Proc. Natl. Acad. Sci. USAVol. 92, pp. 10427-10431, October 1995Cell Biology

Cloning and characterization of a cellular apoptosis susceptibilitygene, the human homologue to the yeast chromosome segregationgene CSE1

(immunotoxin/Pseudomonas exotoxin/tumor necrosis factor/oncogene)

ULRICH BRINKMANN, ELISABETH BRINKMANN, MARIA GALLO, AND IRA PASTAN*Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, Building 37, Room4E16, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255

Contributed by Ira Pastan, July 27, 1995

ABSTRACT We recently isolated human cDNA fragmentsthat render MCF-7 breast cancer cells resistant to cell deathcaused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosisfactor. We report here that one of these fragments is anantisense fragment ofa gene homologous to the essential yeastchromosome segregation gene CSE1. Cloning and analysis ofthe full-length cDNA of the human CSE1 homologue, which wename CAS for cellular apoptosis susceptibility gene, reveals aprotein coding region with similar length (971 amino acids forCAS, 960 amino acids for CSE1) and 59% overall proteinhomology to the yeast CSE1 protein. The conservation of thisgene indicates it has an important function in human cellsconsistent with the essential role of CSE1 in yeast. CAS ishighly expressed in human tumor cell lines and in humantestis and fetal liver, tissues that contain actively dividingcells. Furthermore, CAS expression increases when restinghuman fibroblasts are induced to proliferate and decreaseswhen they are growth-arrested. Thus, CAS appears to play animportant role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

We have used expression cloning to isolate cDNAs containingplasmids that cause MCF-7 breast cancer cells to becomeresistant to immunotoxins (1). These plasmids also render thecells resistant to native Pseudomonas exotoxin (PE) and diph-theria toxin (DT); thus the resistance is due to the action of thetoxin moiety of immunotoxins and not due to reduced expres-sion of the antigen to which the immunotoxin is directed. OnecDNA plasmid (p17 in ref. 1) was particularly interestingbecause the phenotype it produced could not be attributed toknown mechanisms of PE and DT resistance (2, 3). In fact,when cells transfected with this plasmid were exposed to toxin,modification of elongation factor EF2 and inhibition of proteinsynthesis was comparable to toxin-sensitive controls, yet thecells did not die. Thus, this cDNA appears to be involved in amechanism affecting the sensitivity of cells to toxin after theprimary action of the toxin, inhibition of protein synthesis, hasoccurred. The observation that PE and DT can induce apo-ptosis (4-6) and the observation that this plasmid also confersresistance to tumor necrosis factor (TNF) a and ,B indicate thatthis cDNA could mediate resistance by interference withapoptosis (unpublished results). Here we describe that theisolated resistance plasmid that mediates toxin as well as TNFresistance to MCF-7 cells contains an antisense cDNA frag-ment homologous to the yeast CSE1 gene (7). We report thesequence of the human CSE1 homologue and present evidencethat this gene may play a role in cell proliferation.t

MATERIALS AND METHODSExpression Cloning and Immunotoxin Selection of cDNA

Plasmids. Human cDNAs that confer resistance to the immu-notoxin B3(Fv)-PE38KDEL were isolated by expression clon-ing and immunotoxin selection from a cDNA library inpCDM8 (HeLa cDNA expressed from a cytomegaloviruspromoter followed by simian virus 40 poly(A) as described (1).B3(Fv)-PE38KDEL is a fusion protein composed of a trun-cated form ofPE and the Fv region of monoclonal antibody B3that binds to a carbohydrate present on many carcinomas andcancer cell lines-e.g., on MCF-7 cells-and kills such cells (8).pCDM/HE17 (p17 in ref. 1) is a plasmid that confers resis-tance not only to immunotoxin, PE, and DT but also to TNF.

