Proc. Natl. Acad. Sci. USAVol. 93, pp. 136-140, January 1996Cell Biology

Molecular cloning of mesothelin, a differentiation antigen presenton mesothelium, mesotheliomas, and ovarian cancers

(glycosylphosphatidylinositol anchor/plasma membrane/antibody/cell adhesion)

KAI CHANG AND IRA PASTANLaboratory 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, October 9, 1995

ABSTRACT Monoclonal antibody MAb Ki recognizes a40-kDa glycoprotein present on the surface of mesothelialcells, mesotheliomas, and ovarian cancers. We have used MAbKl to isolate a 2138-bp cDNA that encodes this antigen. ThecDNA has an 1884-bp open reading frame encoding a 69-kDaprotein. When the cDNA was transfected into COS and NIH3T3 cells, the antigen was found on the cell surface and couldbe released by treatment with phosphatidylinositol-specificphospholipase C. The 69-kDa precursor is processed to the40-kDa form. The protein has been named mesothelin becauseit is made by mesothelial cells. Mesothelin may play a role incellular adhesion.

Monoclonal antibodies are currently being used to diagnoseand treat cancer (1, 2). To be useful for therapy, the antibodyshould recognize an antigen that is present in large amounts onthe cancer cells and in negligible amounts on normal cells.Alternatively, the antigen can be present in substantialamounts on normal cells, if the normal cells are not compo-nents of an essential organ. This approach has been useful indeveloping new treatments for leukemias and lymphomas.Several differentiation antigens have been identified on lym-phomas and leukemias which are good targets for immuno-therapy because they are not present on the stem cells whichgive rise to differentiated lymphocytes (2). Thus, normallymphocytes that are killed by immunotherapy can be regen-erated. Some examples of lymphocyte antigens of this type areCD 19, CD22, CD25, and CD30 (2, 3). Clearly, it would be veryuseful to have antibodies that recognize differentiation anti-gens on solid tumors, but only a small number of these areavailable. One reason contributing to the paucity of suchantibodies is that efforts to identify differentiation antigens onvarious types of epithelial cells have been relatively modestcompared with the intense efforts made to identify differen-tiation antigens on cells of the hematopoietic system.

Ovarian cancer represents one of the diseases which couldbe treated by immunotherapy, because the ovaries are alwaysremoved during surgery for this disease and reactivity withnormal ovarian tissue is not a problem. Several antibodies thatrecognize differentiation antigens on ovarian cancer cells havebeen generated. One of these is OC125, which recognizes theCA125 antigen (4). CA125 is a high molecular weight glyco-protein that is shed by ovarian cancer cells and has been usefulin the diagnosis of ovarian cancer. However, antibodies toCA125 are not useful for immunotherapy because the CA125antigen is shed into the bloodstream (4). Another is MOV18,which recognizes the folate-binding protein. This protein isabundant in ovarian cancers as well as in some other tumors.Unfortunately, this protein is also abundantly expressed inkidney (5). We have isolated an antibody termed MAb Kl thatreacts with many ovarian cancers and many mesotheliomas.

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.

Like OC125, the antibody also reacts with normal mesothelialcells, but it does not react with other cell types except for weakreactivity with some cells in the trachea (6, 7). The antigenrecognized by MAb KI appears to be a differentiation antigenpresent on mesothelium and is expressed on cancers derivedfrom mesothelium, such as epithelioid type mesotheliomas, aswell as on most ovarian cancers. Thus immunotherapy directedat the CAKI antigen will run the risk of damaging normalmesothelial cells and perhaps cells of the trachea (6-9).

In the ovarian cancer cell line OVCAR-3 as well as HeLacells, the antigen has been shown to be an -40-kDa glyco-protein that is attached to the cell surface by phosphatidyl-inositol. The protein is released when cells are treated withphosphatidylinositol-specific phospholipase C (PI-PLC) (7).We have previously attempted to clone a cDNA encoding theCAK1 antigen but instead have cloned cDNAs encoding twointracellular proteins which also react with MAb KI (10).Neither of these is the cell surface antigen recognized by MAbKI. Here we describe the cloning of a cDNA that encodes theCAKM antigen that is expressed on the cell surface.* We haveexpressed the cDNA in 3T3 cells and characterized the proteinmade by these cells.

