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Cell Growth & Differentiation Vol. 10, 35-42, January 1999
© 1999 American Association for Cancer Research

Netrin-1

Interaction with Deleted in Colorectal Cancer (DCC) and Alterations in Brain Tumors and Neuroblastomas1

Jeffrey A. Meyerhardt2, Karel Caca2, Bradley C. Eckstrand, Gang Hu, Christoph Lengauer, Shripad Banavali, A. Thomas Look and Eric R. Fearon3

Division of Molecular Medicine and Genetics and the Cancer Center, Departments of Internal Medicine [J. A. M., K. C., B. C. E., G. H., E. R. F.], Human Genetics [E. R. F.], and Pathology [E. R. F.], University of Michigan Medical Center, Ann Arbor, Michigan 48109-0638; Molecular Genetics Laboratory, The Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 [C. L.]; and Department of Experimental Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 [S. B., A. T. L.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Netrins, a family of laminin-related secreted proteins, have critical roles in axon guidance and cell migration during development. The deleted in colorectal cancer (DCC) protein has been implicated as a netrin-1 receptor component. The expression and function of netrins in adult tissues remain unknown, and direct interaction of netrin-1 with DCC has not been demonstrated. We cloned the human netrin-1 (NTN1L) gene, mapped it to chromosome 17p12–13, and found that it encodes a 604 amino acid protein with 98% identity to mouse netrin-1 and 50% identity with the Caenorhabditis elegans UNC-6 protein. NTN1L transcripts were detected in essentially all normal adult tissues studied, and markedly reduced or absent NTN1L expression was seen in {approx}50% of brain tumors and neuroblastomas. In one neuroblastoma, missense mutations at highly conserved NTN1L codons were found. Netrin-1 protein could be cross-linked to DCC protein on the cell surface, but it did not immunoprecipitate with DCC in the absence of cross-linking and it failed to bind to a soluble fusion protein containing the entire DCC extracellular domain. Our findings demonstrating NTN1L loss of expression and mutations suggest that NTN1L alterations may contribute to the development of some cancers. Furthermore, the binding of netrin-1 to DCC appears to depend on the presence of a coreceptor or accessory proteins.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The netrins, a family of laminin-related secreted proteins, have critical roles in determining the direction and extent of cell migration and axon outgrowth in the developing nervous system. Netrins were initially discovered through analysis of nematode mutants with defects in the unc-6 gene (1, 2, 3, 4) . Animals with inactivating mutations in unc-6 have defects in the guidance of commissural and motor axons along the dorsoventral body axis. Independent studies to define chemoattractant molecules secreted by the developing floor plate in the vertebrate spinal cord identified two soluble factors closely related to the UNC-6 protein, termed netrin-1 and netrin-2 (5, 6, 7) . Netrins can guide axons along specific trajectories in vitro (7 , 8) , and netrin-1-deficient mice have defective spinal commissural axon guidance, as well as defects in the corpus callosum, hippocampal commissure, and anterior commissure (9) .

Genetic analyses in Caenorhabditis elegans have implicated two cell surface proteins, UNC-5 and UNC-40, in UNC-6 (netrin)-dependent migrations (1 , 2 , 10 , 11) . Although an oversimplistic view, UNC-5 appears to mediate the chemorepulsive effects of netrin on axons and motile cells, and UNC-40 has a primary role in the chemoattractive effects of netrin. Three vertebrate homologues of UNC-5 have been described (12 , 13) . The UNC-5 like proteins have an extracellular domain with two immunoglobulin-like and two thrombospondin type 1 motifs, a single transmembrane region, and a {approx}600-amino acid cytoplasmic domain with homology to the zona occludens-1 junction protein.

