-CSF gene, in the amplified sample.
Other features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.
The present invention relates to a method for diagnosing cancer and/or assessing the state of cancer progression, using an oligonucleotide which essentially contains a nucleic acid sequence of a splice junction site having 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 as G-CSF gene variants fragment generated after post-transcription process by genetic analysis method including a microarray. In other words, the present invention relates to a method for diagnosing cancer and/or assessing the state of cancer progression using G-CSF gene variants obtained by deleting an exon 3 region in G-CSF gene to link an exon 2 region and an exon 4 region, as a diagnostic cancer marker.
Exons 1˜5 of G-CSF gene are normally linked during splicing process in normal human body, however splicing occurs in a form of a variant not having exon 3 in tumor cells or tumor-progressing cells to produce mRNA not having exon 3 (
Molecular biological methods which are used in identifying both genes specifically expressed (or suppressed) in tumor cells and genetic mutation are exemplified by PCR (Bottema, C. D., Mutat. Res., 233:93˜102, 1993; Nelson, D. L., Curr. Opin. Genet. Dev., 1:62˜68, 1991; Pourzand, C. and Cerutti, P., Mutat. Res., 288:113˜121, 1993; Holland, P. M. et al., Proc. Natl. Acad. Sci. USA, 8:7276˜7280, 1991), Single-Stranded Conformation Polymorphism (SSCP, Glavac, D., Hum. Mutat., 19:384˜394, 2002; Strippoli, P. et al., Int. J. Mol. Med., 8:567˜572, 2001), DNA Sequencing Analysis (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74:6463˜5467, 1997), Protein Truncation Test (Hardy, C. A., Methods Mol. Biol., 187:87˜108, 2002), automatic nucleotide sequence analysis (Boutin, P. et al., Hum. Mutat., 15(2):201˜203, 2000), study of loss of heterozygosity (Yang, Q. et al, Clin. Cancer Res., 8:2890˜2893, 2002), study of microsatellite instability (Furlan, D. et al., J. Pathol., 197:603˜609, 2002), gene analysis using MALDI-TOF (Leushner J, Expert. Rev. Mol. Dign., 1:11 ˜18, 2001), gene analysis by hybridization (Wetmur, J. G., Critical Reviews in Biochem. Mol. Biol., 26:227˜259, 1991), gene analysis using DNA chips (Goessl et al., Urology, 58:335˜338, 2001; Zhou et al., Brest Cancer Res. Treat., 66:217˜224, 2001; Korea Pat. Publication No. 2001˜0061173), analysis using protein chips (Pharmacogenomics, 1:385˜393, 2000). Therefore those skilled in the art will understand that they can easily detect the existence of splice junction site of specific variants according to the present invention, generated in post-transcriptional process of G-CSF by properly using well-known molecular biological methods including the above mentioned methods. The present inventors found that the most effective probes capable of detecting the existence of the variants are only probe candidates capable of detecting the existence of splice junction site, thereby inventing a diagnostic method by which the existence thereof can be detected. However, among the above mentioned methods, the detection of specific variants generated during post-transcriptional process of G-CSF according to the present invention is preferably and easily performed by using PCR, hybridization reaction and DNA chip.
To perform cancer diagnosis according to the present invention, a G-CSF gene or variants thereof should first be obtained from tissue specimens or cells. Since a DNA sample for a specific gene is typically obtained from normal tissues or cells at a very small amount, the specific gene should be amplified by PCR and for such amplification, primers suitable for such amplification should be designed. In the present invention, to amplify a part or an entire region of splice junction site of an exon 2 region and an exon 4 region, DNA nucleic acid fragments to be used as primers in PCR for detecting the existence of the splice junction site is required. That is, the primers, as used herein, refer to oligonucleotides capable of amplifying a nucleotide sequence of G-CSF gene, comprising a part or an entire region of the splice junction site of an exon 2 region and an exon 4 region. Those skilled in the art will be able to easily design such primers. Those skilled in the art will be able to easily design such primers. Therefore, all primers capable of amplifying G-CSF gene variants comprising a part or an entire region of the splice junction site, which can be designed by those skilled in the art, are intended to fall within the scope of the present invention.