MATERIALS AND METHODSCells and Antibodies. Human ovarian tumor cell line

OVCAR-3 and cell lines A431, KB, MCF-7, COS-1, WI-38,and NIH 3T3 were obtained from the American Type CultureCollection. Cells were cultured either in RPMI 1640 or Dul-becco's modified Eagle's medium (DMEM) (GIBCO) supple-mented with L-glutamine (2 mM), penicillin (50 units/ml),streptomycin (50 jig/ml), and 5-10% fetal bovine serum (FBS)(GIBCO). NIH 3T3 transfectants were grown inDMEM with theneomycin analogue G418 (GIBCO) at 0.8 mg/ml. Cells wereused when they reached 80-90% confluency, after threewashes with ice-cold phosphate-buffered saline (PBS)(GIBCO). MAb Kl and antibody MOPC-21 have been de-scribed (6) and were used at 5-10 ,ug/ml.

Isolation of the cDNA Clones. The HeLa S3 cDNA library(Clonetech) was screened as described (10) at -50,000 plaque-forming units per 150-mm filter with protein A-purified MAbKI (5 ,ug/ml) and peroxidase-conjugated goat anti-mouse IgG(heavy- and light-chain specific, 10 jig/ml; Jackson Immu-noResearch). Two positive plaques (A6-1 and A6-2) wereisolated and the phages were purified to homogeneity by threeor more rounds of screening. After verification of their spec-ificity with MAb Kl by showing they did not react with acontrol MOPC-21 antibody, single-plaque isolates of A6-1 andA6-2 were used for preparation of 5-10 phage plates, followedby extraction and purification of phage DNA with a A phage

Abbreviations: PI-PLC, phosphatidylinositol-specific phospholipaseC.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. U40434).

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Proc. Natl. Acad. Sci. USA 93 (1996) 137

DNA kit (Qiagen, Chatsworth, CA). Phage DNA was thendigested with EcoRI and the insert was subcloned into theEcoRI site of a pcDNAI/Amp (Invitrogen) vector by a rapidligation protocol (10). Plasmid DNAs were isolated by use ofQiagen's plasmid DNA isolation kit (10). Restriction mappingusing Xho I, EcoRI, Sal I, BamHI, and Nco I, as well as DNAsequencing, revealed that the two plasmid clones (p6-1 andp6-2) had identical 1500-bp inserts.To isolate a longer clone, the insert of p6-1 was purified to

make a cDNA probe (specific activity, 8.5 x 105 cpm/ml) byrandom priming. The HeLa S3 cDNA library was rescreenedby a filter hybridization method (10). Fourteen A clones wereisolated and purified, and their insert sizes were assessed bydigestion with EcoRI. Four large inserts were subcloned intoa pcDNAI/Amp plasmid vector (p9, p13-1, p16, and p18-1). p9contained the largest insert with a long open reading frame.DNA Sequence Analysis. By the use of T3 and T7 promoter

primers and twenty 17-bp synthetic primers, the entire cDNAinsert of p9 was sequenced by the method described by Sangeret al. (11) and an automatic cycle sequencing method (TaqDyeDeoxy Terminator Cycle sequencing kit; Applied Biosys-tems).Northern Blot Analysis. Total RNAs (20 jig) from

OVCAR-3, KB, MCF-7, A431, and W138 cells were electro-phoresed in a 1% agarose gel in Mops buffer with 16.6%formaldehyde and then transferred to a nylon paper. Northernhybridization was done with a method described before (10).The blot was washed and reprobed with a 32P-labeled humanf3-actin cDNA as an internal control to assess the integrity andquantity of the RNA samples loaded.

In Vitro Transcription and Translation. The TNT coupledreticulocyte lysate system, canine pancreatic microsomal mem-brane, 2 jig of plasmid DNAs of pcDICAK1-9, pAPK1 (10),and [3H]leucine were used in an in vitro transcription/transla-tion and translocation/processing experiment according to theprotocol of the manufacturer (Promega). Translation productswere resolved by SDS/10% PAGE under reducing conditions.The proteins were fixed and the unincorporated label wasremoved by soaking the gel three times for 30 min in 200 mlof buffer containing 40% methanol and 10% acetic acid indeionized water. The gels were then soaked for 30 min in 200ml of ENTENSIFY Part A and Part B (NEN). After drying,the translated products were visualized by autoradiography.