A vertebrate homologue of UNC-40, termed DCC,4 was initially identified as a candidate tumor suppressor gene (14) . Although the role of DCC in tumorigenesis is controversial, DCC expression is frequently extinguished in colorectal and other cancers, and clonal somatic mutations in DCC have been found in some cancers (14, 15, 16, 17, 18) . DCC, UNC-40, and other DCC-like proteins have an extracellular domain composed of four immunoglobulin-like and six FN3 repeats, a single transmembrane region, and a cytoplasmic domain of 325 amino acids (4 , 11 , 19, 20, 21, 22, 23) . Several lines of evidence indicate that DCC and related proteins may function as a netrin-1 receptor or a receptor component. C. elegans, Drosophila, and mouse mutant animals with defects in DCC or DCC-related genes have phenotypes that overlap with those of netrin-1-deficient animals (1 , 4 , 9 , 11 , 22 , 24, 25, 26) . Transfection of DCC confers netrin-1 binding upon cells, and antibodies against the extracellular domain of DCC can block netrin-mediated neurite outgrowth in vitro, without affecting netrin-1 binding (27) . Furthermore, the recent studies of Mehlen et al. (28) indicate that DCC has a proapoptotic function in some cell types and that DCC-induced apoptosis can be blocked if the cells are exposed to netrin-1. Nevertheless, evidence of direct binding of netrin-1 to DCC has not been presented.

To study further the relationship between netrin-1 and DCC, we sought to clone and characterize the human netrin-1 (NTN1L) gene. NTN1L encodes a highly conserved 604 amino acid protein, with 98% identity to mouse netrin-1 and 50% identity to the C. elegans UNC-6 protein. NTN1L was found to be expressed in virtually all normal adult tissues. However, markedly reduced or absent NTN1L expression was seen in {approx}50% of brain tumors and neuroblastomas. Sequencing studies of a limited panel of neuroblastomas and brain tumors identified one neuroblastoma with two different NTN1L missense mutations, both present at highly conserved codons. In addition, whereas the netrin-1 protein could be cross-linked to DCC on the cell surface, it did not immunoprecipitate with DCC in the absence of cross-linking, and it did not bind to a soluble fusion protein containing the entire DCC extracellular domain. Inactivation of NTN1L, like DCC inactivation, may contribute to the genesis of some cancers. Furthermore, binding of netrin-1 to DCC is likely to be dependent on a coreceptor or accessory proteins.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Identification and Chromosomal Mapping of a Netrin-1 Homologue.
Our strategy to identify a human netrin-1 homologue used degenerate PCR primers derived from highly conserved sequences in the COOH-terminal domains of the chicken netrin-1, netrin-2, and UNC-6 proteins (6) . Reduced stringency PCR conditions generated a 288-bp fragment from normal human brain cDNA, with {approx}80% nucleotide identity to chicken netrin-1 but only 50% nucleotide identity to chicken netrin-2, implying that the fragment was likely derived from the human homologue of netrin-1 (i.e., NTN1L). The NTN1L fragment was used to screen a human brain stem cDNA library, and a single NTN1L clone containing the 3' region of the open reading frame and untranslated sequences was isolated from {approx}3 x 106 phage clones screened. Preliminary Northern blot studies suggested strong NTN1L expression in adult liver, and additional NTN1L clones were isolated from an adult liver cDNA library. The consensus NTN1L cDNA sequence predicts an open reading frame of 604 amino acids (Fig. 1)Citation .



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Fig. 1. Predicted amino acid sequence of the human NTN1L protein product (hNet-1), and its relationship to netrin-1 proteins from mouse (mNet-1), chicken (cNet-1), and zebrafish (zNet-1); the candidate human netrin-2 homologue (hNet-2); and the C. elegans unc-6 gene. Sequences are indicated in single-letter amino acid code. Amino acid identities are indicated in black and conserved substitutions are in gray. The two codons affected by missense substitutions in neuroblastoma SJNB11 are indicated by arrows (R to H substitution at codon 351 and K to E at codon 489).

 
The human netrin-1 protein is highly conserved compared to netrin-1 proteins from mouse, chicken, and zebrafish, and human netrin-1 also shows {approx}50% amino acid identity to UNC-6 (Table 1)Citation . Previously, the existence of a human netrin-2-like (NTN2L) gene at chromosome 16p13.3 has been reported (29) . However, this gene appears to encode a more distantly related molecule and not netrin-2. Netrin homologues are highly related between any two species (e.g., the human and chicken netrin-1 proteins share 87% amino acid identity), and there is {approx}70% amino acid identity between the netrin-1 and -2 proteins within a given species (e.g., chicken netrin-1 versus chicken netrin-2). Surprisingly, the predicted protein product of the NTN2L gene shows only 52% amino acid identity to human netrin-1 and only 57% amino acid identity to chicken netrin-2. Hence, the findings imply that the previously described human NTN2L gene may actually represent a third netrin-like gene in man rather than a homologue of netrin-2. Additional studies may lead to the identification of a human gene that is a more promising candidate for the netrin-2 homologue.