In accordance with an aspect of the present invention, there is provided a gene microarray or membrane to which a DNA fragment comprising a splice junction site having the 3′-end of an exon 2 linked to the 5′-end of exon 4 of the G-CSF gene is immobilized, which is useful for diagnosis of cancer. The gene microarray includes DNA chips effective in detection of a gene by hybridization including applying to a complementary oligonucleotide probe immobilized on the surface of a slide glass treated with a specific chemical reagent. Non-limiting examples of the membrane, which can be used instead of the slide glass in hybridization, may include all membranes capable of immobilizing DNA fragments; and preferably, nylon and nitrocellulose membranes.
Fixing the probes on the surface of a slide glass and a membrane can be easily achieved by the conventional technique known in the art. In addition, preparation of targets, hybridization and stripping will be performed according to the conventional techniques common in the art.
In another aspect of the present invention, there is included a composition for diagnosis of cancer, comprising a DNA fragment containing a splice junction site having the 3′-end of an exon 2 linked to the 5′-end of exon 4 of G-CSF gene and a diagnostically acceptable conventional carrier. In a further aspect of the present invention, there is included a diagnostic kit comprising a DNA fragment containing a splice junction site having the 3′-end of an exon 2 linked to the 5′-end of exon 4 of the G-CSF gene and a DNA microarray using the DNA fragment.
Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited to these examples, and it is obvious to those of ordinary skill in the field of the present invention that numerous variations or modifications could be made within the spirit and scope of the present invention.
The normal cell lines and tumor cell lines used in Examples of the present invention are given in Table 1, below. The underlined samples have the same result as those of the normal cell lines in Table 1.
The tumor cell lines listed in Table 1 can be obtained from the cell collection centers listed in Table 1. The tumor cell line, obtained from the cancer metastasis research center at College of Medicine, Yonsei University, was prepared as follows. After ascitic fluid was aseptically obtained from advanced cancer patients, supplemented with heparin in an amount of 10 units per ml to prevent clumping of cells and centrifuged at 400×g for 10 min. The precipitated cells obtained by centrifuge were cultured in a 25 cm2 culture flask. In case of containing a large number of erythrocytes, Ficoll-hypaque density gradient centrifugation at 800×g was performed to separate mononuclear cells from erythrocytes, and the obtained mononuclear cell phase was incubated at 37° C. under 5% CO2. After incubation for 1 day (16˜18 hours), the culture medium was centrifuged at 400×g for 10 min, and the precipitated cells were cultured in a new 25 cm2 culture flask. During culturing, cells were observed under a phase contrast microscope, and the culture medium was replaced twice or three times per week. When tumor cell colonies were formed, the tumor cell clusters were obtained by treatment with trypsin-EDTA or by obtaining colony or by using scrapers, or the fluid containing tumor cells was centrifuged to remove normal cells. The resulting pure tumor cells were stored at frozen states according to their passages.
Human leucocyte cells can be obtained as follows. After 8 mL of blood was transferred into 50 mL of Corning tube, 24 mL of RBC lysis buffer was added and the mixture was left to stand at 4° C. for 10 min, while stirring it occasionally. After centrifuging the mixture at 2,000 rpm at 4° C. for 12 min and confirming leucocytic pellet, a supernant was removed. If RBC (red blood cell) was left, said process was repeated. TRIZOL was added to finally obtain leucocytic pellet to separate RNA.
Total RNA was isolated from each tumor cell line, normal cell line and normal tissue using Trizol Reagent (Gibco-BRL, USA). 1 ml of Trizol Reagent was added to a tissue sample ground after quickly freezing using liquid nitrogen, followed by incubation at room temperature for 5 min. 0.2 ml of chloroform was added to the resulting tissue sample, vigorously vortexed for 15 sec and incubated at room temperature for 5 min. After centrifugation at 12,000×g at 4° C. for 15 min, the resultant aqueous phase was transferred to a new tube. An equal volume of isopropanol was added to the tube, and the tube was placed at 4° for 10 min. After centrifugation at 12,000×g at 4° C. for 10 min, the supernatant was carefully discarded, and the pellet was washed with 70% ethanol, followed by centrifugation at 7,500×g at 4° C. for 5 min. After being dried, the RNA pellet was dissolved in RNase-free water.