Expression of the Cloned cDNAs in Mammalian Cells.Transient transfections of COS cells were performed withpcDICAK1-9 (p9) and LipofectAMINE (GIBCO) accordingto the manufacturer's protocol. COS-1 cells were plated a daybefore the experiment at 2.5 x 105 cells per 60-mm dish.Twenty-four microliters of LipofectAMINE and 76 ,ul ofOptiMEM I medium were mixed with 10 jig of pcDNAI/Ampvector or pcDICAK1-9 in 100 p,l of OptiMEM-I medium atroom temperature for 30 min. After the COS-1 cells werewashed twice with OptiMEM-I 2.4 ml of OptiMEMI wasadded and the transfection mixtures were overlaid onto COS-1cells and incubated at 37°C. After 5 hr, 2.6 ml of DMEM with20% FBS was then added into each dish. Forty-eight hoursafter transfection, the dishes were subjected to immunofluo-rescence labeling (6, 7) or other treatments. The insert frompcDICAK1-9 (in pcDNAI/Amp) was also subcloned into apcDNA3 (Invitrogen) vector for stable transfection. Plasmidminipreps were made with Qiagen's Miniprep plasmid DNAkit and orientation of the insert in individual clone wasdetermined by restriction mapping. The resulting plasmid,pcD3CAK1-9, was then used to transfect NIH 3T3, MCF-7,A431, and OVCAR-3 cells by DNA-calcium phosphate pre-cipitation (12). After overnight exposure to the precipitate, thecells were washed with PBS three times and fed with freshDMEM/10% FBS for 2-3 days. Geneticin (G418 sulfate; 0.8mg/ml) was added and the cultures were maintained untilcolonies 2-3 mm in diameter were formed. Colonies were then

transferred into wells of a 96-well plate and then into a 35-mmdish when they were 80% confluent. Transfected cells werescreened by immunofluorescence (6, 7) and positive cells werefurther subcloned by limited dilution as described (6). One ofthe NIH 3T3 transfectant clones, NIH 3T3 K20, was chosen forfurther study. To localize the expression of CAK1, both cellsurface and intracellular immunofluorescence labeling wasalso performed according to methods described before (7).Treatment of Transfected Cells with PI-PLC. CAK1 cDNA-

transfected NIH 3T3 cells (NIH 3T3 K20 cells) were grown in175-mm2 flasks, and when they reached 90% confluency, thecells were washed in PBS three times. The cells were incubatedwith either 5 ml of PI-PLC (1.25 units/ml, from Bacillus cereus;Boehringer Mannheim) or 0.05% trypsin/0.052 mM EDTAfor 30 min at 37°C and 30 min at room temperature withshaking. The supernatants were collected and after centrifu-gation at 1000 x g and concentrated about 10-fold with aCentricon 30 unit (Amicon). The concentrated supernatantswere used in SDS/PAGE and immunoblot analysis. Theenzyme-treated cells can be recultured and the recovery ofCAKI expression can be seen after overnight culture. Treat-ment with PI-PLC was done in a similar manner using 35-mmdiameter dishes followed by immunofluorescence labeling ofthe treated cells (7).Immunoblot Analysis of Transfected NIH 3T3 Cells. Mem-

brane and cytosolic fractions from transfected NIH 3T3 K20cells (10) were subjected to SDS/12.5% PAGE and theresolved proteins were transferred to nitrocellulose. Immuno-blotting was performed as described (8, 10).

RESULTSExpression cloning was used to isolate the CAK1 cDNA. Wepreviously observed that MAb Kl reacts with OVCAR-3 andHeLa cells. Because we were unable to isolate the cDNA froman OVCAR-3 library (10), we screened a HeLa cDNA libraryexpressed in Agtll as described in Materials and Methods. Atotal of 1 X 106 phages were screened and 2 phage clones (A6-1and A6-2) were identified. DNA sequencing showed that bothphages contained the same 1.5-kb insert. The insert hybridizedto mRNA from OVCAR-3 and KB cells (a HeLa subclonewhich also reacts with MAb K1) but not to RNA from manyother cell lines, indicating that the cDNA is specific for cellsreacting with MAb Kl (Fig. 1). The mRNA detected was 2.2kb long, indicating that the insert isolated was not full-length.The insert contained an open reading frame, a stop codon, anda poly(A) tail but the 5' end appeared to be missing. Therefore,the phage library was rescreened with one of the inserts and 14

kb12 3 4 5

9.5-

2A-g

FIG. 1. CAK1 RNA levels in cell lines. Samples (20 j,g) of totalRNA from OVCAR-3 cells (lane 1), MCF-7 cells (lane 2), KB cells (aHeLa subclone; lane 3), A431 cells (lane 4), and W138 cells (lane 5)were resolved by electrophoresis, transferred to nylon paper, andprobed with a 32P-labeled CAK1 cDNA. Hybridization with an actincDNA probe showed that the lanes were equally loaded.