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Table 1 Relationship of human netrin-1 to other vertebrate and invertebrate netrins

 
Using purified P1 clone DNA for FISH analysis, we localized the NTN1L gene to 17p12–13 (Fig. 2)Citation . Subsequent FISH studies demonstrated that the p53 and NTN1L genes were in close proximity in band 17p12–13 (data not shown). The 17p chromosomal region is of particular interest, because neuroblastomas, medulloblastomas, and a number of other cancers demonstrate 17p LOH in many cases without mutation of the remaining p53 allele (30, 31, 32, 33, 34, 35, 36, 37, 38) . These findings imply that additional tumor suppressor gene(s) may reside on 17p.



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Fig. 2. NTN1L maps to chromosome 17p12–13. Hybridization signals of a NTN1L P1 clone (arrow) and a chromosome 17 centromere probe (arrowhead) on a human metaphase are shown.

 
NTN1L Expression and Mutations.
We performed Northern blot studies and detected NTN1L transcripts of {approx}5.0 kb in most normal adult tissues studied, with highest levels of NTN1L expression in heart, small intestine, colon, liver, and prostate (Fig. 3)Citation . The significance of the smaller transcript of {approx}4.4 kb seen in several tissues (e.g., liver) is not clear, but it may reflect alternative splicing in the 5' or 3' untranslated region because no evidence for alternative splicing in the open reading frame was found in our analysis of netrin-1 cDNAs from liver. The expression pattern of NTN1L in human tissues is distinct from that previously reported for netrin-1 in adult tissues of the chicken, in which Northern blot studies revealed detectable levels of netrin-1 expression only in the brain, heart, ovary, skeletal muscle, and thymus (7) . The expression of NTN1L in many different human adult tissues suggests that netrin-1 may have a function outside the developing nervous system distinct from that in mediating axon outgrowth. The unc-6 gene is believed to regulate the migration of a number of nonneuronal motile cell types during development (1, 2, 3, 4 , 11) , and recent studies imply that netrin-1 may act to inhibit DCC-induced apoptosis (28) .



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Fig. 3. NTN1L expression in normal tissues. Northern blots containing 2 µg of poly(A)+ RNA in each lane were hybridized first to a 994-bp SmaI-HindIII NTN1L cDNA probe. After stripping, the blots were rehybridized with a ß-actin cDNA probe. RNAs are from heart (Hrt), brain (Brn), placenta (Pla), lung (Lng), liver (Liv), skeletal muscle (Skm), kidney (Kid), pancreas (Pan), spleen (Spl), thymus (Thy), prostate (Pro), testis (Tst), ovary (Ova), small intestine (S Int), colon (Col), and peripheral blood cells (P Bld). The mobilities (in kb) of molecular weight markers are indicated at the right. Exposure times were 7 days at -80°C for NTN1L and 4 h at room temperature for ß-actin.

 
We carried out RNase protection assays to determine the relative abundance of NTN1L transcripts in various cancer cell lines and tumor xenografts. We studied mainly cancer types known to have frequent 17p LOH but in which p53 mutations have been rarely, if ever, detected, such as neuroblastomas and medulloblastomas. We also studied glioblastomas, in which p53 mutations are found in only a fraction of cases with 17p LOH, and a few epithelial cancer lines. Very reduced or undetectable levels of NTN1L expression were seen in 9 of 16 glioblastoma xenografts, 5 of 7 medulloblastoma xenografts, and 5 of 11 neuroblastoma cell lines (Fig. 4Citation and data not shown). Low levels of NTN1L expression were noted in one of four colorectal cancers and the two breast cancer lines studied (Fig. 4)Citation . No clear-cut correlation between NTN1L and DCC expression was observed in the cancer specimens (data not shown). Southern blot analysis of the tumors and cell lines using a NTN1L cDNA probe did not demonstrate gross deletions or rearrangements of the NTN1L coding region. FISH studies using NTN1L P1 clones to study most of the neuroblastoma cell lines also failed to identify homozygous deletions or gross rearrangements of the NTN1L gene.



