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Pathology – Research and Practice 202 (2006) 67–70

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ORIGINAL ARTICLE

Mutational analysis of proapoptotic ARTS P-loop domain

in common human cancers

Jong Woo Lee, Young Hwa Soung, Su Young Kim, Suk Woo Nam, Won Sang Park,Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee�

Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea

Abstract

Mounting evidence indicates that deregulation of apoptosis is involved in mechanisms of cancer development.ARTS is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizingthe inhibition of inhibitor of apoptotic protein (IAP) on caspases. ARTS contains a P-loop GTP-binding domain,which is essential for the apoptotic function of ARTS. The aim of this study was to ascertain whether a geneticalteration of ARTS gene is involved in the development of human cancers possibly by inactivating the apoptoticfunction of ART. We analyzed the coding region of the P-loop domain of human ARTS gene for the detection ofsomatic mutations in 100 gastric carcinomas, 100 non-small cell lung cancers, and 69 hepatocellular carcinomas using apolymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). However, there was nomutation in the P-loop coding region in the cancers. The data presented here suggest that ARTS P-loop is notfrequently mutated in gastric, lung, and hepatocellular carcinomas, and that apoptosis deregulation in cancers is notdependent on the mutation of ARTS gene.r 2005 Elsevier GmbH. All rights reserved.

Keywords: ARTS; Mutation; Cancer; Apoptosis; P-loop

Introduction

Apoptosis plays critical roles in a wide variety ofphysiological processes during fetal development and inadult tissues [16]. Caspases cause the morphologicalchanges recognized as apoptosis and the biochemicalchanges associated with apoptosis [14,16]. The functionsof caspases are inhibited by a set of proteins, theinhibitor of apoptotic proteins (IAPs), including XIAP,c-IAP1, c-IAP2, and survivin. The IAPs were shown tobind and inhibit caspase-3, -7, and -9, thereby inhibitingapoptosis [1].

e front matter r 2005 Elsevier GmbH. All rights reserved.

p.2005.11.001

ng author. Tel.: 82 2 590 1191; fax: 82 2 537 6586.

ss: suhulee@catholic.ac.kr (S.H. Lee).

Apoptosis-related protein in the TGF-b signalingpathway (ARTS; also known as H5/SEPT4/PNUTL2)is a mitochondrial septin-like protein [8]. ARTSmediates TGF-b-induced apoptosis. ARTS also isimportant for apoptosis induced by a variety ofproapoptotic factors, including etoposide, staurospor-ine, and Fas [4]. Unlike other septin proteins localized inthe actin bundles, ARTS is localized to the mitochon-dria and is released to the cytosol during apoptosis [8].In the cytosol, ARTS binds with XIAP, decreases XIAPprotein levels, and activates caspases [4]. ARTS containsa phosphate-binding site (P-loop or G1 motif;GXSGXGKST) conserved in the septin family and alsofound in many ATP/GTPases, including apoptosisregulators CED-4, Apaf-1, Nod1, and Nod2 [8,15].ARTS mutants with the substitution of amino acids in

ARTICLE IN PRESSJ.W. Lee et al. / Pathology – Research and Practice 202 (2006) 67–7068

the P-loop did not bind to XIAP and failed to induceapoptosis [4,8].

It is now believed that clonal expansion and tumorgrowth are the results of the deregulation of intrinsicproliferation (cell division) and cell death (apoptosis) [6].Failure of apoptosis could allow for the survival oftransformed cells that are prone to undergo furthergenetic damage and play an important role in thepathogenesis of tumors. Either inactivation of pro-apoptotic pathway or activation of anti-apoptoticpathway results in failure of apoptosis, thereby promot-ing tumor cell survival. Apoptosis of cancer cells can bedelayed or blocked by several ways, including somaticmutation and loss of expression of pro-apoptoticmolecules, and expression of apoptosis-inhibitory mo-lecules [6,16]. Somatic mutations of apoptosis-relatedgenes, including the death receptors and caspases, havebeen common in human cancers, and many of themutations were proven to inactivate cell death[7,12,13,17,19]. Similarly, it could be hypothesized thatARTS playing an important role in the apoptosis couldbe mutated and be responsible for the apoptosisresistance of the cancer cells. To date, however, dataon the mutation status of ARTS gene in human cancerhave been lacking. Therefore, we analyzed 269 cases ofcancer tissues of various origins.

Fig. 1. Typical SSCP of ARTS P-loop domain in cancer

tissues. Part of the exon 4 of ARTS gene encoding the P-loop

domain was amplified using a primer set in 5 gastric

carcinomas. SSCPs show no aberrant band as compared to

SSCP from their corresponding normal tissues (Lane N)

obtained from the same patients.