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138 Cell Biology: Chang and Pastan Proc. Natl. Acad. Sci. USA 93 (1996)

1 AGGAATTCCGGTGGCCGGCCACTCCCGTCTGCTGTGACGCGCGGACAGAGAGCTACCGGTGGACCCACGGTGCCTCCCTCCCTGGGATCT91 ACACAGACCATGGCCTTGCAACGGCTCGACCCCTGTTGGTCCTGTGGGGACCGCCCTGGCAGCCTCCTGTTCCTGCTCTTCAGCCTCGGA1 M A L Q R L D P C W S C G D R P G S L L F L L F S L G

181 TGGGTGCATCCCGCGAGGACCCTGGCTGGAGAGACAGGGACGGAGTCTGCCCCCCTGGGGGGAGTCCTGACAACCCCCCATAACATTTCC28 W V H P A R T L A G E T G T E S A P L G G V L T T P H N I 8

271 AGCCTCTCCCCTCGCCAACTCCTTGGCTTCCCGTGTGCGGAGGTGTCCGGCCTGAGCACGGAGCGTGTCCGGGAGCTGGCTGTGGCCTTG58 S L S P R Q L L G F P C A E V S G L S T E R V R E L A V A L

361 GCACAGAAGAATGTCAAGCTCTCAACAGAGCAGCTGCGCTGTCTGGCTCACCGGCTCTCTGAGCCCCCCGAGGACCTGGACGCCCTCCCA88 A Q K N V K L S T E Q L R C L A H R L S E P P E D L D A L P

451 TTGGACCTGCTGCTATTCCTCAACCCAGATGCGTTCTCGGGGCCCCAGGCCTGCACCCGTTTCTTCTCCCGCATCACGAAGGCCAATGTG118 L D L L L F L N P D A F S G P Q A C T R F F S R I T K A N V541 GACCTGCTCCCGAGGGGGGCTCCCGAGCGACAGCGGCTGCTGCCTGCGGCTCTGGCCTGCTGGGGTGTGCGGGGGTCTCTGCTGAGCGAG148 D L L P R G A P E R Q R L L P A A L A C W G V R G S L L S E631 GCTGATGTGCGGGCTCTGGGAGGCCTGGCTTGCGACCTGCCTGGGCGCTTTGTGGCCGAGTCGGCCGAAGTGCTGCTACCCCGGCTGGTG178 A D V R A L G G L A C D L P G R F V A E S A E V L L P R L V721 AGCTGCCCGGGACCCCTGGACCAGGACCAGCAGGAGGCAGCCAGGGCGGCTCTGCAGGGCGGGGGACCCCCCTACGGCCCCCCGTCGACA208 S C P G P L D Q D Q Q E A A R A A L Q G G G P P Y G P P S T811 TGGTCTGTCTCCACGATGGACGCTCTGCGGGGCCTGCTGCCCGTGCTGGGCCAGCCCATCATCCGCAGCATCCCGCAGGGCATCGTGGCC238 W S V S T M D A L R G L L P V L G Q P I I R S I P Q G I V A901 GCGTGGCGGCAACGCTCCTCTCGGGACCCATCCTGGCGGCAGCCTGAACGGACCATCCTCCGGCCGCGGTTCCGGCGGGAAGTGGAGAAG268 A W R Q R S S R D P S W R Q P E R T I L B P B F R R+ E V E K991 ACAGCCTGTCCTTCAGGCAAGAAGGCCCGCGAGATAGACGAGAGCCTCATCTTCTACAAGAAGTGGGAGCTGGAAGCCTGCGTGGATGCG298 T A C P S G K K A R E I D E S L I F Y K K W E L E A C V D A

