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Ugene, a Newly Identified Protein That Is Commonly Overexpressed in Cancer and Binds Uracil DNA Glycosylase

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Ugene, a Newly Identified Protein That Is Commonly Overexpressed in Cancer and Binds Uracil DNA Glycosylase Research Article Ugene, a Newly Identified Protein That Is Commonly Overexpressed in Cancer and Binds Uracil DNA Glycosylase Chunguang Guo,1,4 Xiaodong Zhang,3,4 Stephen P. Fink,2,4 Petra Platzer,2,4 Keith Wilson,7 James K.V. Willson,8 Zhenghe Wang,3,4 and Sanford D. Markowitz1,2,4,5,6 Departments of 1Molecular Biology and Microbiology, 2Medicine, and 3Genetics; 4Case Comprehensive Cancer Center, Case Western Reserve University; 5Case Medical Center; 6Howard Hughes Medical Institute, Cleveland, Ohio; 7PDL BioPHARMA, Redwood City, California; and 8Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas Abstract complex with uracil DNA glycosylase 2 (UNG2), a base excision Expression microarrays identified a novel transcript, desig- repair (BER) enzyme. nated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in Materials and Methods malignant colon cancers. These findings were validated by Cell lines and tissues. VACO cell lines were established and maintained real-time PCR and Northern blot analysis in an independent as previously described (7). DLD1 and SW480 cell lines were obtained from panel of colon cancer cases. In addition, Ugene expression was the American Type Culture Collection. Normal colons, primary colon found to be elevated in many other common cancer types, cancers, and liver metastasis tissues were obtained from the archives of including breast, lung, uterus, and ovary. Immunofluores- University Hospitals of Cleveland under an institutional review board– cence of V5-tagged Ugene revealed it to have a nuclear approved protocol. Total RNA and genomic DNA were prepared as described (8). localization. In a pull-down assay, uracil DNA glycosylase 2 DNA expression microarray analysis. As described previously (9), we (UNG2), an important enzyme in the base excision repair designed custom expression monitoring microarrays using Affymetrix (BER) pathway, was identified as a partner protein that binds GeneChip technology (10). Preparation of samples, hybridization to to Ugene. Coimmunoprecipitation and Western blot analysis GeneChip expression microarrays, and data analysis were all performed confirmed the binding between the endogenous Ugene and as described previously (9). UNG2 proteins. Using deletion constructs, we find that Ugene Rapid amplification of cDNA ends PCR. 5¶ and 3¶ Rapid amplification A binds to the first 25 amino acids of the UNG2 NH2 terminus. of cDNA ends (RACE)–ready cDNAs were generated from 2 g of total We suggest that Ugene induction in cancer may contribute to RNA (V241 cell line) using the 5¶/3¶ RACE kit (Roche). The gene-specific ¶ ¶ the cancer phenotype by interacting with the BER pathway. primers used for 5 RACE were as follows: SP1, 5 -GCGGGACCTAGAG- ¶ ¶ ¶ ¶ [Cancer Res 2008;68(15):6118–26] CTTTTCT-3 ; SP2, 5 -GAGGCAGGTGGAGTTTGAAG-3 ; and SP3, 5 -ATCCC- TTCCCCAGCATTAAG-3¶. The gene-specific primer for 3 RACE was 5¶-ACC- TCATCCTTCCTGCGACG-3¶. Full-length Ugene was PCR amplified from Introduction RACE-ready cDNA using the forward 5¶-CCGACTGAGCCTCTAAAGCGAC-3¶ Cancers of the colon and rectum (CRC) are the second leading and reverse 5¶-TCCTGATTCACAAACTCTTGCTCC-3¶ primers. cause of cancer deaths among adult Americans. Colon cancer Northern blot analysis. Northern blot analysis was performed as develops as a result of the progressive accumulation of genetic and previously described (11) using the entire Ugene coding region as the probe. Southern blot analysis. Total genomic DNA from cell lines and normal epigenetic alterations in key oncogenes and tumor suppressor tissues were digested with PstI, separated by electrophoresis on a 0.8% genes that lead to the transformation of normal colonic epithelium agarose gel, and transferred onto a Zeta-Probe blotting membrane. to adenocarcinoma (1). In addition to primary genetic alterations, 32P-labeled DNA probes were prepared by random primer extension of a the cancer phenotype is also importantly modulated by down- fragment containing the Ugene coding sequence. Equal loading of DNA was stream alterations in the levels of expression of different effector confirmed by rehybridizing blots with a probe designated to the TGFb-RII genes (2–4). Expression microarrays enable comprehensive profil- gene, which is relatively copy number invariant in CRC. ing of the cancer transcriptome and identification of cancer- Human cancer dot blots. Radioactively labeled cDNA probes were specific changes in gene expression (5, 6). synthesized from human Ugene or ubiquitin control cDNA using random In this study, using a global