Oncogene (1997) 14, 1129 ± 1136  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Identi®cation and characterization of neogenin, a DCC-related gene

Je€rey A Meyerhardt1, A Thomas Look4, Sandra H Bigner5 and Eric R Fearon1,2,3

Division of Molecular Medicine & Genetics, Departments of 1Internal Medicine, 2Human Genetics and 3Pathology, University of Michigan Medical Center, Ann Arbor, Michigan; 4Department of Experimental Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee; 5Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA

DCC (deleted in colorectal cancer), a candidate tumor suppressor genes await discovery (Knudson, 1993; suppressor gene located in chromosome band 18q21.2, Fearon, 1995; Lasko et al., 1991; Vogelstein et al., encodes a transmembrane protein of 1447 amino acids. 1989; Sato et al., 1990; Tsuchiya et al., 1992; Neogenin, a protein with nearly 50% amino acid identity Yamaguchi et al., 1992; Seymour et al., 1994; Hahn to DCC, was recently identi®ed because of its dynamic et al., 1995; Cliby et al., 1993; Fujino et al., 1994; Ah- expression in the developing nervous system and See et al., 1994; Knowles et al., 1994; Mitra et al., gastrointestinal tract of the chicken. To explore a role 1994). for the human neogenin (NGN) gene in cancer, we have The existence of a tumor suppressor gene(s) on isolated cDNAs for two alternatively spliced forms of chromosome 18q was ®rst suggested by frequent allelic NGN, encoding proteins of 1461 and 1408 amino acids. losses of 18q in colorectal cancers (Vogelstein et al., Fluorescence in situ hybridization studies (FISH) 1988, 1989). Subsequent studies identi®ed the DCC localized NGN in chromosome band 15q22, a region (deleted in colorectal cancer) gene at 18q21.2 as a infrequently a€ected by alterations in cancer. NGN candidate suppressor gene (Fearon et al., 1990). DCC transcripts of about 7.5 and 5.5 kb were detected in all is an enormous gene spanning greater than 1.35 million adult tissues studied. In contrast to the frequent loss of base pairs (bp) (Cho et al., 1994), and it encodes a 1447 DCC expression, no alterations in NGN expression were amino acid transmembrane protein with four immu- observed in more than 50 cancers studied, including noglobulin like and six ®bronectin type III like glioblastoma, medulloblastoma, neuroblastoma, color- extracellular domains, a single membrane spanning ectal, breast, cervical and pancreatic cancer cell lines region and a 325 amino acid cytoplasmic domain and xenografts. Based on their sequence conservation (Hedrick et al., 1994; Reale et al., 1994). Using and similar expression during development, DCC and sensitive assays, DCC transcripts have been detected NGN may have related functions. However, the in most adult tissues, with highest expression seen in chromosomal location and ubiquitous expression of the developing brain and neural tube (Fearon et al., NGN in various human tumors suggest it is infrequently 1990; Hedrick et al., 1994; Reale et al., 1994; Chuong altered in cancer. et al., 1994; Pierceall et al., 1994; Cooper et al., 1995). DCC expression is reduced or absent in the majority Keywords: tumor suppressor gene; DCC (deleted in of colorectal cancers, though speci®c somatic muta- colorectal cancer) gene; brain tumor; neuroblastoma; tions in DCC have only been identi®ed in a subset of gene expression; chromosome 18q cases (Fearon et al., 1990; Cho et al., 1994; Cho and Fearon, 1995). Loss of DCC expression has also been seen in cancers of the breast, pancreas, endometrium, prostate and brain, as well as male germ cell cancers, Introduction leukemias and neuroblastomas (Cho and Fearon, 1995; Reale et al., 1996), suggesting that DCC inactivation Enormous progress has been made in describing may be an important factor in the development and/or genetic alterations in human cancer cells (Bishop, progression of a variety of cancers besides those arising 1991; Knudson, 1993; Fearon, 1995). The identifica- in the colon and rectum. A tumor suppressor function tion of more than 50 di€erent oncogenes has been for DCC has, in fact, recently been demonstrated in a facilitated by the prior isolation of viral oncogenes, the human squamous cancer cell