To synthesize cDNA from mRNA isolated from each cell line, and human-derived tumor and normal cell line, RT-PCR was performed as follows. 2 μg of total RNA was mixed with 1 μL of an oligo(dT)16-primer, and RNase-free water was added up to a final volume of 11 μL. This mixture was heated at 90° C. for 5 min, and placed on ice, immediately after completion of the heating. After putting 4 μL of a reaction buffer, 2 μL of 10 mM dNTPs, 1 μL of RNase inhibitor and 2 μL of reverse transcriptase into another tube, 8.5 μL of the RNA mixture was added to the pre-mixture tube, followed by incubation at room temperature for 10 min. The reaction mixture was incubated at 42° C. for 90 min, and then at 95° C. for 15 min. Immediately after the incubation at 95° C., the mixture was placed on ice to terminate reaction, thus yielding a cDNA sample.
In order to investigate whether a DNA chip can be used as a tool for detection of a splice junction site of G-CSF mRNA or cDNA, various DNA fragment probes capable of being immobilized on a glass plate was prepared as follows. On probe corresponding to a part of exon 2 of G-CSF, four non-overlapping probes corresponding to exon 3, and one probe corresponding to a part of exon 4, were designed to consist of 20 nucleotides each. Since two different G-CSF mRNAs (human G-CSFa and human G-CSFb mRNAs) are generated by alternative splicing in the exon 2 region (Tshuchiya, M. et al., EMBO J., 5:575˜581, 1986), two types of probes comprising a region corresponding to exon 2 were prepared, based on the two different G-CSF mRNAs. Nucleic acid sequences thereof are shown in Table 2.
To confer ability to be immobilized on a glass plate, when synthesizing all DNA fragment probes, a base having an amino group was inserted to the 3′-end of the probes using an aminolinker column (Cruachem, Glasgrow, Scotland), and slide glass coated with aldehyde residues (CEL Associates, Inc., Houston Tex., USA) were used.
After being dissolved in 3×SSC (0.45 M NaCl, 15 mM C6H5Na3O7, pH 7.0), the DNA probes were immobilized on the slide glass by accumulating the DNA probes using a microarrayer manufactured by the present inventors (Yoon et al., J. Microbiol. Biotechnol., 10:21˜26, 2000), and reacting for over 1 hr under about 55% humidity, and then leaving the glass at room temperature for 6 hrs (
Asymmetric PCR was carried out using mRNA or cDNA isolated from each cell line of Example 2 as a template under the conditions of denaturation at 94° C. for 5 min, 30 cycles of denaturation at 94° C. for 1 min, annealing at 50˜56° C. for 1 min and extension at 72° C. for 30 sec, followed by final extension at 72° C. for 5 min. A primer set used in Asymmetric PCR is as follows. A reverse primer was labeled with FITC for detection.
PCR products were separated on an agarose gel. From the result of electrophoresis, double strand DNA and single stranded DNA fragments were produced in each PCR sample (
After target products amplified by Asymmetric PCR was applied to the DNA chip prepared in Example 3, they were scanned using Scanarray 5000 (GSI Lumonics Inc., Bedford, Mass., USA). To predict results regarding probe, in case of the plasmid having no deletion of exon 3 in G-CSF gene, signals were detected by applying on the DNA chip. In contrast, in case of the exon 3-deleted G-CSF-containing plasmid, signals were detected by applying on the DNA chip, wherein the plasmids have nucleotide sequences of SEQ ID NOs: 26 and 27.
As a result, as shown in
To examine effectiveness of E2E4 for cancer diagnosis, a new type DNA chip 2 was prepared (
To examine whether probes constructed from exon 2 and exon 4 junction region were the most powerful; DNA chip 3 was prepared by designing probes from each nucleotide sequence in each region (
After target products amplified by Asymmetric PCR described in Example 4 was applied to the DNA chip 3 prepared in Example 7 (
As shown in
On the contrary, powerful candidates which can distinguish normal cells is a probe (SEQ ID NO: 8) from exon 3 region, however because signals are shown in almost all samples among cancer samples to which this probe is applied, distinguishing between normal samples and cancer samples is impossible using the existence of this probe. Also, because probes from different site of exon 3 region doesn't show signals in both normal sample and cancer sample due to their weak sensitivity, distinguishing between normal sample and cancer sample is impossible using the probes.