Cloning of CAS cDNA. Plaques (1 x 106) of a Agtll cDNAlibrary from placenta poly(A)+ RNA (Clontech) werescreened by hybridization with a 32P-labeled CAS cDNAfragment. The largest inserts, -2.4 kb, included the 3' end butnot the 5' end of the cDNA. The 5' end was obtained by rapidanalysis of cDNA end procedure (RACE) using 5'-RACE-Ready placenta cDNA (Clontech) as template and the Clon-tech anchor primer 5'-CTGGTTCGGCCCACCTCTGAAG-GTTCCAGAATCGATAG-3' and a specific primer 5'-TAA-TGAGGTCTCTCACAAA-3' positioned 160 bp downstreamof the 5' end of the longest insert. Amplification for 30 cycles(2 min at 94°C/2 min at 60°C/2 min at 72°C, extension for 10sec) using a Perkin-Elmer GeneAmp XL PCR kit resulted ina single 1160-bp fragment with the correct 5' end of the CASbecause the overlap of this fragment 3' end matched with thefirst 160 bp of the longest previously obtained lambda clone 5'end. The A fragments and RACE fragments were cloned intopCRII (Invitrogen).

Sequence Analysis. An Applied Biosystems model 373Asequencer and an Applied Biosystems Dye-Deoxy Terminatorkit were used for sequencing. Homology analyses and motifsearches were performed with the Genetics Computer Grouppackage (Version 8; Madison, WI) or BLAST (9) with theNational Center for Biotechnology Information BLAST net-work service, using updated GenBank, European MolecularBiology Laboratory, Protein Identification Resource, andSwissProt data bases.Northern Analysis. Northern blots containing -2 jig of

poly(A)+ mnRNA of various tissues separated on a formalde-hyde agarose gels (Clontech) were hybridized with 32P-labeledCAS, actin, or glyceraldehyde 3-phosphate dehydrogenase(G3PDH) probes (-109 cpm/,ug) for 20 hr at 50°C in Hybrisol

Abbreviations: PE, Pseudomonas exotoxin; DT, diphtheria toxin;TNF, tumor necrosis factor; CAS, cellular apoptosis susceptibilitygene; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; RACE,rapid analysis of cDNA end procedure.*To whom reprint requests should be addressed.tThe sequence discussed in this paper has been deposited in theGenBank data base (accession no. U33286).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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10428 Cell Biology: Brinkmann et al.

1 (50% formamide; Oncor). Blots were documented on KodakXAR-2 film at -70°C with screen for 20 hr (CAS) or less (actinand G3PDH) and quantitated on a Molecular DynamicsPhosphoImager model 425. RNA levels were measured bycomparing the signals of CAS to actin and G3PDH signals.

Immunoanalysis. Polyclonal anti-CAS antibodies were ob-tained by immunizing rabbits with recombinantly producedand (Ni-)affinity-purified histidine-tagged CAS protein frag-ments (amino acids 1-284 or 327-669, Fig. 1). Details of theimmunization and antibodies will be published elsewhere (U.Scherf, I.P., U.B., unpublished work). Comparison of preim-mune and immune sera show a - 100-kDa protein that isspecifically recognized with immune serum. For CAS quanti-fication by immunoanalysis, cell extracts were adjusted to equalprotein concentrations with Coomassie-stained SDS/PAGE;then equal amounts were transferred to nitrocellulose. CASwas detected with anti-CAS serum, biotin-anti-rabbit IgG,avidin-horseradish peroxidase, and Amersham epichem-iluminescence reagents (Amersham ECL protocol). Signalswere quantified by using an AMBIS Quantprobe image ana-

lyzer.