line (Klingelhutz et al., detection and characterization of translocation break- 1995). points in cancer cells, and the ability of some A protein with roughly 50% amino acid identity to oncogenes to promote tumorigenic growth when DCC, termed neogenin, was identi®ed because of its transferred to nontumorigenic recipient cells. Despite dynamic pattern of expression in the developing much recent attention, only about 15 tumor suppressor nervous system and gastrointestinal tract of the and candidate tumor suppressor genes have been chicken (Vielmetter et al., 1994). Speci®cally, neogenin molecularly cloned (Knudson, 1993; Fearon, 1995). was induced in neural cells immediately prior to cell Results from several independent experimental ap- cycle withdrawal and terminal di€erentiation. To proaches, however, suggest that a sizeable number of further our understanding of NGN and explore the possibility that alterations in neogenin might be present in cancers, we have cloned the human neogenin (NGN) gene. In contrast to DCC, based on its pattern of expression in cancer cells and its chromosomal Correspondence: ER Fearon Received 14 August 1996; revised 4 November 1996; accepted 5 location, NGN appears to be infrequently altered in November 1996 cancer. Neogenin expression in cancer JA Meyerhardt et al 1130 adhesion molecule (N-CAM) family, with four im- Results munoglobulin like and six ®bronectin type III (FN III) like domains. Eight potential asparagine (N)-linked Identi®cation of NGN,aDCC-related gene glycosylation sites (N-X-S/T) were identi®ed in the Given the frequent loss of DCC expression in many approximately 1100 amino acid extracellular region types of cancer, we sought to determine if DCC-related (Figure 1). A single hydrophobic membrane-spanning genes were also inactivated in cancer. A human sequence was found. In the long isoform of NGN,a expression sequence tag (T07322) with over 85% cytoplasmic domain of 338 amino acids with 14 nucleotide identity to chicken neogenin, a DCC- potential phosphorylation sites was observed (Figure related gene, was identi®ed in the GenBank database. 1). Three of the sites are lost in the alternatively spliced Using this sequence as a hybridization probe, we short NGN isoform. Overall, the predicted amino acid isolated cDNA clones from a fetal brain library sequence of human NGN was 86% identical to the spanning 5297 bp and containing the entire human chicken neogenin sequence, with the greatest similarity neogenin (NGN) open reading frame (Figure 1). Two seen in the FN III and cytoplasmic domains (Table 1). alternatively spliced forms of neogenin were previously Comparison of human NGN and DCC revealed that identi®ed in the chicken, the isoforms di€er by the the proteins had identical domain structure and presence or absence of a 159 bp sequence in the roughly 50% identity at the amino acid level (Table neogenin cytoplasmic domain. We identi®ed both 1). The cytoplasmic sequences of NGN and DCC were alternatively spliced forms. NGN protein products of less well-conserved, with only about 37% identity at 1461 and 1408 amino acids were predicted from the the amino acid level. However, their cytoplasmic sequences (Figure 1). domains do not share extensive similarity with any The extracellular domain of human NGN displayed other proteins in the database. features common to members of the neural cell NGN proteins To demonstrate that the NGN cDNAs encoded proteins, Western blot analysis was performed with lysates of Cos-1 cells that had been transiently transfected with expression constructs encoding the two alternatively spliced forms of NGN. To facilitate their detection, vesicular stomatitis virus glycoprotein (VSV-G) epitope tags were fused to the carboxy- termini of each protein. Both cDNAs encoded proteins migrating at about 190 kDa, with the shorter NGN isoform migrating slightly more rapidly (Figure 2). Given the eight potential N-linked glycosylation sites in the NGN extracellular domain and the fact that the 1447 amino acid DCC protein migrates at roughly 175 ± 190 kDa (Hedrick et al., 1994; Reale et al., 1994), the apparent molecular masses were in good agreement with those predicted from the NGN sequences.

