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[CANCER RESEARCH 59, 2023–2028, May 1, 1999] Advances in Brief

HRad17, a Human Homologue of the Schizosaccharomyces pombe Checkpoint Gene rad17, Is Overexpressed in Colon Carcinoma1

Shideng Bao, Mau-Sun Chang, Daniel Auclair, Yapin Sun, Youbin Wang, Wei-Kong Wong, Jinyang Zhang, Yuan Liu, Xiuqi Qian, Rebecca Sutherland, Cristina Magi-Galluzi, Ellen Weisberg, Edmond Y. S. Cheng, Luning Hao, Hidefumi Sasaki, Michael S. Campbell, Stine-Kathrein Kraeft, Massimo Loda, Kin-Ming Lo, and Lan Bo Chen2 Dana-Farber Cancer Institute [S. B., M-S. C., D. A., Y. S., Y. W. W-K. W., J. Z., R. S., C. M-G., E. W., L. H., H. S., M. S. C., S-K. K., L. B. C.] and Beth Israel Deaconess Hospital [M. L.], Harvard Medical School, Boston, Massachusetts 02115, and Shionogi BioResearch Corporation, Lexington, Massachusetts 02421 [Y. L., X. Q., E. Y. S. C., K-M. L.]

Abstract structural homologues in mammalian systems or only some of them. Significant homologies between rad3 and human ATM and ATR (6), Using the palindromic PCR-cDNA display method, we have cloned a fission yeast and human chk1 (7), and human and yeast rad1 (8), as novel gene overexpressed by human colon carcinoma relative to normal well as numerous other pairs, have been reported. However, because colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint the most thoroughly studied mammalian checkpoint component, p53, genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces does not have an apparent structural homologue in yeast, it remains cerevisiae. This novel gene designated as hRad17 is localized to chromo- possible that mammals share some but not all of the checkpoint some 5q12,13.1, a region known to be deleted in a variety of human machinery found in yeast. Mutations in human checkpoint proteins cancers. Promoter region and one pseudogene of hRad17 have been iden- such as p53, ATM/ATR, and Bub1 that have been found in tumors tified. Whereas the increased expression of hRad17 by human colon cannot account for the majority of expected checkpoint aberrations carcinomas may be related to the known resistance of these cells to associated with human cancer, given the multistep hypothesis of DNA-damaging agents during therapy, the deletion of hRad17 in a variety cancer etiology. We have used a novel palindromic PCR-cDNA of cancers may predispose them to increased rate of mutation and height- display method to identify a human homologue of yeast checkpoint ened sensitivity to DNA-damaging agents, including radiation and anti- proteins rad17 of S. pombe and RAD24 of S. cerevisiae. This novel cancer drugs. human gene, designated as hRad17, is overexpressed by colon carci- Introduction noma relative to normal colon. Its chromosomal localization at 5q12,13.1 suggests that a variety of human cancers would have a Cell cycle checkpoints are essential in eukaryotes for ensuring high deletion in this gene. Increased expression of this gene might be fidelity transmission of genetic information from one generation to the responsible for increased resistance to DNA-damaging agents by next. They include DNA damage checkpoints, DNA replication cancer cells; its decreased expression, on the other hand, could lead to checkpoints, spindle assembly checkpoints, and cytokinesis check- higher rate of mutation as well as increased sensitivity to radiation and points (1). Cell cycle checkpoints have been best studied in yeast. A chemotherapy. series of checkpoint genes required for DNA damage and/or DNA replication checkpoints have been identified in both fission and bud- Materials and Methods ding yeast (2). The rad17 gene of Schizosaccharomyces pombe is one Palindromic PCR-cDNA Differential Display. Surgical samples of hu- of several essential checkpoint components including rad1, rad3, man testis and prostate were placed in liquid nitrogen as soon as possible after rad9, rad17, hus1, and cut5/rad4 that are absolutely required for removal. The frozen tissue was ground into fine particles, and total RNA was prevention of mitosis after DNA damage in fission yeast (3). Func- prepared using the guanidine isothiocyanate method of Chirgwin et al. (9). The tional loss of any one of these genes abolishes the radiation-induced palindromic primer TCCTTAGAAC was used with the Superscript First