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Fig. 4. NTN1L expression in cancer tissues. RNase protection assay of NTN1L gene expression in cancer cell lines and xenografts. Shown are the protected 291-bp NTN1L riboprobe fragments following incubation of 20 µg total RNA from cell lines and xenografts and subsequent digestion with RNase T2. A control ß-actin riboprobe indicated equivalent RNA loading in all lanes (data not shown). Glioblastoma xenograft samples are in Lanes 1–16 and medulloblastoma xenografts are in Lanes 17–22. Colorectal and breast carcinoma samples are: Lane 23, LoVo; Lane 24, WIDR; Lane 25, DLD1; Lane 26, Hct-116; Lane 27, MDA-MB-361; Lane 28, SKBR3. Neuroblastoma cell line samples are: 29, SJNB4; Lane 30, SJNB6; Lane 31, SJNB7; Lane 32, SJNB8; Lane 33, SJNB10; Lane 34, SJNB11; Lane 35, SJNB12; Lane 36, SJNB14; Lane 37, SJNB17; Lane 38, SJNB20; Lane 39, IMR32.

 
To determine whether localized mutations in NTN1L might be present in cancers, we undertook direct sequencing analysis of NTN1L transcripts in a subset of the neuroblastomas and brain tumors. Using an RT-PCR approach, we sequenced the entire NTN1L open reading frame in seven cancers with readily detectable levels of NTN1L transcripts: three neuroblastomas, two glioblastomas, and two medulloblastomas. In one of the neuroblastoma cell lines (SJNB11), we obtained evidence that the NTN1L alleles harbored missense mutations at highly conserved codons. One mutation was an Arg-to-His substitution at codon 351 and the other was a Lys-to-Glu substitution at codon 489 (Fig. 1)Citation . These two codons are conserved in all vertebrate netrins, as well as in the C. elegans UNC-6 protein. Hence, both NTN1L alleles may be inactivated by missense mutations in this cancer. Taken together with the frequent alterations in NTN1L gene expression, the data suggest that the NTN1L gene may be inactivated by somatic mutations in some cancers.

Netrin-1 Binding to DCC.
As reviewed above, genetic and cell biological studies imply that DCC may function as a receptor or a component of a receptor for netrin-1. Nevertheless, definitive evidence of a direct physical interaction between netrin-1 and DCC has not yet been provided. Because previous studies had demonstrated that a recombinant chicken netrin-1 protein with a c-myc epitope tag at its COOH-terminal region retained biological activity in in vitro studies (7) , we generated a mammalian expression vector encoding a human netrin-1 protein with a COOH-terminal c-myc epitope tag. This vector was transfected into several mammalian cell lines, and the recombinant netrin-1 protein was found to be stably expressed and secreted into the medium (data not shown). Similar to findings reported previously (7 , 27) , we found that much of the netrin-1 protein was present in the cell lysate and that the addition of heparin to the medium increased the abundance of netrin-1 in the medium (data not shown).

We first attempted to demonstrate an interaction between DCC and netrin-1 using chemical cross-linking with the membrane-insoluble, cleavable cross-linking reagent DTSSP and subsequent immunoprecipitation with an antibody against the DCC cytoplasmic domain. Cells coexpressing netrin-1 and full-length DCC were treated with DTSSP. Following immunoprecipitation with the DCC antibody and cleavage of the cross-link, the c-myc-tagged netrin-1 protein was detected with an antibody against the c-myc epitope (Fig. 5Citation , right, Lane 3). A mutant DCC protein (DCC-TB) lacking the majority of the DCC extracellular domain (namely, the four immunoglobulin domains and FN3 domains 1–4) failed to cross-link to netrin-1 (right, Lane 4), implying that sequences in this region of the DCC extracellular domain were required for interaction with netrin-1. Cross-linking and immunoprecipitation studies in which cDNAs for netrin-1 or DCC were not transfected (Fig. 5Citation , right, Lanes 1 and 2) or in which netrin-1 was coexpressed with other transmembrane proteins, such as E-cadherin or a chimeric fusion protein containing the five immunoglobulin-like repeats of the CSF-1 receptor extracellular domain fused to the DCC transmembrane and cytoplasmic domains (Fig. 5Citation , right, Lane 5), confirmed the specificity of the cross-linking of netrin-1 to the DCC extracellular domain. No netrin-1 protein was immunoprecipitated with DCC, unless cells were treated with the DTSSP cross-linking reagent prior to lysis and immunoprecipitation (data not shown). Essentially identical cross-linking results were obtained in both 293 and COS-1 cells.