Chromosomal localization of NGN Data from allelic loss studies of various cancers suggest

that a number of chromosomal regions contain novel

„—˜le I ƒequen™e ™omp—risons of the hum—n xqxD ™hi™ken

neogenin —nd hum—n hgg proteins

—

7 emino —™id identity with hum—n xqx

˜

ghi™ken neogenin

„ot—l protein VSFV7

sgElike dom—ins @RA USFS7

px type sss dom—ins @TA WHFH7

„r—nsmem˜r—ne dom—in WIFQ7

gytopl—smi™ dom—in

VVFS7

Figure 1 The predicted 1461 amino acid sequence (in single letter rum—n hgg

„ot—l protein

code) of NGN. The eight cysteines (C) in the four immunoglo- RWFS7

sgElike dom—ins @RA

bulin like domains are marked by circles, and the conserved RTFT7

px type sss dom—ins @TA

tryptophan (W) and tyrosine (Y) residues in the six FN type III SUFQ7

„r—nsmem˜r—ne dom—in

domains are boxed. The eight potential N-linked glycosylation THFW7

gytopl—smi™ dom—in

sites in the extracellular domain are indicated by solid arrows; the QUFQ7

— ˜

gomp—rison presumed membrane-spanning region is underlined; and the 14 gomp—rison with the long isoform of hum—n xqxF

potential phosphorylation sites in the cytoplasmic domain are to —minoEtermin—l region of ™hi™ken neogenin ˜—sed on the

et —lFD IWWRA whi™h —ppe—rs to ˜egin indicated by open arrows. The sequences in the NGN cytoplasmic pu˜lished sequen™e @†ielmetter

xqx sequen™e domain, absent in the alternatively spliced form, as boxed —t —mino —™id QS when —ligned with the hum—n Neogenin expression in cancer JA Meyerhardt et al 1131 tumor suppressor genes awaiting identi®cation (re- losses of 15q have been infrequently observed in cancer viewed in Fearon, 1995 and Lasko et al., 1991). Using (Lasko et al., 1991; Vogelstein et al., 1989; Sato et al., ¯uorescence in situ hybridization, we observed speci®c 1990; Tsuchiya et al., 1992; Yamaguchi et al., 1992; hybridization of a human NGN P1 clone to all four Seymour et al., 1994; Hahn et al., 1995; Cliby et al., 15q chromatids in 20 of 20 metaphase spreads analysed 1993; Fujino et al., 1994; Ah-See et al., 1994; Knowles (example shown in Figure 3). In these 20 metaphases, et al., 1994; Mitra et al., 1994). Though one recent there were no signals on any other chromosome. study suggested that 15q allelic losses were common in Localization of NGN to band 15q22 was based on metastatic cancers of the breast, colon and lung, the the position of the ¯uorescence signals relative to losses were restricted to proximal 15q and did not chromosome landmarks. In previous studies, allelic include the 15q22 region (Wick et al., 1996). Hence, based on its location and the allelotype studies carried out thus far, NGN is not likely to be frequently a€ected by allelic losses in cancer. 123