G2 arrest. All of these genes except cut5/rad4 are also required for the Strand cDNA Synthesis kit (Life Technologies, Inc.) to reverse transcribe post replication checkpoints, which prevents mitosis in response to ϳ100 ng of RNA from human testis and prostate specimens. This was DNA replication blocks. It has been reported that deletion of rad17 followed by PCR (94°C, 30 s; 40°C, 1 min 40 s; 72°C, 1 min; 40 cycles) using 35 resulted in an 8-fold increase in the rate of chromosome loss (4), the same primer and Taq DNA polymerase with 2 mM MgCl2 and S-labeled 35 suggesting a role for rad17 in the maintenance of genomic integrity. dATP. The S-labeled cDNA was then run on a 6% polyacrylamide gel and autoradiographed. RAD24 in Saccharomyces cerevisiae appears to be the counterpart of cDNA fragments of interest were then eluted from the dried gels. The gel rad17 in S. pombe and is one of several key checkpoint genes slice was soaked in 100 ␮l of distilled water for 10 min, boiled for 15 min, and including RAD9, RAD17, RAD24, MEC3, MEC1, TEL1, and RAD53 spun at 14,000 rpm for 2 min. The supernatant was transferred to a fresh tube, that are indispensable to mitotic cell cycle arrest and transcriptional followed by the addition of 10 ␮lof3M sodium acetate, 5 ␮l of glycogen (10 activation in response to DNA damage (1). RAD24, RAD17, and mg/ml), and 450 ␮l of 100% ethanol. The sample was then incubated at Ϫ70°C MEC1 are also required for meiotic checkpoints in budding yeast (5). for 30 min and spun at 14,000 rpm for 10 min. The supernatant was removed, It is still unknown whether all of the yeast’s checkpoint proteins have and the pellet was washed with 200 ␮l of 85% ethanol. The final sample was resuspended in 10 ␮l of distilled water. PCR was performed on the eluted fragments using the same conditions Received 12/17/98; accepted 3/17/99. The costs of publication of this article were defrayed in part by the payment of page described above. PCR products were then run on a 1.5% agarose gel and charges. This article must therefore be hereby marked advertisement in accordance with purified using the Qiaex II Agarose Gel Extraction kit (Qiagen, Chatsworth, 18 U.S.C. Section 1734 solely to indicate this fact. CA). 1 This work has been supported by grants from the National Cancer Institute. 2 Northern Blot Analysis. Gel-purified hRad17 palindromic PCR fragment To whom requests for reprints should be addressed, at Department of Pathology, 32 Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: (617) 632- was converted into P-labeled probes for Northern analysis using a random- 3386; Fax: (617) 632-4470; E-mail: [email protected]. primed labeling kit (Boehringer). Multiple tissue Northern blots (Clonetech) 2023