A Homology of human CAS and yeast CSE1

1 MELSDANLQTLTEYLKKTLDPDPAIRRPAEKFLESVEGNQNYPLLLLTLL

1 ..MSD..LETVAKFLAESVIASTA..KTSERNLRQLETQDGFGLTLLHVI

51 EKSQDNV.IKVCASVTFKNYIKRNWRIVEDEPNKICEADRVA.IKANIVH

45 ASTNLPLSTRLAGALFFKNFIKRKW..VDENGNHLLPANNVELIKKEIVP

99 LMLSSPEQIQKQLSDAISIIGREDFPQKWPDLLTEMVNRFQSGDFHVING

93 LMISLPNNLQVQIGEAISSIADSDFPDRWPTLLSDLASRLSNDDMVTNKG

149 VLRTAHSLFKRYRHEFKSNELWrEIKLVLDAFALPLTNLFKATIELCSTH11 .111:111:1. 1:1:11: 1111111.1. 1: 11:1.. 1..

143 VLTVAHSIFKRWRPLFRSDELFLEIKLVLDVFTAPFLNLLKTVDEQITAN

199 ANDASALRILFSSLILISKLFYSLNFQDLPEFWEGNMETWMNNFHTLLTL

193 ENNKASLNILFDVLLVLIKLYYDFNCQDIPEFFEDNIQVGMGIFHKYLSY

249 DNKLLQTDDE.EEAGLLELLKSQICDNAALYAQKYDEEFQRYLPRFVTAI

243 SNPLLEDPDETEHASVLIKVKSSIQELVQLYTTRYEDVFGPMINEFIQIT

298 WNLLVTTGQEVKYDLLVSNAIQFLASVCERPHYKNLFEDQNTLTSICEKV

293 WNLLTSISNQPKYDILVSKSLSFLTAVTRIPKYFEIFNNESAMNNITEQI

348 IVPNMEFRAADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFF

343 ILPNVTLREEDVELFEDDPIEYIRRDLEGSDTDTRRRACTDFLKELKEKN

398 EGPVTGIFSGYVNSMLQEYAKNPSVNWKHKDAAIYLVTSLASKAQTQKHGI . II .I I: .: .: : :: :I . :II I II I I III.I.II .:.. .

393 EVLVTNIFLAHMKGFVDQYMSDPSKNWKFKDLYIYLFTALAINGNITNAG

448 ITQANELVNLTEFFVNHILPDLKSANVNEFPVLKADGIKYIMIFRNQVPK:. .111.1...1I1.11 .: :1.:11.1111:.1

443 VSSTNNLLNVVDFFTKEIAPDLTSNNIPHI.ILRVDAIKYIYTFRNQLTK

498 EHLLVSIPLLINHLQAGSIVVHTYAAHALERLFTMRGPNN..ATLFTAAE5.:I: 0:1: . 11.:..11.1 11 .:1:::1:1:.1. 1:1 .:

492 AQLIELMPILATFLQTDEYVVYTDAAITIEKILTIRESNTSPAFIFHKED

546 IAPFVEILLTNLFKALTLPGSS ..... ENEYIMKAIMRSFSLLQEAIIPY1. .1111.11: : .111 11::11 :11

542 ISNSTEILLKNLIALILKHGSSPEKLAENEFLMRSIFRVLQTSEDSIQPL

591 IPTLITQLTQKLLAVSKNPSKPHFNHYMFEAICLSIRITCKANPAAVVNF

592 FPQLLAQFIEIVTIMAKNPSNPRFTHYTFESIGAILNYTQRQNLPLLV..

641 EEALFLVFTEILQNDVQEFIPYVFQVMSLLLETHKNDIPSSYMALFPHLL

640 DSMMPTFLTVFSEDIQEFIPYVFQIIAFVVE. QSATIPESIKPLAQPLL

Proc. Natl. Acad. Sci. USA 92 (1995)

RESULTSA Plasmid That Renders MCF-7 Cells Resistant to PE, DT,

and TNF Contains a Human cDNA Fragment Homologue ofYeast CSE1 Gene. pCDM/HE17 is a plasmid containing a700-bp HeLa cDNA that was isolated by a combination ofexpression cloning and immunotoxin selection from a HeLacDNA library transfected into MCF-7 breast cancer cells. Thisplasmid renders MCF-7 cells -10-fold less sensitive toward aPE-derived immunotoxin, as well as to native PE and DT (1).Sequence analysis of pCDM/HE17 reveals that it contains a436-bp cDNA fragment that is homologous to the yeast CSEIchromosome segregation gene (ref. 7; 45% protein identity,66% similarity in this portion, Fig. 1). This cDNA fragment,which contains nt 2100-2536 of the full-length clone (Gen-Bank accession no. U33286), is present in an "inverse" orien-tation, suggesting that transcription from the cytomegaloviruspromoter of pCDM/HE17 generates antisense RNA.pCDM8/HE17 also contains 264 bp of an unrelated sequencefused to the antisense fragment. This fusion appears to be alibrary-ligation artifact because this sequence is not related tothe sequence of the full-length CSE1 homologue isolated from