— — — NGN and DCC expression in normal and neoplastic tissues Using Northern blot analysis, NGN transcripts of 220 — about 7.5 and 5.5 kb were detected in all normal adult tissues studied (Figure 4a and data not shown). In t 97 — a d — Hrt — Brn — Pla — Lng — Liv — Skm — Kid — Pan — Spl — Thy — Pro —Tst — Ova In —S — Col Bl —P

9.5 66 — —7.5 —4.4 Neogenin —2.4

46 — —1.3

Western blot detection of NGN proteins. Lysates were Figure 2 β-actin prepared from Cos-1 cells transfected with pcDNA3 mammalian expression vectors containing NGN cDNAs for the long (lane 1) or short (lane 2) isoforms, each tagged with a VSV-G epitope, or a pcDNA3 vector lacking a cDNA insert (lane 3). NGN proteins appeared to migrate at roughly 190 kDa, with the shorter NGN isoform migrating slightly faster. The relative mobility of pre- b stained marker proteins is indicated at the left (in kDa) — Hrt — Brn — Pla — Lng — Liv — Skm — Kid — Pan — Spl — Thy — Pro —Tst — Ova — S Int — Col — P Bld

9.5 —7.5

—4.4 DCC —2.4

—1.3

β-actin

Figure 4 Northen blot analysis of NGN and DCC expression. Northern blots containing approximately 2 mg of Poly(A)+ RNA in each lane were hybridized to NGN (a)orDCC (b) cDNA probes. Following hybridization to NGN or DCC, the blots were stripped and rehybridized with a b-actin cDNA probe. The lanes contain RNA from heart (Hrt), brain (Brn), placenta (Pla), lung (Lng), liver (Liv), skeletal muscle (Skm), kidney (Kid), pancreas Figure 3 NGN maps to chromosome 15q22. (a) The hybridiza- (Pan), spleen (Spl), thymus (Thy), prostate (Pro), testis (Tst), tion of a NGN P1 clone (arrow) and a chromosome 15 ovary (Ova), small intestine (S Int), colon (Col) and peripheral centromere probe (arrowhead) to human metaphase chromo- blood cells (P Bld). The mobility (in kb) of molecular weight somes is shown markers is indicated at the right Neogenin expression in cancer JA Meyerhardt et al 1132 previous studies, DCC transcripts have been detected protein, with similar structure to DCC and 50.2% in most normal adult tissues (Fearon et al., 1990; Reale amino acid identity. NGN transcripts were found to be et al., 1994). However, their very reduced abundance expressed in all normal adult tissues studied. In has often necessitated very sensitive detection methods,