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1999 American Association for Cancer Research. HUMAN Rad17 IN COLON CANCER were hybridized with the 32P-labeled probe as described (10). Filters were then differentially between normal and malignant colon tissues. The exposed to X-ray film. mRNAs isolated from normal colon and colorectal carcinoma tissues In Situ RNA Hybridization. In situ RNA hybridization was performed as were reverse transcribed to cDNA and amplified by PCR, all using a described previously (10). A 690-bp fragment corresponding to the COOH- single palindromic primer. After electrophoresis and autoradiography, Ј terminus and 3 region of hRad17 was excised using PstI and cloned in cDNA patterns derived from the testis and prostate were compared pBluescript (Stratagene). Riboprobes were generated with T7 and T3 RNA and analyzed for differences. As shown in Fig. 1A, one cDNA polymerase. DNA Sequencing. Reamplified PCR products were purified from an aga- fragment amplified with the palindromic primer appeared to be pres- rose gel and subcloned into TA vectors (pCR 2.1 Invitrogen). DNA was then ent at significantly higher levels in colorectal carcinoma tissue as alkaline denatured followed by neutralization and precipitation, annealing with compared to normal colon (arrow). This cDNA was recovered from sequencing primers and sequencing using the Sequenase Version 2.0 kit the gel and re-amplified by PCR with the same palindromic primer. A (United States Biochemical, Cleveland, OH). Sequencing was performed on both strands of the PCR products. cDNA and Genomic Library Screening. The full-length clone of hRad17 was obtained by screening a human fibroblast ␭ZAP II cDNA (Stratagene) library using standard library screening procedures. Mouse and green monkey rad17 homologues were obtained by screening 3T3 and COS cell cDNA libraries, respectively. The hRad17 genomic clones were isolated from a human peripheral blood lymphocyte genomic library. The promoter region and a pseudogene were identified after DNA sequencing. FISH.3 Mitotic chromosome spreads of human lymphocytes were prepared according to standard procedures. The XhoI/XbaI hRad17 cDNA probe was biotinylated using the BRL BioNick labeling kit. Slides were baked at 55°C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 2ϫ SSC for 2 min at 70°C, followed by dehydration in ethanol. The probe was denatured at 75°C for 5 min in 50% formamide and 10% dextran sulfate. After overnight hybridization with the probe, slides were washed, labeled with DAPI, and visualized. Assignment of map position was achieved by superim- posing the FISH signal with the DAPI-banded chromosomes (11). Generation of Antibodies and Immunohistochemical Staining. We overexpressed full-length HRad17 as a fusion protein with GST in Escherichia coli and purified it by binding to glutathione-conjugated beads. HRad17 was released from GST-beads by treatment with thrombin, and the HRad17-GST fusion protein was eluted by excess glutathione. Mice were immunized with purified HRad17 to produce a series of monoclonal antibodies recognizing HRad17. Detailed characterization of these antibodies will be presented elsewhere. Specimens of normal colon, colorectal carcinoma tissues, and mouse testis were fixed in 3.7% formalin and embedded in paraffin. Routine sections were cut 5 ␮m thick, dried at 40°C for 4 h, cleared in xylene, and rinsed in ethanol. The immunohistochemical staining was performed by an automated Ventana 320/ES immunohistochemistry robot. Endogenous peroxidase was quenched with methanol/peroxide solution and blocked with normal horse serum. Sec- tions were incubated with a diluted anti-HRad17 monoclonal antibody for 20 min. A peroxidase-conjugated secondary antibody was applied and visualized with 3Ј,3Ј-diaminobenzidine as a substrate. A negative control was run simul- taneously with preimmune immunoglobulins.

Results The differential display method typically uses an oligo-dT(NN) primer for reverse transcription, followed by PCR with the same primer and a random 10-mer (12). The resulting amplified cDNA fragments tend to represent the 3Ј untranslated regions of mRNAs. We have developed a modification of this technique that uses a single palindromic primer for both mRNA reverse transcription and subse- quent PCR reactions. This modification takes advantage of our ob- servation that palindromic sequences occur more frequently within open reading frames than in untranslated regions and with a frequency 10-fold greater than would be predicted by chance alone.4 Thus, the cDNA fragments identified are much more likely to represent the protein coding regions of genes. This technique, termed palindromic PCR cDNA differential dis- Fig. 1. Identification of a cDNA differentially expressed between normal and colo- play, was used to identify and isolate cDNA fragments expressed rectal carcinoma tissues. A, autoradiogram of cDNA fragments derived from colorectal carcinoma (T) and normal colon (N) tissue biopsies cDNA by PCR amplification with the palindromic primer TCCTTAGAAC. Arrows, cDNA present at significantly different 3 The abbreviations used are: FISH, fluorescence in situ hybridization; DAPI, 4Ј,6- levels in the two tissues. B, the palindromic PCR cDNA fragment was recovered, diamidino-2-phenylindole; GST, glutathione S-transferase; ORF, open reading frame. reamplified by PCR with the same primer, and electrophoresed. C, sequence of the PCR 4 A. Lin and L. B. Chen, manuscript in preparation. fragment. Underlines, palindromic motif. 2024