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Fig. 5. Cross-linking of netrin-1 to the DCC extracellular domain. Indicated above the lanes are the specific cDNA constructs that were transfected into 293 cells. Forty-eight h after transfection, cells were subjected to a 30-min treatment with 3,3'-dithiobis(sulfosuccinimidyl)propionate, a membrane-insoluble, cleavable cross-linking reagent. Cells were then harvested by scraping and lysed in radioimmunoprecipitation assay buffer. A portion of the lysate was placed in loading buffer with 2-mercaptoethanol, cleaving the cross-link, and then subjected to SDS-PAGE and ECL-Western blot analysis with an antibody against the c-myc 9E10 epitope tag at the COOH terminus of netrin-1 (left). The remainder of each lysate was immunoprecipitated with either a polyclonal rabbit antiserum against the DCC cytoplasmic domain or a monoclonal antibody against the E-cadherin extracellular domain. The immunoprecipitates were then placed in loading buffer with 2-mercapto-ethanol (which cleaves the cross-link), and then subjected to SDS-PAGE and ECL Western blot analysis with one of the following antibodies: a monoclonal antibody against the DCC cytoplasmic domain (middle, Lanes 1–5); a monoclonal antibody against the E-cadherin extracellular domain (middle, Lane 6); or the 9E10 anti-myc epitope antibody (right, Lanes 1–6). The myc-tagged netrin protein (Net-myc), indicated by an arrow, was detected in the immunoprecipitates only when coexpressed with full-length DCC (DCC-FL). No netrin-1 cross-linking was seen with a truncated DCC protein lacking immunoglobulin repeats 1–4 and FN3 repeats 1–4 (DCC-TB); a chimeric protein containing immunoglobulin repeats 1–5 of the CSF-1 receptor and the DCC transmembrane and cytoplasmic domains (CSF-DCC); or human E-cadherin. The mobility of selected molecular weight markers is shown at the left of each gel, and the mobilities of the transmembrane proteins are indicated (*). Note that the goat antimouse immunoglobulin secondary antibody detected the mouse immunoglobulin heavy chain in the E-cadherin immunoprecipitate (Ig).

 
We subsequently undertook studies to determine whether netrin-1 could bind stably to DCC in solution. The recombinant c-myc-tagged netrin-1 protein was expressed in 293 cells either alone or together with a recombinant DCC protein in which the DCC extracellular domain had been fused to the Fc region of IgG1. Both the netrin-1 and DCC fusion proteins were abundant in the supernatants of transfected cells (Fig. 6Citation , left). Although incubation of the concentrated medium on protein A-agarose beads allowed highly efficient recovery of the DCC fusion protein, the recombinant netrin-1 protein was not copurified with DCC (Fig. 6Citation , right). Taken together with the results of the cross-linking studies, the data indicate that netrin-1 binding to DCC may require the presence of a coreceptor or other accessory proteins.



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Fig. 6. Netrin-1 fails to bind to the DCC extracellular domain in solution. Left, an ECL-Western blot analysis of concentrated medium from transfected 293 kidney cells; right, an ECL-Western blot analysis of proteins recovered directly from the media after incubation with protein A beads. Transfections were carried out with plasmids encoding the DCC-immunoglobulin Fc fusion protein (DCC-Ig) and the myc epitope-tagged human netrin-1 protein. Transfected cells were incubated in the absence or presence of heparin (2 µg/ml), as indicated. 24 h after transfection, cells were placed in serum-free medium for an additional 24-h period. After a 3-fold concentration of the medium, the DCC-immunoglobulin fusion protein was recovered directly on protein A beads. Bound proteins were electrophoresed and subjected to ECL-Western blot analyses. ECL-Western analyses were carried out using a combination of the c-myc monoclonal antibody 9E10 and the DCC extracellular domain monoclonal G92–13. No detectable netrin-1 protein was recovered on the protein A beads, despite its abundant levels in the conditioned medium. The lanes at left show the abundance of the proteins in one-fifth volume of the concentrated medium prior to its protein A incubation.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Netrins are a family of laminin-related proteins with critical roles in axon guidance and cell migration in vertebrate and invertebrate development (2, 3, 4 , 6 , 9 , 24 , 25) . Genetic and cell biological studies have implicated two distinct families of transmembrane proteins, the DCC/UNC-40 like molecules and the UNC-5 like molecules, in the response to the netrin cues that guide axons and cells along specific trajectories (10, 11, 12, 13 , 22 , 26 , 27) . Nevertheless, prior to this study, the identity of the human NTN1L gene, its expression pattern in normal and neoplastic adult tissues, and the nature of the interaction between its encoded protein product and DCC were unknown. Here, we present studies that provide new insights into the NTN1L gene at chromosome 17p12–13 and its highly conserved 604-amino acid protein product.