such as reverse transcription polymerase chain reaction

xqx —nd hgq expression in tumor xenogr—fts —nd

(RT ± PCR). Although previous studies have suggested „—˜le P

—

that DCC transcripts were only detectable by Northern ™ell lines

blot analysis in adult brain (Fearon et al., 1990; ˆenogr—ftGgell line xqx hgg

Cooper et al., 1995), we were able to detect DCC qlio˜l—stom—s

SR CC transcripts of about 10 kb and/or 7 kb in many adult CG±

PRS

CC tissues studied (Figure 4b and data not shown). Of CC

PSW

CCC

note, in testis, we also detected abundant levels of ±

PUH

CC

DCC transcripts of altered size (5.5 and 4.0 kb). These CG±

QIU CC C

altered DCC transcripts have not been characterized in QPH CCC ±

QTV CC detail. CC

QWU CC

We carried out ribonuclease (RNase) protection ±

QWV

CCC

studies to determine the relative abundance of NGN CC RHV

CCC

and DCC transcripts in cancers. In glioblastomas, CC RHW CC C

RRQ CCC NGN expression was detected in all specimens studied C

RWQ

CC (Figure 5, Table 2), while DCC expression was not ±

SRP

CCC

detected in upwards of 40% of the specimens (Table 2), C

STI

CC

con®rming previous results (Ekstrand et al., 1995). C

STT CC ±

NGN expression was also detected in all seven TRH CC CG±

medulloblastoma xenografts studied, but DCC expres- wedullo˜l—stom—s

QRI CC

sion was absent in two of the seven tumors (Table 1). C

QVR

CCC

In additional RNase protection and RT ± PCR studies, ± RPS

CCC

NGN expression was detected in all other cancer lines C RVU CC ±

SII CCC studied, including neuroblastoma, breast, colorectal, C

SST

CCC pancreatic, and cervical cancers (Figures 5 and 6, Table C

TWH CCC 2). In contrast, DCC expression was not detectable in CC

the majority of these lines (Figure 6 and Table 2). xeuro˜l—stom—s ƒtxfEU CC CG±

ƒtxfEV CCC CG±

ƒtxfEIH CC ±

ƒtxfEII

CC

Discussion ±

ƒtxfEIU CC C

sw‚QP CC CC

Neogenin, a protein with roughly 50% amino acid golore™t—l

hvhI CG± identity to DCC was ®rst identi®ed because of its CCC

vo†o

C dynamic regulation in the developing nervous system CCC

‡sh‚

±

and gastrointestinal tract of the chicken (Vielmetter et CCC

r™tIIT

±

al., 1994). To further explore the role of the DCC/ CCC

‚uy CCC C

Neogenin family of proteins in cancer, we have cloned fre—st

ƒuf‚Q ± the human Neogenin (NGN) gene and localized it to CCC

wheEwfEQTI ±

chromosome 15q22. NGN encodes a 1461 amino acid CCC

wheEwfEPQI CCC ±

€—n™re—ti™

es€gI CCC C

ge€exP

CCC

Probe ± €—n™I

CCC

Only C ƒuVTFVT CCC ±

¨

€TR

CCC

NGN ± Protected ¨

€TPT

CCC

Fragments γ-actin ±

€TIIU CC ± NGN ¨ gervi™—l

γ-actin ¨ rev— CCC C

r„Q CCC ±

ƒire C 1234567891011 12 13 141516171819 20 212223 ±

g—ski

CCC

Figure 5 Ribonuclease (RNase) protection assay of NGN ±

gRss CC

expression in cancer. Samples were glioblastoma xenografts CG±

gQQe CCC

(lanes 1 ± 10) and colorectal (lanes 11 ± 14), breast (lanes 15 and C

—

16), and neuroblastoma (lanes 17 ± 20) cancer cell lines. Ten mgof ixpression studied in the m—jority of s—mples ˜y ‚x—se prote™tion

xqx primers —nd two sets of RNA from a negative control rat cell line, Rat1, was loaded in —s well —s ‚„ ± €g‚ with two sets of

primers @see pigures S —nd TAF „he rel—tive levels of xqx —nd lane 21. Approximately 500 c.p.m. of the undigested 425 bp NGN hgg

expression in the tumor spe™imens were s™ored using the and 375 bp g-actin riboprobes were loaded in lanes 22 and 23, hgg

respectively. The relative mobilities of the protected 312 bp NGN following systemX @±A no expression dete™ted ˜y either ‚x—se

and 275 bp g-actin fragments are indicated. The speci®c prote™tion —ndGor ‚„ ± €g‚ studiesY @CG±A no dete™t—˜le expression

xenografts and cells lines were: lane 1 - 397; lane 2 - 398; lane ˜y ‚x—se prote™tionD ˜ut f—int ‚„ ± €g‚ sign—ls dete™tedY @CA low

3 - 408; lane 4 - 409; lane 5 - 425; lane 6 - 443; lane 7 - 493; lane 8 level expression dete™ted ˜y ‚x—se prote™tion —ndGor ‚„ ± €g‚Y

- 542; lane 9 - 561; lane 10 - 566; lane 11 - DLD1; lane 12 - LoVo; @CCA moder—te expression dete™ted ˜y ‚x—se prote™tion —ndGor

lane 13 - WIDR; lane 14 - Hct 116; lane 15 - SKBR3; lane 16 - ‚„ ± €g‚Y @CCCA high level expression dete™ted ˜y ‚x—se MDA-MB-361; lane 17 - SJNB-14; lane 18 - SJNB-17; lane 19 - prote™tion —ndGor ‚„ ± €g‚ SJNB-20; lane 20 - IMR32 Neogenin expression in cancer JA Meyerhardt et al 1133 12345678910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

NGN

DCC

β-actin

Figure 6 RT ± PCR assay of NGN and DCC expression in cancer. Shown are Southern blots of the RT ± PCR products generated with NGN cytoplasmic domain and DCC extracellular domain primers, and ethidium bromide-stained b-actin RT ± PCR products. Pancreatic cancer cell lines and xenografts in lanes 5 ± 7, respectively; cervical cancers in 8 ± 13; breast cancers in 14 and 15; colorectal cancers in 16 ± 18; neuroblastomas in 19 and 20; glioblastomas in 21 ± 26; and control samples in lanes 27 ± 30. The speci®c lines were: lane 1 - AsPC1; lane 2 - CAPAN2; lane 3 - Panc 1; lane 4 - Su86.86; lane 5 - Px4; lane 6 - Px26, lane 7 - Px117; lane 8 - HeLa; lane 9 - HT3; lane 10 - SiHA; lane 11 - Caski; lane 12 - C4II; lane 13 - C33A; lane 14 - MDA-MB-231; lane 15 - MDA-MB- 361; lane 16 - LoVo; lane 17 - Hct116; lane18 - DLD1; lane 19 - SJNB7; lane 20 - IMR32; lane 21 - GBM 54; lane 22 - GBM 398; lane 23 - GBM 408; lane 24 - GBM 640; lane 25 - GBM 317; lane 26 - GBM 270; lane 27 - no RNA (negative control for RT and PCR); lane 28 - liver cDNA library; lane 29 - fetal brain cDNA library; lane 30 - no cDNA (control for PCR). The exposure times of all NGN lanes was 2 h and the exposure time of all DCC lanes was 6 hours