691 QPVLWERTGNIPALVRLLQAFLERGSNTIASAAADKIPGLLGVFQKLIAS.1 :1 .1111:.11..:. :..:: .:..11:11:1111

688 APNVWELKGNIPAVTRLLKSFIKTDSSIFP.....DLVPVLGIFQRLIAS

741 KANDHQGFYLLNSIIEHMPPESVDQYRKQIFILLFQRLQNSKTTKFIKSF

733 KAYEVHGFDLLEHIMLLIDMNRLRPYIKQIAVLLLQRLQNSKTERYVKKL

791 LVFINLYCIKYGALALQEIFDGIQPKKFGMVLEKIIIPEIQKVSGNVEKK

783 TVFFGLISNKLGSDFLIHFIDEVQDGLFQQIWGNFIITTLPTIGNLLDRK

841 ICAVGITNLLTECPPMMDTEYTKLWTPLLQSLIGLFELPEDDTIPDE.EH1. :1: 1::.: ..:::..1..1 .:.1:1: ....::

833 IALIGVLNMVIN.GQFFQSKYPTLISSTMNSIIETASSQSIANLKNDYVD

890 FIDIEDTPGYQTAFSQLAFAGKKEHDPVGQMV..NNPKIHLAQSLHMLST.::1:...:..V11.1. :.1. 11:.:: I..::.:1:.1:....882 LDNLEEISTFGSHFSKLVSISEKPFDPLPEIDVNNGVRLYVAEALNKYNA

938 ACPGRVPSMVSTSLNAEALQYLQGYLQAASVTLL-:....: . 1.1. I...1 :.

932 ISGNTFLNTILPQLTQENQVKLNQLLVGN*.....

971 CAS

960 CSE1

B Repetitive Sequences in CAS

500506529553582602623651669689707730750773805821849868890906935955

LL VSI pLL INHLQ A GSLF TMRG P NNLL TNLFK A LTLLL QEAII P YILL AVSKN P SKCL SIRIT C KANIL QNDVQ E FILL LETHK N DILL QPVLW E RTLL QAFLE R GSLL GVFQK L IALL NSIIE H MPLL FQRLQ N SKAL QEIFD G IQVL EKIII P EILL TECP P MMLL QSLIG L FELFl DIEDT P GYLA FAGKK E HDXL STAC P GRVAL QYLQ G YL

I VVHTYAAHALERA TLFTAAEIAPFVEIP GSSENEYIMKAIMRSFSP TLITQLTQKP HFNHYMFEAIP AAVVNFEEALFLVFTEP YVFQVMSP SSYMALFPHG NIPALVRN TIASAAADKIPGS KANDHQGFYP ESVDQYRKQIFIT TKFIKSFLVFINLYCIKYGALP KMFGMQ KVSGNVEKKICAVGITND TEYTKLWTPP EDDTIPDEEHQ TAFSQP VGQMVNNPKIHLAQSLHP SMVSTSLNAEQ AASVT LL

FIG. 1. Sequence of CAS and homology to CSE1. The CAS cDNA sequence from placenta is deposited in GenBank (accession no. U33286).The initially isolated (HeLa) CAS antisense plasmid pCDM/HE17 (p17 in ref. 1) contains cDNA from positions 2536-2100, amino acids 662-807.(A) Protein sequence of CAS aligned to CSE1 from yeast. (B) The C terminus of CAS appears composed of consecutive repetitions of similarsequence stretches containing double leucines and prolines. Note that the repetitive units are not only similar at the leucine and proline positionsbut also throughout the whole repeat unit.

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human placenta cDNA and also is not linked to the gene inHeLa cells, as shown by hybridization of Southern blots ofHeLa and placenta DNA cleaved with various enzymes (datanot shown). Furthermore, this unrelated sequence does nothave an open reading frame and, therefore, is noncoding, andits expression cannot be detected by Northern blot analysis inany human tissue. We reasoned, therefore, that the resistancemediated by pCDM/HE17 is due to antisense RNA to thehuman CSEJ homologue, which we call GAS (cellular apo-'ptosis susceptibility gene; U.B., unpublished work).

