1999 Media Guide

1999 Men\u27s Track and Field Media Guide, George Fox College

Investigation of <i>CDO1</i> expression with Cancer Profiling Arrays.

<p>Cancer profiling arrays II was performed to compare <i>CDO1</i> expression between tumor (T) and matched normal control (N) tissues of multiple tissue types. The array was hybridized with the <i>CDO1</i> cDNA probe labeled with ³²P-α-deoxycytidine triphosphate according to the manufacturer’s protocol. Tissue type and the number of cases with down-regulation of <i>CDO1 vs.</i> total cases are indicated. Arrow, downregulation of <i>CDO1</i> in tumor compared to normal tissue. Ubiquitin cDNA (Ubi) was used as a control. n/d, not determined.</p

Immunohistochemical analysis of CDO1 in colon cancer tissue microarray.

<p>Expression level was indicated as -, absent or faint expression; +, moderate expression; ++, expression; +++, strong expression.</p><p>CDO1 positivity (+) was counted in samples with over moderate expression.</p><p>P values from Fisher’s exact test performed in Normal vs. adenocarcinoma in BC05118, adenocarcinoma in CO1922,</p><p>or mucinous adenocarcinoma in CO1922. *P<0.05 considered significant.</p>a<p>Cancer adjacent normal colonic tissue that excluded 5 cases of smooth muscle and 4 cases of disrupted tissue (not determined).</p>b<p>A case (fibrous tissue) was excluded.</p>c<p>Ten cases of adenocarcinoma without grade and two cases squamous cell carcinoma were excluded,</p><p>but 38 cases of mucinous adenocarcinomas were included.</p>d<p>Tumor grade was not considered.</p

Additional file 1 of High-risk HPV infection-associated hypermethylated genes in oropharyngeal squamous cell carcinomas

Additional file 1: Supplementary Figure S1. ROC Curves for determining optimal cut-off values between HPV-positive OPSCCs (n=50) and other samples including HPV-negative OPSCCs (n=44) and control samples (n=33). Supplementary Figure S2. Disease-specific survival curves of 94 OPSCCs depending on various clinicohistological factors, including HPV status

Additional file 2 of High-risk HPV infection-associated hypermethylated genes in oropharyngeal squamous cell carcinomas

Additional file 2: Supplementary Table S1. Candidate promoter methylated genes. Supplementary Table S2. Primers and probes for QMSP. Supplementary Table S3. Association of methylation markers with clinicopathological data (n=94). Supplementary Table S4. Classification of cases based on p16 and RB Staining. Supplementary Table S5. Correlation of QMSP values with p16 and RB expression (Mann-Whitney U test)

Sensitivity and specificity of CDO1 methylation in multple types of human cancer.

<p>TaqMeth V is expressed as mean ± SD, and TaqMeth V is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044951#s4" target="_blank">Materials and Methods</a>.</p>a<p>P value was derived from Wilcoxon-Mann-Whitney test of ^aPT vs. PN;</p>b<p>PT vs. NN only in lung.</p><p>PT, tumor tissue from cancer patients; PN, normal tissue from cancer patients; NN, normal tissue from non-cancer patients.</p><p>AUROC is expressed as mean ± SD, and optimal cut-off values were calculated from ROC analysis.</p><p>Methylation level below the cut-offs was considered as unmethylated and over the cut-offs were as methylated.</p>c<p>P value in ROC analysis;</p>d<p>P value in Fisher’s exact test.</p><p>Sensitivity, positive methylation/total tumor cases; Specificity, negative methylation/total normal cases.</p

<i>CDO1</i> mRNA levels in different types of cancer. A

<p>, Expression of <i>CDO1</i> in cell lines was examined by RT-PCR or qRT-PCR analyses. m, mock treatment. a, 5-Aza-dC treatment (5 µM for three days). β-actin was used as a loading control. Methylation status of <i>CDO1</i> promoter in each cell line was examined and indicated as M for methylation, U for unmethylation, and M/U for co-existence of methylated and unmethylated alleles. <i>CDO1</i> was completely methylated in all cancer cell lines since only cytosine peaks were observed in CpGs sequenced (100% methylation) while it was not methylated in HEK293 since only thymidine peaks were observed (0% methylation). <i>CDO1</i> was partially methylated in MCF-12A since both methylated and unmethylated alleles were observed in 10 CpGs of the <i>CDO1</i> promoters examined. When a cytosine peak were compared with a thymidine peak in each CpG of the 10 CpGs, cytosine peaks were dominant (methylated), but since these “methylated CpGs” were found in less than 50% of total CpGs (10/34), it was considered as “methylation-negative” according to the criteria described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044951#s4" target="_blank">Materials and Methods</a>. <b>B</b>. qRT-PCR was performed in cDNAs derived from patients with colon, breast, esophagus, bladder and stomach cancer (T) and patients without cancer (NN) (upper). Relative expression (Fold) was calculated by comparing the ratios of mRNA expression of <i>CDO1</i> to an internal control gene, β-actin. The <i>CDO1</i> expression level was determined in 10 lung cancer patients and 10 patients without cancer (lower). 2?-()*100, the expression of <i>CDO1</i> relative to β-actin calculated based on the threshold cycle (C_t) as 2^−ΔCt (ΔCt = C_{t,<i>CDO1</i>} - C_t,β-actin). Experiments were done in duplicate, and values indicate means ± SD. *, <i>P<0.05</i> in <i>T-</i>test. <b>C</b>, The <i>CDO1</i> expression level was examined in five pairs (A ∼ E) of matched cDNA prepared from patients with colon and lung cancer. PT, primary tumor; PN, matched normal tissues.</p