Target sample was amplified by Asymmetric PCR using a plasmid having exon 2 region of type A as a template as described in Example 4 and the target sample was applied to DNA chip 3 (
Total RNA from each cancer cell lines, normal blood and normal tissues was isolated using TRIZOL® REAGENT (GIBCO-BRL, USA). In case of blood, it was isolated using TRIZOL® LS REAGENT (GIBCO-BRL, USA). To prepare a sample, blood and LS REAGENT are added in a ratio of 1:3. According to case, blood sample was previously diluted in a ratio of 1:1, then REAGENT can be added in a ratio of 1:3. 0.75 mL of TRIZOL LS Reagent was added to 0.25 mL of blood sample (or diluted blood sample) and RNA can be extracted according to protocol. In case of tissues, 1 mL of Trizol reagent was added to a tissue sample ground after quickly freezing using liquid nitrogen to isolate RNA according to protocol.
The resulting tissue sample added with 1 mL of Trizol Reagent was incubated at room temperature for 5 min. The resulting tissue sample was supplemented with 0.2 mL of chloroform, vigorously mixed for 15 sec, and incubated at room temperature for 5 min. After centrifugation at 12,000×g at 4° C. for 15 min, the resultant aqueous phase was transferred to a new tube. An equal volume of isopropanol was added to the tube, and the tube was placed at 4° C. for 10 min. After centrifugation at 12,000×g at 4° C. for 10 min, the supernatant was carefully discarded, and the pellet was washed with 70% ethanol, followed by centrifugation at 7,500×g at 4° C. for 15 min. After being dried, the RNA pellet was dissolved in RNase-free water.
To synthesize cDNA from mRNA isolated from each cell line, and human-derived tumor and normal cell line and to amplity G-CSF gene, RT-PCR was performed as follows. 1˜2 μL of total RNA and 8 μL of ONE-STEP PCR premix (Intron Inc., Korea) were mixed with primers of SEQ ID NOs: 28 and 29 in Table 5, and RNase-free water was added up to a final volume of 20 μL. Then, G-CSF gene can be directly amplified from RNA by carrying out an amplification reaction under the condition described in Table 5. GAPDH was amplified using primers of SEQ ID NO: 30 and SEQ ID NO: 31 and it was used as a control for RNA amplification.
hG-CSF was amplified using 1˜2 μL of first PCR product as template, which was amplified by ONE-STEP PCR method (Table 2), based on 50 μL of total reaction volume with primers of SEQ ID NO: 32 and SEQ ID NO: 33, wherein SEQ ID NO: 33 was labeled with fluorescence (Cy5 or different kind of fluorescence). Asymmetric PCR which has a big difference in addition ratio of forward primer (SEQ ID NO: 32) and reverse primer (SEQ ID NO: 33) from 1:5 to 1:10 was secondarily performed to obtain final amplification products.
GAPDH can be also obtained by labeling reverse primer (SEQ ID NO: 31) with fluorescence to perform an amplification reaction, as described the above.
DNA chip was prepared by mixing each probe (E2E4a and E2E4b) in 3×SSC spotting solution at a concentration of 50 μM (
PCR products from normal individuals and patients amplified using primers of SEQ ID NO: 32 and SEQ ID NO: 33 were hybridized to the DNA chip according to Example 8 and Example 9 (
As shown in
As described and proven above in detail, the present invention provides an oligonucleotide essentially containing a nucleic acid sequence of a splice junction site having the 3′-terminal end of exon 2 region linked to the 5′-terminal end of exon 4 region of a G-CSF gene, a diagnostic kit for cancer diagnosis containing the oligonucleotide and a method for diagnosing cancer using the nucleic acid molecule. According to the present invention, cancer can be quickly and exactly diagnosed using variation of a G-CSF gene.
Although a specific embodiment of the present invention has been described in detail, those skilled in the art will appreciate that this description is merely a preferred embodiment and is not construed to limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the accompanying claims and equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2006-0006279 | Jan 2006 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR07/00300 | 1/18/2007 | WO | 00 | 11/14/2008 |