Cloning of GAS cDNA from Human Placenta and ItsHomology to Yeast CSE1. To obtain a complete GAS codingsequence, we used the GAS cDNA fragment from the resis-tance plasmid (p17 in ref. 1) as probe to screen a humanplacenta cDNA library in Xgtll. We isolated several cDNAinserts, the longest being -~2.4 kb. A full-length clone isexpected to be -~3.1 kb from the mRNA size in Northern blots(Fig. 2). Because all cDNA clones obtained were from the 3'end of the cDNA and thus lacking the 5' end of GAS, weisolated the 5' end of the cDNA by RAGE (see Materials andMethods). The nucleotide sequence of GAS is deposited inGenBank, and the deduced amino acid sequence of GAS andits homology to GSE1 is shown in Fig. 1A. The GAS cDNAfrom HeLa used as a screening probe had the same sequenceas the human placenta cDNA and corresponds to nt 2100-2536of the full-length clone. The coding regions of human GAS andyeast CSEI are of approximnately the same size, for 971 and 960amino acids, respectively, and their sequence is similar overtheir whole length with some small gaps. The overall homology(protein similarity) is 59%, and in some portions, the homol-ogy is >75% with 50% identity. Data base analyses (updatedGenBank, SwissProt, Protei'n Identification Resource, Euro-pean Molecular Biology Laboratory) showed that several shortexpressed sequence tags correspond to the 3' end and one tothe 5' end of GAS cDNA. No other protein except GSE1 hadsignificant homology to GAS. Standard motif search programs(Genetics Gomputer Group MOTIFS) did not reveal any se-quence motif that would allow a prediction of the possiblemolecular function of GAS. However, visual inspection and"manual" motif searches (homology searches-with GAS se-quence-derived peptides) revealed that GAS contains someshort sequence stretches with similarity to the phosphorylation

Human Tissues

sites of the mitogen-activated protein kinases ERKi andERK2 (named for extracellular signal-regulated kinase) and apossible phosphorylation site in transcription factor TFIID(refs. 11 and 12; positions 9-37 of GAS), a basic region thatmight be a nuclear localization or DNA-binding site (positions372-385, also noticed in GSE1, ref. 7), and a His-Xaa2-His-Xaa6-Gys-Xaa6-Gys sequence at position 613-630 that mightform a Zn finger. Furthermore, the G-terminal portion of GASappears to be composed of repetitions of a double leucine andproline-containing sequence (Fig. 1B). The possible functionof these sequences remains to be evaluated.

Expression of GAS in Human Tissues and Cancer CellLines. The extensive homology illustrated in Fig. 1 indicatesthe GAS and CSEJ genes are very conserved and may havesimilar and important functions. This result is consistent withthe observation that in yeast CSEJ is an essential gene, andhomozygous CSE1 mutations are lethal. CSEJ has a role in cellproliferation or division (chromosome segregation, ref. 7). Todetermine whether GAS also may have a role in cell prolifer-ation we analyzed its expression in human tissues and in tumorcell lines by Northern blot analyses. We found that the size ofits mRNA is %ZZ'3.1 kb. GAS mRNA is present in many tissues(Fig. 2). Expression is very high in testis and fetal liver, whichcontain many actively proliferating cells, and elevated intissues that contain some proliferating cells-e.g., lymphoidtissues. Expression is also slightly elevated in skeletal muscle.Very low expression is detected in peripheral blood, whichdoes not contain proliferating cells. We also examined severaldifferent tumor cell lines, including SW480 (colon), A-549(lung), and HL-60, K-562, Molt-4 and Raji (leukemias andlymphomas). These lines all contain high levels of GAS mRNA(Fig. 2). A quantitation of GAS expression, normalized to theexpression of actin or G3PDH, is shown in Fig. 3. These dataindicate that GAS is preferentially expressed in proliferatingcells'and, therefore, might play a role- in cell proliferation.