addition, NGN transcripts were also detected in and Tessier-Lavigne, 1995). Given the predicted virtually all cancer tissues and cell lines studied, similarity of UNC-40 to DCC and NGN, the findings including many tumors in which DCC expression was suggests that DCC and NGN may play important roles not detected. in mediating directional cell migration in the develop- Loss of DCC expression in cancers does not ing nervous system. establish that DCC is a tumor suppressor gene. What role, if any, would DCC, NGN and the netrins Nonetheless, the data are consistent with the possibi- be expected to play in other tissues? Moreover, how lity. Unfortunately, because of its very large size (i.e. would loss of DCC function contribute to the altered 41.35 million bp), de®nitive examination of the DCC phenotype of cancer cells, particularly if NGN function locus for alterations a€ecting its expression in cancer is retained in the cells? As shown above, DCC and cells, such as somatic mutations or increased methyla- NGN are both expressed at low levels in virtually all tion of its regulatory sequences, has not been possible. adult tissues. We have cloned the human netrin-1 gene In contrast to DCC, the chromosomal location and and have found it expressed in all adult tissues ubiquitous expression of NGN in cancer suggest that it surveyed (Meyerhardt et al., unpublished observa- is unlikely to be a suppressor gene. The ®ndings tions). In addition, netrin-1 transcripts can be detected presented here also imply that, despite their extensive in many human cancer cell lines, including those sequence similarity, the biologic functions of DCC and derived from colorectal tumors (Meyerhardt et al., NGN in cell growth regulation and cancer may be unpublished observations). The e€ects of netrins on distinct. epithelial cells are poorly understood. However, it is Recent studies of genes regulating cell migration and tempting to propose that netrins may provide growth axon guidance in the developing nervous system have inhibitory or di€erentiation cues to epithelial cells. provided interesting new clues into DCC and NGN Given the substantial di€erences between the DCC and function. The C. elegans unc-40 gene encodes a NGN cytoplasmic sequences, cancer cells that have lost transmembrane protein with identical domain struc- DCC function may fail to respond appropriately to ture to DCC and NGN and about 25% amino acid netrin signals, despite retaining NGN expression. identity with each (Chan et al., 1995). Though it may Alternatively, DCC alterations may contribute to also have other functions, unc-40 is necessary for the defects in the migratory properties of cancer cells or appropriate circumferential migration of a subset of their failure to respect tissue boundaries. Indeed, such cells and axons in the developing nematode (Hedge- an e€ect might account for the apparently more cock et al., 1990). unc-40 is believed to function in the aggressive and metastatic growth properties of some same pathway as another gene termed unc-6 (Hedge- cancer cells lacking DCC expression (Cho and Fearon, cock et al., 1990; Culotti, 1994). unc-6 encodes a 1995; Reale et al., 1996). secreted protein bearing signi®cant similarity to the amino-terminal region of the B2 chain of laminin, an extracellular matrix protein (Ishii et al., 1992) and two vertebrate homologues of unc-6, termed netrin-1 and Materials and methods netrin-2 have recently been identi®ed (Sera®ni et al., 1994; Kennedy et al., 1994). The netrin proteins were Cloning of NGN initially identi®ed and puri®ed because of their ability A 312 basepair (bp) polymerase chain reaction (PCR) to promote the outgrowth of commissural axons, but product corresponding to a human expression sequence tag they also appear to function as chemoattractants for (T07322) with 85% identity to chicken neogenin was commissural axons (Kennedy et al., 1994; Kennedy ampli®ed from an oligo-dT primed human fetal brain Neogenin expression in cancer JA Meyerhardt et al 1134 library (Stratagene Cloning Systems, La Jolla, CA) using GTCTGCTGGCTGATTCTG-TGTT-3'). Three P1 clones two oligos (5'-TTACGCCATTGGTTATG-3' and 5'-CAC- were isolated (DMFC-HFF#1-113-H12, -531-A11 and - CATCAGGATTACGTG-3') derived from the ends of the 1421-D6). Puri®ed DNA from clone #113-H12 was labeled sequence tag. The PCR product was labeled with [32P]dCTP with digoxigenin-11-dUTP (Boehringer Mannheim, India- by random priming and used to screen the fetal brain napolis, IN) by nick translation. Phytohemagglutinin- library. Approximately 26106 plaques were lifted onto stimulated human peripheral blood lymphocytes from a Hybond N+ nylon ®lters (Amersham, Arlington Heights, normal donor were used as the source of metaphase IL). The ®lters were hybridized and washed as described chromosomes. Labeled DNA was hybridized overnight at (Vogelstein et al., 1987). A total of 19 independent clones 378C to ®xed metaphase chromosomes in a solution were isolated by multiple rounds of hybridization selection. containing sheared human DNA, 50% formamide, 10% Phagemids were rescued by in vivo excision with the dextran sulfate, and 26SSC. Speci®c hybridization signals ExAssist/SOLR system provided with the library. were detected by incubating the slides with ¯uorescein- conjugated sheep anti-digoxigenin antibodies (Boehringer Mannheim). The chromosomes were then counterstained DNA sequencing with 4,6-diamidino-2-phenylindole (DAPI) and analysed. Both strands of overlapping, double-stranded phagemid The assignment of NGN to chromosome 15 was con®rmed clones, containing the entire open reading frame of NGN by co-hybridization of the NGN P1 clones and a (GenBank #U61262), were sequenced in their entirety using biotinylated chromosome 15 centromere-speci®c probe a combination of external and internal primers and (D15Z) (Oncor, Inc., Gaithersburg, MD). In this case, exonuclease III/mung bean nuclease deletions (Strata- probe signals were detected by incubating the slides with gene). Plasmid DNA was prepared using Qiagen Spin ¯uorescein-conjugated sheep anti-digoxigenin antibodies as Plasmid Kit (Qiagen, Inc., Chatsworth, CA) and sequenced well as a Texas red avidin conjugate (Vector Laboratories, by the dideoxy chain termination method using Sequenase Burlington,CA).TheNGN gene was further localized on 2.0 (U.S. Biomedical Corp., Cleveland, OH) and a chromosome 15 by comparing the position of ¯uorescein modi®ed protocol (Kraft et al., 1988). Sequencing signals to chromosome landmarks, such as the centromere, reactions were electrophoresed on 6% polyacrylamide telomere, and heterochrome and euchromatin boundaries (19 : 1 acrylamide : bis-acrylamide)/8.0 M urea/16TBE (Franke, 1994). gels. After drying, the gels were exposed to X-OMAT ®lm (Eastman Kodak, Rochester, NY). RNA isolation Brain tumor xenografts were established from primary NGN expression constructs human glioblastomas and medulloblastomas and propa- Full-length cDNAs encoding each of the two alternatively gated in nude mice as previously described (Schold et al., spliced forms of NGN were constructed from the over- 1983). Pancreatic xenografts were established from lapping fetal brain cDNAs. PCR was used to fuse a pancreatic adenocarcinomas (Hahn et al., 1995). Human vesicular stomatitis virus glycoprotein (VSV-G) epitope tag neuroblastoma cell lines SJNB-7, -8, -10, -11 and -17 were (YTDIEMNRLGK) to the carboxyterminus of each of the derived from advanced stage primary tumors at St. Jude two full-length NGN cDNAs. The modi®ed cDNAs were Children's Research Hospital. All other cell lines were sequenced to verify that no errors had been introduced. purchased from ATCC. Total RNA was isolated from The tagged cDNAs were subcloned into the pcDNA3 minced brain tumor xenograft tissues or pelleted tumor mammalian expression vector (Invitrogen Corp., San cells, using Trizol reagent (Gibco BRL Life Technologies) Diego, CA). or the RNAgents RNA isolation system (Promega, Madison, WI).