Increased cell growth by <i>CDO1</i> knockdown.

<p><b>A</b>, siRNAs targeting <i>CDO1</i> mRNA (siR-1 ∼ -4) and a non-targeting control siRNA (siR-Cont) were transfected into <i>CDO1</i>-expressing HEK293 cells, and <i>CDO1</i> gene knockdown was examined by RT-PCR analysis (left) and cell growth was determined by the MTT assay (right). β-actin was used as a loading control. Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, <i>P<0.05</i> in <i>T-</i>test. Both siR-1 and 2 reduced the <i>CDO1</i> mRNA, but only siR-2 displayed increased HEK293 cell growth (left). <b>B</b>, The morphology of HEK293 cells were examined under a phase-contrast microscope after siRNA-transfected cells were grown on Matrigel beds for 3 days. MG alone, a picture of Matrigel without cells. Scale bar, 500 µm. <b>C</b>, HepG2 cells express <i>CDO1</i>, but do not harbor the gene methylation (data not shown). <i>CDO1</i> siRNA (−1 and −2) or control siRNA were transfected into HepG2 cells, and RT-PCR and MTT assays were performed. N, cells without transfection. Increased cell growth was observed in cells transfected only with siR-2. <b>D</b>, Colony focus assays were performed in HepG2 cells after transfection with siR-2 and controls. Colonies were grown for 13 days and stained with crystal violet solution. After air-drying, colonies were counted (left) and photographed (right). Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, <i>P<0.05</i> in <i>T-</i>test. <b>E</b>, The <i>in vitro</i> cell invasion assay was performed in the HepG2 cells after transfection with siR-2 and controls. Cells were incubated for 16 hrs, and after fixation and staining, invading cells were counted at 100 X magnification (left). Cell growth for 16 hrs determined by MTT assay was not significant (right).</p

Methylation of the <i>CDO1</i> promoter in multiple types of human cancer. A

<p>, Quantitative methylation levels of <i>CDO1</i> were determined in primary tissues derived from breast, esophagus, lung, bladder, and stomach. TaqMan methylation values (TaqMeth V) is described in Materials and Method. PT, primary tumor; PN, matched normal tissues; NN, normal tissues from non-cancer patients; CL, cell lines. Lines indicate the optimal cut-off value for each tissue. Arrow, MCF-12A, a non-tumorigenic cell line, harbored a low level of <i>CDO1</i> methylation (TaqMeth V, 6.2). All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results. <b>B</b>, ROC analysis (PT <i>vs</i>. PN) of <i>CDO1</i> in multiple human cancers. Solid line, <i>CDO1</i>; dashed line, no discrimination.</p

Immunohistochemical analysis of <i>CDO1</i> in colon and esophagus cancer tissue array. A

<p>, Strong expression of <i>CDO1</i> in non-malignant colon tissues. The expression of <i>CDO1</i> protein was positive in 6 out of 6 patients. NN1, a patient without cancer, and two more cases are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044951#pone.0044951.s002" target="_blank">Figure S2A</a></b>. <b>B</b>, A group of samples were derived from a single patient consist of colon adenocarcinomas (AD), matched cancer adjacent normal appearing tissue (NAT) and matched cancer adjacent tissues (Adjacent). <i>CDO1</i> expression was investigated in a total of 36 groups of samples. Patients were numbered arbitrarily (Pt1 ∼ Pt3). CRC Pt1, a patient with CRC. Two more cases are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044951#pone.0044951.s002" target="_blank">Figure S2B</a></b>. <b>C</b>, <i>CDO1</i> expression in ESCC. PT, ESCC; PN, matched normal appearing tissues. ESCC Pt1, a patient with ESCC. Three more cases are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044951#pone.0044951.s002" target="_blank">Figure S2C</a></b>. <b>D</b>, <i>CDO1</i> expression in colon adenocarcinoma (AD) with different tumor grades. Mu-AD, mucinous adenocarcinomas. <b>E</b>, <i>CDO1</i> expression in ESCC with different tumor grades. EAD, esophageal adenocarcinomas; Me-ESCC, metastatic ESCC.</p