Expression of CAS in Proliferating and Resting HumanFibroblasts. To investigate further the possible role of GAS incell proliferation, we analyzed whether GAS expression isinfluenced by or correlates with the proliferation status ofhuman fibroblasts. To do that, active'ly growing WI-38 fibro-blasts (in 10% serum) were shifted to 0.15% serum for 3 days.This serum starvation leads to growth arrest. The cells were

Cancer Cell Lines

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G3PDH

FIG. 2. Expression of CAS in human tissues and some cancer cell lines. Northern blots containing -2 tLg of poly(A)l RNA in each lane werehybridized with GAS-specific probe or actin and G3PDH control probes that had comparable specific activities. Films were exposed at - 70'C withscreen for 18 hr with the GAS probe and 6 hr with the controls. Control hybridizations show that approximately equal amounts ofRNA were loadedfor most tissues, except for skeletal mu'scle, which could not be exactly quantitated due to an additional actin band and apparently elevated G3PDH.Some tissues that appear in duplicate demonstrate reproducibility of the results. ca, Carcinoma.

Cell Biology: Brinkmann et al.

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10430 Cell Biology: Brinkmann et al.

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PhosphoImager. Relative expression was calculated as follows: The CAS/control ratio found in most tissues (mean of prostate, small intestine, colon,heart, brain, lung, and pancreas) was defined as a basal level and set to 1. Testis, which is the highest tissue, is set to 100; the others tissues arerelative to these.

then returned to 10% serum, in which the cells resumed cellproliferation until they reached confluency, when growth wasagain arrested. Protein samples were obtained from the cellsunder the different growth conditions and analyzed by immu-noblot with polyclonal rabbit anti-CAS antibodies. The relativeamount of CAS protein, which migrates as an -100-kDaprotein, was determined by image analyses of the immunoblots(Fig. 4; see Materials and Methods). We found that CAS

%Serum: 10% 0.15% 10%

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FIG. 4. CAS expression in resting and proliferating WI-38 cells.Equal amounts of total cell extracts were separated on a reducingSDS/4-15% PAGE stained with Coomassie blue to demonstrateequal loading and then analyzed by immunoblot and image quantifi-cation of the immunoblot. Polyclonal rabbit anti-CAS antibodies wereused to detect CAS, which appears as 100-kDa protein. WI-38 cellswere growth-arrested by serum starvation and then induced to pro-liferate by serum addition. Cells on day 0 are grown in 10% serum andsampled just before serum downshift; their CAS level was set to 100%.Values on days 1 and 3 are from cells in low serum (0.15%) for theindicated time; on day 3 after taking the serum-starved sample, cellswere returned to 10% serum and then sampled on days 4, 6, and 7.

protein levels are high in cells that are actively growing in 10%serum and decline rapidly upon growth arrest caused by serumstarvation. Furthermore, the amount of CAS protein increaseswhen the resting cells are induced to proliferate by addition of10% serum. Finally, CAS levels decline again when the cellsbecome confluent and, therefore, growth-arrested. These re-sults confirm a correlation of CAS expression and cell prolif-eration.

DISCUSSIONWe have previously isolated a plasmid containing a humancDNA that can render MCF-7 breast carcinoma cells resistantto the cytotoxic effects of PE and DT (1) as well as to TNF-aand TNF-,3 (unpublished results). This plasmid causes resis-tance by rendering cells less sensitive to toxin and TNF-mediated apoptosis (unpublished results). It contains an an-tisense cDNA fragment that is a homologue to the yeast CSEJgene, an essential gene involved in cell division (7). This humanCSE1 homologue, called CAS gene, is of the same size andshows remarkable homology to the yeast CSE1 gene product.This result suggests it might be a gene with an essential andconserved function, which according to the function describedfor the yeast CSE1 gene would be in chromosome segregation(cell division). This hypothesis is supported by the observationthat the CAS gene is preferentially expressed in human tissuescontaining proliferating cells and in proliferating tumor celllines and that CAS expression correlates with cell proliferationin human WI-38 fibroblasts. We conclude that CAS might havetwo functions: one in cell death induced by toxins and TNF andone in cell proliferation or cell division.