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1999 American Association for Cancer Research. HUMAN Rad17 IN COLON CANCER single band was detected (Fig. 1B), and the cDNA was then subcloned to screen a human fibroblast ␭ZAPII cDNA library. Four overlap- and sequenced. Sequence analysis revealed that the 523-bp fragment ping clones were isolated and sequenced, and one contained a (Fig. 1C) contained a partial ORF of 174 amino acids. A database complete ORF. The 2010-bp ORF encodes a 670-amino acid search suggested that the PCR fragment contained a putative ATP- protein with a predicted molecular mass of about 75 kDa (GenBank binding site and shared limited homology with replication factor C accession no. AF112263). It is a basic hydrophilic protein with a subunits. putative ATP-binding site and two potential nuclear localization To confirm that the expression of the gene represented by the PCR signals, 340RPRKK346 and 358KRRKK364, suggesting that this may fragment is much higher in colorectal carcinoma tissues, in situ RNA be a nuclear protein. The PCR fragment described above corre- hybridization on biopsies from 10 normal colon and 10 colorectal sponds to amino acids 59–233 of the ORF. carcinoma tissues, all from different patients, was performed. High The GenBank database analysis revealed that the ORF shared expression of the gene represented by the PCR fragment was observed significant homology at the amino acid level with rad17 of S. pombe in 9 of 10 colorectal carcinoma tissues (Fig. 2B), whereas very little (26% identity; 51% similarity) and RAD24 of S. cerevisiae (22% expression was found in normal colon (Fig. 2A). These results con- identity; 46% similarity). A comparison of the proteins is shown in firmed that the PCR fragment-containing gene is indeed overex- Fig. 3C. This level of similarity suggested that our novel ORF is likely pressed by colorectal carcinoma tissues. To evaluate expression levels to be a human homologue of these yeast proteins. Thus, we designated in other tissues, a filter containing RNAs from multiple human tissues it as Hrad17. Since our finding, another group specifically looking for was subjected to Northern blot analysis with the same PCR fragment. the human homologue of S. pombe rad17 has independently identified As shown in Fig. 2C, among various normal tissues examined, only the same gene as Hrad17 (13). Although they reported 49% identity testis expresses a high level of this mRNA. to S. pombe rad17 at both the DNA and amino acid levels, we were Identification of the Palindromic PCR Clone as hRad17. To unable to reproduce such a high degree of homology in our analyses. obtain a full-length cDNA, the PCR fragment was used as a probe The sequence homology between hRad17 and S. pombe rad17 appears to be higher than that between the two yeast homologues. All three proteins are basic hydrophilic proteins with a similar size. We have also cloned the mouse and African green monkey (GenBank accession no. AF106067) homologues of the hRad17 gene. Both are very similar to the human sequences (Fig. 3A). In addition, in the course of genomic cloning and sequencing of hRad17, we have identified the promoter region (GenBank accession no. AF106065) and a pseudogene located at 7p21 (GenBank accession no. AF106066) for hRad17. Chromosomal Localization of hRad17. A cDNA probe was used to determine the chromosomal localization of the Hrad17 gene by FISH. Fig. 3, A and B, shows chromosomes from human lymphocytes labeled with the complete XbaI/XhoI Hrad17 cDNA as a probe. Detailed analysis of the chromosomal DAPI banding patterns (11) allowed assignment of the hRad17 gene to chromosome 5, region q12–q13.2. Because a hRad17 pseudogene (GenBank accession no. AF106066), was identified during the cloning of hRad17 genomic DNA, the chromosomal localization was repeated using a 3.0-kb PCR probe from the intron located at position 1488 relative to hRad17 cDNA. This experiment confirmed the localization of hRad17 to chromosome 5q12–13.2 (data not shown). The chromosomal localization of hRad17 reported here differs from that reported previously, which has assigned the same gene to chromosome 4q13.3–21.2 (13). Expression of HRad17 Protein in Colon, Colorectal Carcinoma, and Testis. Full-length HRad17 was expressed as a fusion protein with GST and purified. Mice were immunized with the highly purified HRad17, and monoclonal antibodies were generated. These monoclonal antibodies recognize HRad17 protein by Western blot analysis as well as HRad17 produced by in vitro transcription-translation driven by HRad17 full-length cDNA. Characterization of these monoclonal antibodies will be presented elsewhere. Immunohistochemistry was performed on paraffin sections of colorectal carcinoma tissues (Fig. 4A). Again, high expression of HRad17 was detected in sections from colorectal carcinoma cells biopsies (Fig. 4A, left side of panel) when compared with normal cells (Fig. 4 A, right side of panel), with staining being observed mainly in the nucleus. HRad17 staining detected here was completely abolished when anti-HRad17 monoclonal antibodies were first incubated with excess of highly purified HRad17 (data not shown). Analysis of the HRad17 staining pattern in testis sections indicates Fig. 2. Expression of hRad17 mRNA in colon cancer and in normal tissues. In situ that the protein is present primarily in the nuclei of spermatogonia, RNA hybridization was performed on sections from normal (A) and colorectal carcinoma (B) biopsies using the hRad17 palindromic PCR fragment as a probe. C, HRad17 cDNA spermatocytes, and spermatids (Fig. 4, B and C). As for human colon probe was used for Northern blot analysis of various normal human tissues. sections, the staining again appears as distinct clusters within the 2025