Western blot analysis Northern analysis Transfections of Cos-1 cells (American Type Culture Collection [ATCC], Rockville, MD) were performed with Northern blots of normal human adult tissues (approxi- Lipofectamine (Gibco BRL Life Technologies, Gaithers- mately 2 mgofpoly(A)+ RNA loaded per lane) were burg, MD) per the manufacturer's instructions. Protein purchased from ClonTech (ClonTech Laboratories, Inc., extracts were prepared from the cells 48 h after transfec- Palo Alto, CA). Hybridization were performed according tion as previously described (Pierceall et al., 1994; to the manufacturer's instructions using a 486 bp 32P- Ekstrand et al., 1995). Following electrophoresis on an labeled NGN cDNA probe (corresponding to amino acids 8% SDS-polyacrylamide gel and transfer to Immobilon-P 330 ± 491) or a 4.35 kb 32P-labeled DCC cDNA probe membranes (Millipore, Bedford, MA) with a semidry (Hedrick et al., 1994). Following hybridization, blots were electroblotter (Bio-Rad, Hercules, CA), tagged proteins washed with 26 SSC/0.5% SDS for 45 min at room were detected with a polyclonal rabbit anti-VSV-G temperature, with a subsequent increased stringency wash antiserum (MBL International, Watertown, MA) and a of 0.16SSC/0.1% SDS for 30 min at 508C. Blots were donkey anti-rabbit IgG coupled to horseradish peroxidase stripped per the manufacturer's instructions, and reprobed (Pierce Biochemicals, Rockford, IL). Antibody complexes with a 32P-labeled 2.0 kb cDNA fragment of b-actin, were detected by enhanced chemiluminescence (ECL) provided by ClonTech. (Amersham, Arlington Heights, IL) and subsequent exposure to Kodak X-OMAT ®lm. Ribonuclease protection assay Ribonuclease (RNase) protection assays were performed P1 clone isolation and ¯uorescence in situ hybridization (FISH) essentially as described (Pierceall et al., 1994; Ekstrand et The p1 library (DMPC-HFF#10) was screened by al., 1995). A NGN riboprobe was generated from pAMP1- Genome Systems, Inc. (St Louis, MO) with two NGN T07322, a plasmid containing a 312 bp NGN cDNA primers derived from sequences at the end of the coding fragment (corresponding to amino acids 771 ± 873). The region and the downstream 3' untranslated region DCC riboprobe has been previously described (Ekstrand et (nucleotides 4465 ± 4645; sense oligo 5'-GAGATGGCC- al., 1995). To control for loading, g-actin and b-actin CACCTGGAAGGAC-3' and antisense oligo 5'- riboprobes were used. The g-actin riboprobe has been Neogenin expression in cancer JA Meyerhardt et al 1135 described (Ekstrand et al., 1995), and the b-actin riboprobe Ampli®cations were performed using the following was prepared from the pTRI-b-actin-125-human plasmid conditions: hotstart followed by 35 cycles of 948C645 s, construct (Ambion, Inc., Austin, TX). Probes were labeled 558C645 s and 728C62.5 min. One-®fth of each reaction with [32P]CTP, and following puri®cation through an was electrophoresed on 1.2% agarose gels and visualized with acrylamide gel, 1.06106 c.p.m. of the NGN and u.v. light following ethidium bromide staining. The identities 2.06105 c.p.m. of the g-actin or b-actin riboprobe were of the DCC and NGN products were con®rmed by Southern incubated with 10 mg of RNA. Similarly, 1.06106 c.p.m. of transfer and hybridization with their respective 32P-labeled the DCC riboprobe and 2.06105 c.p.m. of the g-actin cDNA probes. A set of b-actin primers was used to riboprobe were hybridized overnight with 20 mgofRNA. independently con®rm the ®rst strand cDNA reaction. For Non-hybridizing sequences were digested with RNase T2 all samples studied, the results with the two sets of NGN (Gibco BRL Life Technologies). Protected fragments were primers were concordant. Similarly, concordant results were recovered by ethanol precipitation and electrophoresed on obtained with the two sets of DCC primers. a denaturing polyacrylamide sequencing gel. After drying the gel, autoradiography was carried out with X-OMAT ®lm and intensifying screens. Abbreviations DCC - deleted in colorectal cancer; NGN - human RT ± PCR assay neogenin; FN III - ®bronectin type III, Ig - immunoglobu- lin; bp - base pair; kb - kilobase pair; RNase - ribonuclease; Total RNA was treated with two units of RNase-free RT ± PCR - reverse transcription polymerase chain reac- DNase (Boehringer Mannheim). First-strand cDNA was tion; VSV-G - vesicular stomatitis virus glycoprotein; kDa - prepared from three micrograms of RNA using AMV kilodalton; ECL - enhanced chemiluminescence. reverse transcriptase (Promega) and random hexamers. One tenth of the cDNA was used for each PCR with primer pairs derived from the human DCC and Acknowledgements NGN sequences. Extracellular domain NGN primers The authors thank Drs Scott E Kern and Kathleen R Cho (corresponding to amino acids 329 ± 491) were NGN329S- for providing pancreatic and cervical cancer RNA samples, 5'-TTGAAGCTCAAGCAGAGCTTACAG-3' and NGN- respectively; Virginia Valentine and Dr David N Shapiro 491A-5'-GACTGGTATTCTCAACACGTTCC-3'. NGN for excellent technical assistance with the FISH studies; cytoplasmic domain primers (amino acids 1128 ± 1239) Gang Hu, David Siemieniak and Kaye Ho€man for were NGN1128S-5'-GTACCCGTCGTACCACCTCTCA- computer assistance; and Dr Kathleen R Cho for CC-3' and NGN1239A-5'-CATCATTTTTGGTCTCATT- comments on the manuscript. This work was supported CCTCG-3'. DCC extracellular domain primers (amino by NIH Grants CA70097 (ERF), CA71907 (ATL), acids 93 ± 221) were DCC902S-5'-CAAATGGGTCTCT- CA23009 (ATL), CA21765 (ATL), CA43722 (SHB), and GCTGATAC-3' and DCCEX3A-5'-TCTTGAGCTGGC- NS20023 (SHB); DAMD Grant 17-94-J-4366 (ERF), and TGGATTTCGAGC-3'. DCC cytoplasmic domain primers the American Lebanese Syrian Associated Charities of St. (amino acids 1110 ± 1309) were DCK3090S-5'-CACAGT- Jude Children's Research Hospital (ATL). JAM is a GCTGGTAGTGGTCAT-3' and DCK4504A- 5'-TTGGG- predoctoral fellow of the Howard Hughes Medical TTGATGGTCCTTCACTCAC-3'. Institute.