Evidence for a function of CAS in cell proliferation is itshomology to the yeast CSE1 gene. In yeast, CSE1 is involvedin cell division and was identified due to a mutant chromosomesegregation phenotype (7). The mechanism of action of theCSE1 gene product is not yet established. Recently it wasfound that CSE1 mutants interfere with B-type cyclin degra-dation (13). Examination of the sequence of CSE1 and CAS bycomputer programs designed to identify consensus sequencesfor specific functions has not identified any motifs that unam-biguously could assign a function to CAS. Chromosome seg-regation genes can be cell cycle check points (10, 14). The factthat CSE1 is essential in yeast indicates that CSE1 and CAS

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might have such a function. This activity would also explain itsconservation in higher eukaryotes. The fact that CAS is highlyexpressed in human tissues containing proliferating cells (moststrongly in testis), in tumor cell lines, and in normal cellsinduced to proliferate by serum addition also suggests a rolefor the human CSE1 homologue in cell proliferation.

It is interesting that a cDNA that was originally isolatedbecause its expression in an antisense orientation interfereswith toxin and TNF-mediated cell death (hence the name CASfor cellular apoptosis susceptibility gene) turns out to beassociated with cell proliferation. However, it is feasible thatgenes, such as CSE1, that are involved in cell division in yeast,may in higher eukaryotes be "switch points" to decide whethera cell should undergo apoptosis. A connection between cellproliferation and apoptosis has been demonstrated for MYC,which can induce cell proliferation as well as make cellssusceptible to apoptosis; for p53, which blocks cell growth andinduces apoptosis; and for BCL2, which stimulates cell growthand prevents apoptosis (10, 15, 16). Genes that regulateapoptosis and cell growth can also function as oncogenes,suggesting a possible role of CAS in cancer.

We thank D. Lipman for help in sequence homology searches andDr. Uwe Scherf (Laboratory of Molecular Biology, National CancerInstitute) for anti-CAS antibodies. For M.G. this publication is apartial fulfillment of the requirements for a Ph.D. from GeorgeWashington University.

1. Brinkmann, U., Brinkmann, E. & Pastan, I. (1995) Mo. Med. 1,206-216.

2. Carroll, S. F. & Collier, R. J. (1987) J. Bio. Chem. 262, 8707-8711.

3. Fendrick, J. L., Iglewsky, W. J., Moehring, J. M. & Moehring,T. J. (1992) Eur. J. Biochem. 205, 25-31.

4. Kochi, S. K. & Collier, R. J. (1993) Ep. Cell Res. 208, 296-302.5. Chang, M. P., Bramhall, J., Graves, S., Bonavida, B. & Wisniesky,

B. J. (1989) 1. Bio. Chem. 264, 15261-15267.6. Morimoto, H. & Bonavida, B. (1992) J. Immunol. 149, 2089-

2094.,j;. t.7. Xiao, Z., McGrew, J. T., Schroeder, A. J. & Fitzgerald-Hayes, M.

(1993) Mo. Cell. Biol. 13, 4691-4702.8. Brinkmann, U., Pai, L. H., FitzGerald, D. J., Willingham, M. &

Pastan, I. (1991) Proc. Natl. Acad. Sci. USA 88, 8616-8620.9. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman,

D. J. (1990) J. Mol. Biol. 215, 403-410.10. Hartwell, L. H. & Kastan, M. B. (1994) Science 266, 1821-1828.11. Gonzalez, F. A., Raden, D. L., Rigby, M. R. & Davis, R. J. (1992)

FEBS Lett. 304, 170-178.12. Sekiguchi, T., Nohiro, Y., Nakamura, Y., Hisamoto, N. &

Nishinoto, T. (1991) Mol. Cell. Biol. 11, 3317-3325.13. Irniger, S., Piatti, S., Michaelis, C. & Nasmyth, K. (1995) Cell 81,

269-277.14. Nugroho, T. T. & Mendenhall, M. D. (1994) Mo. Cell. BioL. 14,

3320-3328.15. Hermeking, H. & Eick, D. (1994) Science 265, 2091-2093.16. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, 0. &

Korsmeyer, S. J. (1991) Cell 67, 879-888.

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