Materials and methods

Methacarn-fixed tissues of 100 gastric carcinomas,100 non-small cell lung cancers, and 69 hepatocellularcarcinomas were randomly selected for this study.Approval was obtained from the Catholic Universityof Korea, the College of Medicine’s institutional reviewboard. Informed consent was provided according to theDeclaration of Helsinki. The gastric carcinoma samplesconsisted of 39 diffuse-type, 32 intestinal-type, and 29mixed-type gastric carcinomas based on Lauren’sclassification, and 22 early and 78 advanced gastriccarcinomas according to the depth of invasion. The non-small cell lung cancers consisted of 52 squamous cellcarcinomas, 43 adenocarcinomas, and five large cellcarcinomas. The hepatocellular carcinomas were grade I(n ¼ 8), grade II (n ¼ 30), and grade III (n ¼ 31)according to Edmondson’s and Steiner’s criteria [2].

Malignant cells and normal cells were selectivelyprocured from hematoxylin- and eosin-stained slidesusing a 301/2 gauge hypodermic needle (Becton Dick-inson, Franklin Lakes, NJ) affixed to a micromanipu-lator as described previously [11]. In this study, primarylesions, but not the meatstatic lesions, were analyzed fordetection of mutations. DNA was extracted using amodified single-step DNA extraction method as de-scribed previously [11].

Because the P-loop domain of ARTS binds withXIAP and plays an essential role in apoptosis [4,8], weanalyzed the ARTS mutation in the exon 4 encoding thewhole P-loop domain. Genomic DNAs from tumor cellsand normal cells from the same patients were amplifiedwith a primer pair covering the exon 4 of humanARTS gene. The primer sequences were as follows(forward and reverse, respectively): exon 4 (50-cagtg-gagttgctttttggctat-30 and 50-caagtctgcctgattgtcctctc-30).Numbering of cDNA of ARTS was done with respectto the ATG start codon (NM_004574). Radioisotope([32P]dCTP) was incorporated into the polymerase chainreaction (PCR) products for detection by autoradio-gram. PCR and single strand conformation polymorph-ism (SSCP) analyses were carried out as describedpreviously [14]. After SSCP, bands showing mobilityshifts were cut out from the dried gel and re-amplifiedfor 30 cycles using the same primer sets. PCR productswere sequenced using the cyclic sequencing kit (Perkin-Elmer, Foster City, CA).

Results and discussion

Genomic DNAs isolated from normal and tumortissues obtained from the same patients throughmicrodissection were analyzed for the detection ofmutations in ARTS gene encoding the P-loop domain(amino acid residues 132–140) using PCR-SSCP analy-sis. However, the SSCP from the whole samplesanalyzed did not reveal any aberrantly migrating bandcompared to the wild-type bands from the normaltissues (Fig. 1). To confirm the SSCP results, werepeated the experiments twice, including tissue micro-dissection, PCR, SSCP, and direct DNA sequencinganalysis to ensure the specificity of the results, andfound that the data were consistent (data not shown).

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We have previously identified several pro-apoptoticgene mutations, including death receptors (Fas andTRAIL receptor2), caspases (caspase-3, -7, -8 and -10),and Bcl-2 family (Bid and Bad) in many types of humancancers [7,9,10,12,13,17–20], suggesting that genes en-coding the proteins in the apoptotic machinery arepopular targets for the somatic mutations in cancer cells.Because ARTS behaved like a component of theapoptotic machinery, we expected to detect some ARTSmutations in the cancer samples. However, we detectedno ARTS P-loop domain mutation in the samples,suggesting that proapoptotic gene ARTS mutations arenot common in the cancers, and that mutational eventsin ARTS gene do not contribute to the development ofcommon human cancers, including gastric, lung, andhepatocellular carcinomas.

Somatic mutations of apoptosis-related genes fre-quently occurred in specific domains important inapoptosis-signaling. For example, most of the deathreceptor mutations have been found in the deathdomain, and caspase gene mutations frequently affectedprotease subunits [7,12,13,17,19]. Because the P-loopdomain is conserved in some proapoptotic genes, suchas Apaf-1, Nod1 and CED-4, and is essential for theARTS-mediated apoptosis, we focused on the muta-tional analysis of ARTS gene in the P-loop domain. It ispossible that, in addition to the P-loop domain,mutations might exist that may promote the survivalof the cancer cells by inactivating the function of ARTS.Also, there may be other ways of ARTS inactivation inthe cancer cells. For instance, the expression of ARTS islost in 70% of acute lymphoblastic leukemias, andforced overexpression of ARTS restored apoptoticactivity in the cells [3]. In acute lymphoblastic leuke-mias, promoter methylation of ARTS gene contributedto loss of ARTS expression, and lack of ARTSexpression in the cancer cells might inhibit apoptosis.By contrast, there were opposite data on ARTSexpression in the tumors, showing that astrocytomasstrongly expressed ARTS protein, whereas normalastrocytes did not express ARTS [5]. To ascertainwhether loss of ARTS expression is involved in thepathogenesis of common human carcinomas, includinggastric, lung, and liver carcinomas, the expression statusof ARTS in cancers should be analyzed in future studies.

Acknowledgment

This work was supported by funds from KOSEF(R01-2004-000-10463-0).

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