References

Ah-See KW, Cooke G, Pickford IR, Soutar D and Balmain Fujino T, Risinger JL, Collins NK, Liu F-S, Nishii H, A. (1994). Cancer Res., 54, 1617 ± 1621. TakahashiH,WestphalE-M,BarrettJC,SasakiH, Bishop JM. (1991). Cell, 64, 235 ± 248. Kohler MF, Berchuck A and Boyd J. (1994). Cancer Chan S, Su M-W, Culotti JG and Hedgecock EM. (1995). Res., 54, 4294 ± 4298. Worm Breeder's Gazette, 13, 48. Hahn SA, Seymour AB, Shamsul Hoque ATM, Schutte M, Cho KR and Fearon ER. (1995). Curr. Opin. Gen. Devel., 5, da Costa LT, Redston MS, Caldas C, Weinstein Cl, Fisher 72 ± 78. A, Yeo CJ, Hruban RH and Kern SE. (1995). Cancer Res., Cho KR, Oliner JD, Simons JW, Hedrick L, Fearon ER, 55, 4670 ± 4675. Presinger AC, Hedge P, Silverman GA and Vogelstein B. Hedgecock EM, Culotti JG and Hall DH. (1990). Neuron, 2, (1994). Genomics, 19, 525 ± 531. 61 ± 85. Chuong C-M, Jiang T-X, Yin E and Widelitz RB. (1994). Hedrick L, Cho K, Fearon ER, Wu T-C, Kinzler KW and Dev. Biol., 164, 383 ± 397. Vogelstein B. (1994). Genes Dev., 8, 1174 ± 1183. Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Ishii N, Wadsworth WG, Stern BD, Culotti JG and Keeney G, Podratz KC and Jenkins RB. (1993). Cancer Hedgecock EM. (1992). Neuron, 9, 873 ± 881. Res., 53, 2393 ± 2398. Kennedy TE and Tessier-Lavigne M. (1995). Curr. Opin. in Cooper HM, Armes P, Britto J, Gad J and Wilks AF. (1995). Neurobiology, 5, 83 ± 90. Oncogene, 11, 2243 ± 2254. Kennedy TE, Sera®ni T, de la Torre JR and Tessier-Lavigne Culotti JG. (1994). Curr. Opin. Genet. Devel., 4, 587 ± M. (1994). Cell, 78, 425 ± 435. 595. Klingelhutz AJ, Hedrick L, Cho KR and McDougall JK. Ekstrand BC, Mans®eld TA, Bigner SH and Fearon ER. (1995). Oncogene, 10, 1581 ± 1586. (1995). Oncogene, 11, 2393 ± 2402. Knowles MA, Elder PA, Williamson M, Cairns JP, Fearon ER. (1995). Clinical Oncology.Abelo€MD, Shaw ME and Law MG. (1994). Cancer Res., 54, Armitage JO, Lichter AS and Niederhuber JE. (eds). 531 ± 538. Churchill Livingstone: New York, pp.11 ± 40. Knudson AG. (1993). Proc.Natl.Acad.Sci.USA,90, Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, 10914 ± 10921. Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kraft T, Tardi€ J, Krauter KS and Leinwand LA. (1988). Kinzler WK and Vogelstein B. (1990). Science, 247, 49 ± Biotechniques, 6, 544 ± 546. 56. Lasko D, Cavenee WK and Nordenskjold M. (1991). Annu. Franke U. (1994). Cytogenet. Cell Genet., 65, 206 ± 219. Rev. Genet., 25, 281 ± 314. Neogenin expression in cancer JA Meyerhardt et al 1136 Mitra AB, Murty VVS, Li RG, Pratap M, Luthra UK and Vielmetter J, Kayyem JF, Roman JM and Dreyer WJ. Chaganti RSK. (1994). Cancer Res., 54, 4481 ± 4487. (1994). J. Cell Biol., 127, 2009 ± 2020. Pierceall WE, Reale MA, Candia AF, Wright CVE, Cho KR Vogelstein B, Fearon ER, Hamilton SR, Preisinger AC, and Fearon ER (1994). Dev. Biol., 166, 654 ± 665. Willard HF, Michelson AM, Riggs AD and Orkin SH. Reale MA, Hu G, Zafar AI, Getzenberg RH, Levine SM and (1987). Cancer Res., 47, 4806 ± 4813. Fearon ER. (1994). Cancer Res., 54, 4493 ± 4501. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger Reale MA, Reyes-Mugica M, Pierceall WE, Rubinstein MC, AC, Leppert M, Nakamura Y, White R, Smits A and Bos Hedrick L, Cohn SL, Nakagawara A, Brodeur GM and J. (1988). N. Eng. J. Med., 319, 525 ± 532. Fearon ER. (1996). Clin. Cancer Res., 2, 1097 ± 1102. Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger Sato T, Tanigami A, Yamakawa K, Akiyama F, Kasumi F, AC, Nakamura Y and White R. (1989). Science, 244, 207 ± Sakamoto G and Nakamura Y. (1990). Cancer Res., 50, 211. 7184 ± 7189. Wick W, Petersen I, Schmutzler RK, Wolfarth B, Lenartz D, Schold SC, Hullard DE, Bigner SH, Jones TR and Bigner Bierho€ E, Hummerich J, Muller DJ, Stangl AP, DD. (1983). J. Neurooncol., 1, 5 ± 14. Schramm J, Wiestler OD and von Diemling A. (1996). Sera®ni T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM Oncogene, 12, 973 ± 978. and Tessier-Lavigne M. (1994). Cell, 78, 409 ± 424. Yamaguchi T, Toguchida J, Yamamuro T, Kotoura Y, Seymour AB, Hruban RH, Redston M, Caldas C, Powell Takada N, Kawaguchi N, Kaneko Y, Nakamura Y, SM, Kinzler KW, Yeo CJ and Kern SE. (1994). Cancer Sasaki MS and Ishizaki K. (1992). Cancer Res., 52, 2419 ± Res., 54, 2761 ± 2764. 2423. Tsuchiya E, Nakamura Y, Weng S-Y, Nakagawa K, Tsuchiya S, Sugano H and Kitagawa T. (1992). Cancer Res., 52, 2478 ± 2481.