1081 GCCCTGCTGGCCACCCAGATGGACCGCGTGAACGCCATCCCCTTCACCTACGAGCAGCTGGACGTCCTAAAGCATAAACTGGATGAGCTC328 A L L A T Q M D R V N A I P F T Y E Q L D V L K H K L D E L

1171 TACCCACAAGGTTACCCCGAGTCTGTGATCCAGCACCTGGGCTACCTCTTCCTCAAGATGAGCCCTGAGGACATTCGCAAGTGGAATGTG358 Y P Q G Y P E S V I Q H L G Y L F L K M S P E D I R K W N V

1261 ACGTCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCGACAAAGGGCACGAAATGAGTCCTCAGGCTCCTCGGCGGCCCCTCCCACAGGTG388 T S L E T L K A L L E V D K G H E M S P Q A P R R P L P Q V

1351 GCCACCCTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACACCCTGACCGCCTTCTACCCTGGGTACCTG418 A T L I D R F V K G R G Q L D K D T L D T L T A F Y P G Y L

1441 TGCTCCCTCAGCCCCGAGGAGCTGAGCTCCGTGCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTGTGACCCAAGG448 C S L S P E E L S S V P P S S I W A V R P Q D L D T C D P R

1531 CAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGTCCGAATACTTCGTGAAGATCCAGTCCTTCCTGGGT478 Q L D V L Y P K A R L A F Q N M N OS E Y F V K I Q S F L G

1621 GGGGCCCCCACGGAGGATTTGAAGGCGCTCAGTCAGCAGAATGTGAGCATGGACTTGGCCACGTTCATGAAGCTGCGGACGGATGCGGTG508 G A P T E D L K A L S Q Q N V 8 M D L A T F M K L R T D A V

1711 CTGCCGTTGACTGTGGCTGAGGTGCAGAAACTTCTGGGACCCCACGTGGAGGGCCTGAAGGCGGAGGAGCGGCACCGCCCGGTGCGGGAC538 L P L T V A E V Q K L L G P H V E G L K A E E R H R P V R D

1801 TGGATCCTACGGCAGCGGCAGGACGACCTGGACACGCTGGGGCTGGGGCTACAGGGCGGCATCCCCAACGGCTACCTGGTCCTAGACCTC568 W I L R Q R Q D D L D T L G L G L Q G G I P N G Y L V L D L

1891 AGCGTGCAAGAGACCCTCTCGGGGACGCCCTGCCTCCTAGGACCTGGACCTGTTCTCACCGTCCTGGCACTGCTCCTAGCCTCCACCCTG598 S V Q E T L S G T P C L L G P G P V L T V L A L L L A S T L

1981 GCCTGAGGGCCCCACTCCCTTGCTGGCCCCAGCCCTGCTGGGGATCCCCGCCTGGCCAGGAGCAGGCACGGGTGATCCCCGTTCCACCCC628 A

2071 AAGAGAACTCGCGCTCAGTAAACGGGAACATGCCCCCTGCAGACAAAAAAAAAAAAAAAAAAAAAAAA 2138

FIG. 2. Nucleotide sequence and deduced amino acid sequence of the CAK1-9 cDNA. The nucleotide sequence (upper line) and the deducedamino acid sequence (lower line) of the CAK1-9 cDNA are listed. The translation of CAK1-9 starts at nucleotides 100-102 (ATG) and terminatesat 1986-1988 (TGA). The putative signal peptide is underlined and a typical hydrophobic sequence for glycosylphosphatidylinositol anchorage isdoubly underlined. A possible furin cleavage sequence, RPRFRR, is underlined and the cleavage site is shown by an arrow. There are four potentialN-linked glycosylation sites (in boldface letters). A variant polyadenylylation signal (AGTAAA) is present 22 bp upstream from the poly(A) tail.The original p6-1 cDNA sequence spans nucleotides 721-2138.

new phages with cDNA inserts of various sizes were isolated.The largest insert (no. 9) was 2138 bp long and when sequencedcontained an open reading frame of 1884 bp (Fig. 2). Itcontains a typical Kozak sequence (13) (AXXATGG) fol-lowed by an open reading frame that encodes a 69 kDa protein.The sequence was not present in various data bases examined(EMBL/GenBank release 89.0, June 1995). Because theCAKi antigen was originally found to be about 40 kDa in size,several experiments were carried out to determine whetherclone 9 encoded CAK1.