Despite the fact that netrins have been hypothesized to function predominantly in axon outgrowth and cell migration during development, our studies indicate that the human NTN1L gene is broadly expressed in adult tissues as well as in certain cancer cell lines. These observations suggest that netrin-1 may have a function(s) distinct from that in mediating axon outgrowth and cell migration. Indeed, the recent studies of Mehlen et al. (28) indicate that, in some cell types, such as 293 cells and Caco-2 colorectal cancer cells, DCC may generate an antiapoptotic signal in the presence of netrin-1 and a proapoptotic signal in its absence. Similar to its axon outgrowth function, the role of netrin-1 in apoptosis appears to depend on its interaction with DCC (28) . Consistent with this proposal, we obtained evidence that netrin-1 could be cross-linked to DCC on the surface of 293 and COS-1 cells. However, recombinant human netrin-1 failed to immunoprecipitate with DCC in the absence of cross-linking and it failed to bind to the DCC extracellular domain in solution. Thus, a coreceptor or accessory proteins may be required for binding of netrin-1 to DCC. Whether DCC forms hetero-oligomers with UNC-5-like proteins to confer netrin-1 binding or whether DCC requires other unknown cell surface or matrix-associated proteins for netrin-1 binding will require further studies.

Although the specific mechanisms through which netrin-1 uses the DCC transmembrane protein to mediate its effects on various cell types remain poorly defined, it is of interest that NTN1L expression was markedly reduced or undetectable in {approx}50% of the neuroblastomas and brain tumors studied. In sequencing studies of NTN1L transcripts from a limited panel of cancers, missense mutations at highly conserved NTN1L codons were found in one neuroblastoma. Our findings on NTN1L loss of expression and mutations suggest that NTN1L inactivation, like DCC inactivation (15 , 18 , 39, 40, 41) , may contribute to the development of some cancers, such as neuroblastomas and brain tumors. The chromosomal localization of the NTN1L gene at 17p12–13 is also noteworthy. Specifically, neuroblastomas, medulloblastomas, and some glioblastomas have been found to have LOH of one p53 allele and the distal region of 17p, without detectable mutations in the retained p53 allele, implying that additional tumor suppressor genes may exist on 17p (30, 31, 32, 33, 34, 35, 36, 37, 38) . The fact that netrin-1 expression shows no clear-cut correlation with DCC expression in the cancer cell lines studied here suggests that patterns of expression and interactions between DCC, DCC-related proteins (e.g., neogenin), netrins, and other potential DCC ligands may be complex in normal and neoplastic tissues. Moreover, the relationship of UNC-5-like molecules and other transmembrane receptors and downstream signaling molecules in DCC-mediated apoptosis is not yet known. Further work should help to establish whether changes in the sequence and/or expression of NTN1L are causally related to cancer development. The function of netrins and their candidate receptors in development and in normal and cancerous tissues will likely result from a combination of biochemical, cell biological, and genetic approaches, and studies of NTN1L will shape this research.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cloning of Human NTN1L cDNAs.
Normal human brain tissue was generously provided by Dr. Phil Kisch (University of Michigan, Ann Arbor, MI). Total RNA was isolated using Trizol reagent per the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). cDNA was synthesized from {approx}5 µg of total RNA using oligo(dT)12–18 and avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.). Degenerate primers corresponding to amino acids 507–513 and 597–603 of the chicken netrin-1 sequence were used for PCR with the following conditions: initial 10 cycles of reduced stringency cycling of 94°C for 45 s, 45°C for 60 s, and 72°C for 90 s and 30 additional cycles with an annealing temperature of 55°C. An amplified PCR fragment of about 290 bp was subcloned into pAMP1, using the CloneAmp system (Life Technologies, Inc.). The fragment had 80% nucleotide identity to chicken netrin-1. This human netrin-1-like (NTN1L) fragment was labeled with [32P]dCTP by random priming and used to screen {approx}3 x 106 plaques of an oligo(dT)-primed human brain stem cDNA library (Stratagene Cloning Systems, La Jolla, CA). Plaques were lifted onto Hybond N+ nylon filters (Amersham, Arlington Heights, IL), and the filters were hybridized at 60°C as described (42) . A single hybridizing NTN1L clone was isolated following three rounds of hybridization selection, and the phagemid was rescued by in vivo excision using the ExAssist/SOLR system provided with the library. The 5'-most region of this brain stem NTN1L clone was radiolabeled and used to screen {approx}1 x 106 plaques from a random-primed human liver cDNA library, generously provided by Dr. James Anderson (Yale University, New Haven, CT). Manual and automated sequencing was carried out on both strands of rescued phagemids to construct the full-length open reading frame and flanking sequences. The human NTN1L cDNA sequence was submitted as GenBank accession no. U75586.