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Fig. 4. Localization of HRad17 protein in normal colon, colorectal carcinoma, and in the testis. A, para- fin section from a colorectal cancer tumor biopsy was labeled with anti-HRad17 antibody. Colorectal carcinoma cells (C)ontheleft of the panel show brown labeling in the nucleus, whereas normal cells (n)on the right show no staining. B, a mouse testis section was labeled with anti-HRad17 antibody. C, the anti- HRad17 antibody was preincubated with purified recombinant HRad17 protein before being used to label mouse testis. Myoid cells (m), spermatogonia (s), spermatocytes (sc), and spermatids (sp) are indicated by arrowheads.

nucleus. Interestingly, HRad17 is absent in mature spermatozoa (Fig. logues of known proteins in different organisms. Recently, this strat- 4B). Thus, HRad17 is present throughout the earlier stages of sper- egy was used successfully to clone a human homologue of the S. matogenesis but not in the terminally differentiated spermatozoa. pombe rad17 checkpoint gene, designated Hrad17 (13). HRad17 staining described here was also completely abolished by We identified the same protein by a different approach. Using the preincubation of the antibody with purified recombinant HRad17 palindromic PCR-cDNA differential display method, we compared protein, demonstrating the specificity of the labeling (Fig. 4C). the mRNA expressions between normal and cancerous human colon. One of the genes we found to be expressed at much higher levels in Discussion colon carcinoma was hRad17. The basis for increased expression Conservation of cell cycle machinery across incredibly diverse remains to be elucidated. We have cloned ϳ2.0 kb of the hRad17 eukaryotes is by now well established. Clearly, the proteins and promoter region (GenBank accession no. AF106065) and found pu- mechanisms involved in imposing order on the process of cell division tative responsive elements for known oncogenic transcription factors are of the highest importance and arose early in the course of evolu- such as c-myb and TCF-1 that are highly expressed in colon carci- tion. Such similarities in proteins can be exploited to identify homo- noma tissues and thought to contribute to the etiology of this disease

Fig. 3. Chromosomal localization and sequence of hRad17. A, mitotic chromosomes from human lymphocytes were hybridized with an hRad17 cDNA probe. The location of the probe appears in yellow and is indicated by an arrow. B, the same field showing the DAPI-stained chromosomes. C, amino acid sequence comparison of hRad17, African green monkey rad17, and mouse rad17 (this work) with S. cerevisiae RAD24 (U18922) and S. pombe rad17 (X91889). 2027