In Vitro Translation. Insert 9 was cloned into a pcDNAI/Amp vector to make pcDICAK1-9 and used in the TNTreticulocyte system. A 69-kDa protein was produced (Fig. 3,lane 1). In the presence of pancreatic microsomes a slightlylarger protein was observed (lane 2), indicating that theprotein had been inserted into microsomes and glycosylated.As a control, a cDNA encoding a 30-kDa cytosolic protein thatalso reacts with MAb Ki (10) was subjected to the sameanalysis. The size of that protein was unaffected by thepresence of microsomes (lanes 3 and 4).

Expression in Cultured Cells. pcDICAK1-9 was transfectedinto COS cells for transient expression. Fig. 4 shows thespecific MAb Kl labeling pattern of COS cells transfected withinsert 9. In nonpermeabilized cells, a typical cell surfacefluorescent pattern was detected (Fig. 4A). In permeabilizedcells, strong staining of the Golgi region was evident (Fig. 4B).No cytosolic staining was detected. Also, no immunoreactivity

was detected in cells transfected with vector without insert(Fig. 4C) or with control inserts (data not shown). Thus, insert9 encodes a cell surface protein that is also present in the Golgicompartment.

Size and Processing ofCAKI Antigen. To determine the sizeof the antigen produced by cells transfected with insert 9, NIH3T3 cells were transfected with pcD3CAK1-9 to make stablecell lines. Stably transfected clones were produced as describedin Materials and Methods and the presence of antigen on thesurface was confirmed by immunofluorescence. Then mem-

1 2 3 4m - + - +

kDa

-68r -43_II~P -29

-18

FIG. 3. In vitro translation of CAK1 cDNA. Plasmids pcDICAK1-9(lanes 1 and 2) and pcDIAPK1 (lanes 3 and 4) were used in a TNTcoupled reticulocyte lysate system in the presence (+) or absence (-)of pancreatic microsomal membrane (m). The products were resolvedby SDS/10% PAGE under reducing conditions and autoradiographed.

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FIG. 4. Immunofluorescence of CAKI -9-transfected cells.pcDNAI/Amp vectors with insert 9 (A and B) or without insert (C andD) were transfected into COS cells. Two days later, the cells wereimmunocytochemically labeled with antibody MAb Ki at 4°C (forsurface labeling, A) or at 23°C (for intracellular labeling, B) andphotographed. Arrows indicate the typical Golgi distribution of therecombinant CAKI in the transfected cells. (X145.)

brane and cytosolic fractions were prepared from NIH 3T3K20 cells and from OVCAR-3 cells, subjected to SDS/PAGE,and analyzed by immunoblotting with MAb KI (Fig. 5). Aspreviously reported, the major reactivity in OVCAR-3 cellswas with a doublet of about 40 and 43 kDa that was present inmembranes but not in the cytosol. In the transfectants, twobands of equal intensity were detected in the membranefraction; one of about 40 kDa and a second of about 71 kDa.No signal was detected in the cytosol. These data suggest thatCAKI is made as a high molecular weight precursor that isprocessed by proteolysis to an 40-kDa form.

Nature of Cell Surface Attachment. To determine whetherCAKI was attached to the transfectants via a phosphatidyl-inositol linkage as it is in OVCAR-3 cells (7), the NIH 3T3transfectant cell line K20 was treated with PI-PLC for 60 min.Fig. 6A shows the strong cell surface labeling pattern inuntreated cells. Fig. 6C shows that fluorescence was absentafter treatment with PI-PLC. Fig. 6 B and D show phase-contrast images before and after treatment, respectively. Thetreated cells were still attached to the dish but were slightlyaltered in shape. The medium from PI-PLC-treated cells wasconcentrated, subjected to SDS/PAGE, and analyzed withMAb KI. A band of about 70 kDa was detected (data notshown), but no lower molecular weight bands were detected.

1 2 3 4 5 6

kDa

68- '~

43-4._i

FIG. 5. Immunoblot analysis of OVCAR-3 cells and NIH 3T3 cellstransfected with CAKI-9 cDNA. Approximately 100 jig of membranefraction (lanes 1 and 3) or cytosolic fraction (lanes 2 and 4) of thetransfected NIH 3T3 (pcD3CAK1) and mock control (pcD3) andmembrane (lane 5) or cytosolic fraction (lane 6) of OVCAR-3 cellswere electrophoresed and immunoblotted with MAb KI.