gene expression profiling array, primer labeling followed by probe purification on CHROMA SPIN + STE-100 we identified a previously uncharacterized gene, called Ugene, columns (BD Biosciences). Hybridization of the cancer profiling array with human Ugene probes and washings of the array were done according to the which is overexpressed in malignant colon cancers. We further manufacturer’s recommendations (BD Biosciences). The hybridized cancer show that Ugene is frequently elevated in most malignant tumor profiling arrays were then exposed to the phosphorimaging screens and types. In addition, we provide experimental evidence showing scanned with a Storm 840 PhosphorImager. We then stripped this same that Ugene protein is localized within the nucleus and forms a membrane and hybridized it with human ubiquitin cDNA probe to show equal sample loading. Ugene real-time PCR. Primers and a fluorogenic hybridization probe Note: Supplementary data for this article are available at Cancer Research Online were designed using the Primer3 software (12). Ugene was amplified using (http://cancerres.aacrjournals.org/). 400 nmol/L of forward primer 5¶-CTGTCTTCTTTCCTGCAACAAC-3¶ and Z. Wang and S.D. Markowitz contributed equally to this work. ¶ ¶ Requests for reprints: Sanford D. Markowitz, Howard Hughes Medical Institute reverse primer 5 -TAGGACGTTTACACCTGTGGAG-3 and detected using Laboratory, Wolstein Research Building, 3rd Floor, Mailstop 7285, Case Western fluorogenic hybridization probe 5¶-/56-FAM/ATAAACTGCCTGGCTGT- Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-7285. Phone: 216-368- GAAACATCCAG/3BHQ_2/-3¶. h-2-Microglobulin (B2M) was amplified using 1976; Fax: 216-368-8928; E-mail: [email protected]. Â I 0.2 of the human B2M TaqMan primer/probe kit (Perkin-Elmer 2008 American Association for Cancer Research. A doi:10.1158/0008-5472.CAN-08-1259 Biosciences). Each PCR was carried out in triplicate in a 25- L volume using TaqMan Assay Mastermix (Applied Biosystems) for 8 min at 95jC, Cancer Res 2008; 68: (15). August 1, 2008 6118 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Ugene Induction in Cancers followed by 50 cycles of 95jC for 15 s, 57jC for 30 s, and 72jC for 30 s. vectors are as follows: for Ugene, forward 5¶-ACCTCATCCTTCCTGCGACG- The level of Ugene expression was determined as the ratio of Ugene: 3¶ and reverse 5¶-TCTAATACACTCCTCTGCTGAGAT-3¶; for UNG2, forward B2M = 2(CT B2M-CT Ugene). 5¶-ATGGGCGTCTTCTGCCTTG-3¶ and reverse 5¶-CAGCTCCTTCCAGT- Construction of expression/deletion vectors. The coding sequence of CAATG-3¶. FLAG-tagged constructs were similarly made by adding the Ugene (Ugene-p/Ugene-q, XM_001133365) and UNG2 (NM_080911) was complimentary FLAG tag sequence with a stop codon (5¶-TTACTTGT- PCR amplified and cloned into the eukaryotic expression vector pcDNA3.1/ CATCGTCGTCCTTGTAGTC-3¶) at the 5¶ end of the reverse primer. UNG2 V5/His-TOPO (Invitrogen) to generate COOH-terminal V5-tagged Ugene/ deletion constructs were generated by blunt ligation of the PCR products, UNG2 expression vectors. The primer sequences for constructing the amplified using V5-tagged UNG2 expression vector as a template, with the Figure 1. Ugene mRNA expression in normal and cancer samples. A, expression of U46258 on GeneChip microarrays. Shown for comparison are analyses of RNA samples from normal colon epithelium; colon adenomas; primary colon cancers of stages II, III, and IV; colon cancer hepatic metastases; and colon cancer cell lines. Horizontal bars, median expression values within each group. Average intensity of 25 units corresponds to null expression. B, real-time PCR measurement of Ugene transcript expression in 11 normal colon epithelial samples versus 13 colon cancer cell lines. Ugene values are normalized against expression of the housekeeping gene, B2M. Black bars, mean value for each group. C, the ratio of Ugene expression in colon cancer versus matched normal colon mucosa, as measured by real-time PCR in 20 patients. Ugene values are again normalized against B2M. Values represent averages of six replicates. D, cDNA dotblotanalysis of Ugene expression level in matched tumor and normal tissue blots from the various organs. N, normal; T, tumor. Numbers below each blot represent the number of tumors with >2-fold increase in Ugene expression relative to their matched normal [Ugene (")] versus the number of total samples analyzed (total #). Each L-shaped box of three dots represents normal tissue (left), primary tumor (top right), and metastases (bottom right) from the same patient. Equal sample loadings over all lanes were confirmed by rehybridizing the blot to a ubiquitin probe (data not shown). www.aacrjournals.org 6119 Cancer Res 2008; 68: (15). August 1, 2008 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2008 American Association
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