(14, 15). Whether these factors are involved in the overexpression of for the appropriate response to damaged DNA. This could result in hRrad17 in colorectal cancer remains to be determined. damaged DNA going unchecked and unrepaired. Malfunctions in Although a functional role for HRad17 has not yet been established, these pathways could constitute a critical turning point in the genesis its homology to yeast checkpoint proteins suggests it may play a part of cancer; loss or mutation of a checkpoint gene would generate still in cell cycle control. HRad17 is also abundantly expressed in the more mutations, leading to the most intriguing aspect of human testis. The role of the testis is to produce billions of sperm cells cancers, genomic instability. continually. An organ with such a high rate of cell division would be expected to require high levels of proteins involved in cell cycle References control. In addition, it is the only organ in adults where cells under- 1. Elledge, S. J. Cell cycle checkpoints: preventing an identity crisis. Science (Wash- going meiosis can be found. The HRad17 protein shows a striking ington DC), 274: 1664–1672, 1996. pattern of localization within the cells of the seminiferous tubule. It is 2. Murray, A. W. The genetics of cell cycle checkpoints. Curr. Opin. Genet. Dev., 5: found throughout the early stages of spermatogenesis, including cells 5–11, 1995. 3. Bentley, N. J., and Carr, A. M. DNA structure-dependent checkpoints in model undergoing both meiotic and mitotic divisions. The only cells that lack systems. Biol. Chem., 378: 1267–1274, 1997. HRad17 are the terminally differentiated spermatozoa, cells that do 4. Griffiths, D. J., Barbet, N. C., McCready, S., Lehmann, A. R., and Carr, A. M. Fission not divide any further. Interestingly, RAD24, the budding yeast ho- yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins. EMBO J., 14: 5812–5823, 1995. mologue of HRad17, plays a critical role in a meiotic checkpoint as 5. Lydall, D., Nikolsky, Y., Bishop, D. K., and Weinert, T. A meiotic recombination well. RAD24, in combination with RAD17 and MEC1, prevents the checkpoint controlled by mitotic checkpoint genes. Nature (Lond.), 383: 840–843, first meiotic division until meiotic recombination is complete (5). 1996. 6. Bentley, N. J., Holtzman, D. A., Flaggs, G., Keegan, K. S., DeMaggio, A., Ford, J. C., ATM and ATR (5), human proteins similar to MEC1, have been shown Hoekstra, M., and Carr, A. M. The Schizosaccharomyces pombe rad3 checkpoint to localize to synaptonemal complexes in meiotic cells. They are also gene. EMBO J., 15: 6641–6651, 1996. abundantly expressed in testis (16). There is ample evidence that 7. Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z., Piwnica-Worms, H., and Elledge, S. J. Conservation of the chk1 checkpoint pathway in mammals: linkage of human homologues of yeast proteins form similar complexes and DNA damage to cdk regulation through cdc25. Science (Washington DC), 277: function in similar pathways to those of the yeast proteins (3, 8). Thus, 1497–1501, 1997. HRad17 may interact with ATM and/or ATR as part of a human 8. Parker, A. E., Van de Weyer, I., Laus, M. C., Oostveen, I., Yon, J., Verhasselt, P., and Luyten, W. H. A human homologue of the Schizosaccharomyces pombe rad1ϩ pathway analogous to the RAD24/RAD17/MEC1 in budding yeast. checkpoint gene encodes an exonuclease. J. Biol. Chem., 273: 18332–18339, 1998. The consequences of overexpression of HRad17 by human colon 9. Chirgwin, J. M., Przybyla, E. A., MacDonald, R. J., and Rutter, W. J. Isolation of carcinoma are unknown. However, it may confer increased resistance biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem- istry, 27: 5294–5299, 1979. of colon cancer cells to DNA-damaging agents including radiation 10. Wang, F-L., Wang, Y., Wong, W-K., Liu, Y., Addivinola, F. J., Liang, P., Chen, and alkylating drugs. It has been proposed that in yeast, rad17 and L. B., Kantoff, P. W., and Pardee, A. B. Two differentially expressed genes in normal RAD24 play a role in the DNA-damage checkpoint (17). Arrest of the prostate tissue and in carcinoma. Cancer Res., 56: 3634–3637, 1996. 