FIG. 6. Removal of the recombinant CAKI by PI-PLC treatmentof the transfected NIH 3T3 cells as assessed by immunofluorescence.The transfected NIH 3T3 K20 cells were treated with PI-PLC andlabeled with MAb KI as described in Materials and Methods. A and Bshow NIH 3T3 K20 cells without PI-PLC treatment. C and D dem-onstrate that the CAKI signal was completely abolished after PI-PLCtreatment. B and D are phase-contrast images. (x145.)

DISCUSSIONThis paper describes the molecular cloning of the CAKIantigen, which is found on mesothelium, mesotheliomas, ovar-ian cancers, and some squamous cell carcinomas. We havenamed this antigen mesothelin to reflect its presence onmesothelial cells. One unexpected feature of mesothelin is thatits cDNA encodes a 69-kDa protein, whereas the antigenpresent on OVCAR-3 cells, used to isolate MAb Ki, has amolecular mass of 40 kDa. The DNA sequence and thededuced amino acid sequence of CAKI are shown in Fig. 2.The cDNA is 2138 bp long and contains an open reading frameof 1884 bp. The protein it encodes contains 628 amino acidswith a calculated molecular mass of 69,001 Da. A homologyanalysis was performed with nucleotide or amino acid se-quences and none was detected with EMBL/GenBank ac-cessed by the Genetics Computer Group program. The proteincontains four potential N-linked glycosylation sites, NXS orNXT, that are shown in boldface letters. A typical signalsequence is not present at the amino terminus. However, ashort hydrophobic segment is located 15 amino acids from thefirst methionine (Fig. 2). This sequence might function as asignal sequence for membrane insertion, because the proteinis found on the cell surface (Figs. 4 and 5) and is inserted intomicrosomes during cell-free translation (Fig. 3). Also presentis a putative proteolytic processing site, RPRFRR, beginningat amino acid 293 (Fig. 2). This site is recognized by furin, aprotease important in the processing of several membraneproteins as well as in the activation of Pseudomonas anddiphtheria toxins (14). The 40-kDa form appears to be derivedfrom a 69-kDa precursor by several processing steps. These aresummarized in Fig. 7. Initially, mesothelin is made as a 69-kDapolypeptide with a hydrophobic tail which is probably removedand replaced by phosphatidylinositol (7). After glycosylation atone or more of its four putative N-linked glycosylation sites, itis cleaved by a protease to yield the 40-kDa fragment (ordoublet) found on the surface of OVCAR-3 cells and a smaller(31-kDa) fragment. The latter could be released into themedium and/or further degraded. The amino-terminal frag-ment has recently been detected in the medium of OVCAR-3cells (unpublished data). In transfected NIH 3T3 and MCF-7

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1 100 200 300 400 500 600 AAN I I I I I I I C

Precursor Form

77kDS.P. RR Kl Binding Portion

CHO

MembraneI. Intact7lkD

H. P.

CHO CHO CHO

RRForms

CHO

II. Cleaved-4OkD (-10kD CHO)

III. Shed3lkD

CHO CHO CHO

M

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CHOI I

CHO CHO

CHO

FIG. 7. Different forms of the CAK1 tumor antigen. S.P., putative signal peptide; H.P., glycosylphosphatidylinositol (GPI)-anchorage-dependenthydrophobic peptide; CHO, carbohydrates; M, membrane; AA, amino acids.

cells, we find approximately equal amounts of the 70-kDa and40-kDa proteins. We originally detected the 40-kDa form inOVCAR-3 and HeLa cells and did not notice a larger form.Reexamination of the OVCAR-3 and HeLa cell gels reveals atrace amount of the 70-kDa precursor (7). We have not yetexamined the size of mesothelin in human tissues or cancersamples or in other human cell lines to determine its size or thenature of its processing.

Mesothelin is one of many proteins and glycoproteins thatare attached to the cell surface by phosphatidylinositol. Severalfunctions have been ascribed to these molecules. Some arereceptors involved in cell signaling; others are involved incellular recognition and/or adhesion (15, 16). Glycosylphos-phatidylinositol-linked proteins may interact with tyrosinekinases (17, 18). Currently we have very little information onthe function of mesothelin. One possibility is that it has a rolein adhesion, since CAKi transfects are more slowly removedfrom culture dishes than nontransfected cells (unpublisheddata). Mesothelial cells are extremely flat and regulate thetraffic of molecules and cells in and out of the peritoneal cavity.Mesothelin may have a role in these processes. Mesothelincould also be responsible for the adhesion and implantation ofovarian carcinoma cells that frequently occur throughout theperitoneal cavity. The isolation of a cDNA clone that encodesmesothelin will make investigation of these phenomena pos-sible.

Mesothelin is very abundant in normal mesothelial cellsfrom which malignant mesotheliomas and ovarian cystadeno-carcinomas are derived. These two types of tumors share aunique biological characteristic that distinguishes them fromother solid tumors. In the early stages, both types of tumorsspread aggressively throughout the peritoneal (or thoracic)cavity and invade locally but do not metastasize distallythrough the lymphatic system or the bloodstream. In fact, manypatients succumb to their cancer before distant metastasesdevelop. Very little is known about the molecules involved inthe dissemination of mesotheliomas and ovarian cancers.Mesothelin may have a role in this process, since cells over-expressing mesothelin have altered adhesive properties (un-published data) and mesothelin expression is diminished inpoorly differentiated ovarian cancers (8, 9). Investigation ofmesothelin should not only advance our knowledge of me-

sothelial cell function but also promote our understanding ofthe role of this glycoprotein in carcinogenesis, invasion, andmetastasis of ovarian cancers and malignant mesotheliomas.

We thank Elizabeth Brinkmann for performing DNA sequencingand Althea Jackson for expert editorial assistance. This work is thesubject of a patent application, the rights of which belong to theNational Institutes of Health.

1. Mach, J.-P., Pelegrin, A. & Buchegger, F. (1991) Curr. Opin.Immunol. B, 685-693.

2. Grossbard, M. L., Press, 0. W., Appelbaun, F. R., Beinstein, I. D.& Nadler, L. M. (1992) Blood 80, 863-878.

3. Engert, A., Burrows, F., Jung, W., Tazzari, P. L., Stein, H.,Pfreundschuh, M., Diehl, V. & Thorpe, P. (1990) CancerRes. 50,84-88.

4. Bast, R., Klug, T. L., St. John, E., Jenison, E., Niloff, J. M.,Lazarus, H., Berkowitz, R. S., Leavitt, T., Griffith, T., Parker, L.,Zurawski, V. R. & Knapp, R. C. (1983) N. Engl. J. Med. 309,883-887.

5. Campbell, I. G., Jones, T. A., Foulkes, W. D. & Trowsdale, J.(1991) Cancer Res. 51, 5329-5338.

6. Chang, K., Pastan, I. & Willingham, M. C. (1992) Int. J. Cancer50, 373-381.

7. Chang, K., Pai, L. H., Batra, J. K., Pastan, I. & Willingham, M. C.(1992) Cancer Res. 52, 181-186.

8. Chang, K., Pastan, I. & Willingham, M. C. (1992) Int. J. Cancer51, 548-554.

9. Chang, K., Pai, L. H., Pass, H., Pogrebniak, H. W., Tsao, M.-S.,Pastan, I. & Willingham, M. C. (1992) Am. J. Surg. Pathol. 16,259-268.

10. Chang, K. & Pastan, I. (1994) Int. J. Cancer 57, 90-97.11. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad.

Sci. USA 74, 5463-5467.12. Chen, C. & Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752.13. Kozak, M. (1987) Nucleic Acids Res. 5, 8125-8148.14. Chiron, M. F., Fryling, C. M., FitzGerald, D. J. (1994) J. Biol.

Chem. 269, 18169-18176.15. Dustin, M. L., Selvaraj, P., Mattaliano, R. J. & Springer, T. A.

(1987) Nature (London) 329, 846-848.16. Stiernberg, J., Low, M. G., Flaherty, L. & Kincade, P. W. (1987)

J. Immunol. 38, 3877-3884.17. Stefanova, I., Horjsi, V., Ansotegui, I. J., Knapp, W. & Stock-

inger, H. (1991) Science 254, 1016-1019.18. Pandey, A., Shao, H., Marks, R. M., Polverini, P. J. & Dixit,

V. M. (1995) Science 268, 567-569.

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