11. Heng, H. H., and Tsui, L. C. Modes of DAPI banding and simultaneous in situ cell cycle in G2 phase after DNA damage is believed to promote cell hybridization. Chromosoma, 102: 325–332, 1993. viability by allowing time for DNA repair before entry into mitosis. 12. Liang, P., and Pardee, A. B. Differential display of eukaryotic messenger RNA by Agents that abrogate G arrest or mutations in genes that regulate the means of the polymerase chain reaction. Science (Washington DC), 257: 967–971, 2 1992. G2 checkpoint tend to sensitize cells to DNA-damaging agents (18, 13. Parker, A. E., Van de Weyer, I., Laus, M. C., Verhasselt, P., and Luyten, W. H. 19). If the human homologue of S. pombe rad17 acts in a similar Identification of a human homologue of the Schizosaccharomyces pombe rad17ϩ fashion, targeting overexpressed HRad17 might render colon cancer checkpoint gene. J. Biol. Chem., 273: 18340–18346, 1998. 14. Torelli, G, Venturelli, D., Colo, A., Zanni, C., Selleri, L., Moretti, L., Calabretta, B., cells more sensitive to DNA-damaging agents. It has been shown that and Torelli, U. Expression of c-myb protooncogene and other cell cycle-related genes in normal and neoplastic human colonic mucosa. Cancer Res., 47: 5266–5269, 1987. UCN-01, a potent abrogator of the G2 checkpoint, does sensitize HT-29 colon carcinoma cells to ␥-rays (20). 15. Mayer, K., Hieronymus, T., Castrop, J., Clevers, H., and Ballhausen, W. G. Ectopic activation of lymphoid high mobility group-box transcription factor TCF-1 and The hRad17 gene is located on chromosome 5q12–13.1. This is in overexpression in colorectal cancer cells. Int. J. Cancer, 72: 625–630, 1997. agreement with the localization of hRad17 determined by another 16. Keegan, K. S., Holtzman, D. A., Plug, A. W., Christenson, E. R., Brainerd, E. E., group.5 Although no human disease has been associated with deletion Flaggs, G., Bentley, N. J., Taylor, E. M., Meyn, M. S., Moss, S. B., Carr, A. M., Ashley, T., and Hoekstra, M. F. The ATR and Atm protein kinases associate with of this specific chromosomal location, thousands of cases of human different sites along meiotically pairing chromosomes. Genes Dev., 10: 2423–2437, cancers are reported to have deletions spanning this region. Contrary 1996. to colon cancer where overexpression may be responsible for resist- 17. Lydall, D., and Weinert, T. G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair. Mol. Gen. Genet., 256: ance to DNA-damaging agents, lack of HRad17 may contribute to the 638–651, 1997. genesis of other types of both sporadic and hereditary human cancers. 18. Lau, C. C., and Pardee, A. B. Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc. Natl. Acad. Sci. USA, 79: 2942–2946, 1982. Because rad17 and RAD24 are essential for post-DNA damage check- 19. Weinert, T. A., and Hartwell, L. H. The RAD9 gene controls the cell cycle response points in yeast, HRad17 may also play a similar role in humans. If so, to DNA damage in Saccharomyces cerevisiae. Science (Washington DC), 241: a loss of HRad17 could compromise checkpoint pathways responsible 317–322, 1988. 20. Wang, Q., Fan, S., Eastman, A., Worland, P. J., Sausville, E. A., and O’Connor, P. M.

UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted 5 F. Dean, Rockefeller University, personal communication. p53. J. Natl. Cancer Inst., 88: 956–965, 1996.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1999 American Association for Cancer Research. HRad17, a Human Homologue of the Schizosaccharomyces pombe Checkpoint Gene rad17, Is Overexpressed in Colon Carcinoma

Shideng Bao, Mau-Sun Chang, Daniel Auclair, et al.

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