FISH.
The human P1 library (DMPC-HFF 1) was screened by Genome Systems, Inc. (St. Louis, MO) using primers to amplify a 231-bp fragment of NTN1L: forward, 5'-GACTGGTGGAAGTTCACTGT-3'; and reverse, 5'-GTGTCCCGCCACTGGATCAC-3'. Three P1 clones containing NTN1L sequences were isolated (DMPC-HFF 1-537-F4, DMPC-HFF 1-864-H3, and DMPC-HFF 1-1139-B10). Purified P1 DNAs were used to localize the NTN1L gene to chromosome 17p12–13 using FISH, as described previously (43 , 44) . Definitive chromosomal assignment was made by hybridizing the NTN1L P1 DNAs together with a chromosome 17 centromere-specific probe (D17Z1) or a p53-specific probe.

Northern Analysis.
Northern blots containing 2 µg of poly(A)+ RNA from normal human tissues were obtained from Clontech (Palo Alto, CA). Hybridizations were performed at 42°C according to manufacturer’s instructions, using two different netrin-1 cDNA probes. One probe was a 994-bp SmaI-HindIII fragment from the 5' half of the netrin-1 cDNA, and the other probe was a 620-bp HindIII-AccIII probe from the 3' half of the NTN1L cDNA. Blots were washed with 2x SSC-0.5% SDS for 45 min at room temperature, followed by an increased stringency wash with 0.1x SSC-0.1% SDS for 30 min at 50°C. Blots were stripped per the manufacturer’s recommendation and reprobed with a 32P-labeled 2.0-kbp fragment of ß-actin, provided by Clontech.

RNase Protection Assays.
Brain tumor xenografts established in nude mice were a generous gift from Dr. Sandra Bigner (Duke University, Durham, NC). The SJNB human neuroblastoma cell lines were established from primary tumors obtained at St. Jude Children’s Research Hospital. All other cell lines were obtained from American Type Culture Collection (Manassas, VA). Total RNA from the xenografts and neuroblastoma, colon, and breast cell lines was isolated using Trizol reagent (Life Technologies, Inc.). RNase protection assays were performed essentially as described (18 , 45) . The NTN1L riboprobe was generated from pHNET1185, a plasmid containing 291-bp NTN1L cDNA fragment (corresponding to amino acids 507–603). The ß-actin-125 probe was purchased from Ambion (Austin, TX). After purification through a polyacrylamide gel, {approx}1 x 106 cpm of NTN1L and 2 x 105 cpm of ß-actin antisense transcripts were combined and hybridized overnight with 20 µg of total RNA. Nonhybridizing sequences were digested with RNase T2 (Life Technologies, Inc.). Protected fragments were recovered by ethanol precipitation and electrophoresed on a denaturing polyacrylamide sequencing gel. After drying, gels were exposed to X-OMAT film (Eastman Kodak, Rochester, NY).

Expression Constructs.
A full-length NTN1L cDNA was constructed from the overlapping brain stem and liver cDNAs. A PCR-based strategy was used to fuse a c-myc epitope tag (amino acid sequence EQKLISEEDL) to the COOH terminus of the full-length NTN1L cDNA. The sequence of the modified cDNA was verified and the myc epitope-tagged NTN1L cDNA was subcloned into the pcDNA3 mammalian expression vector (Invitrogen, San Diego, CA), generating the expression construct pNET1-myc. The DCC expression construct pCMV/DCC-S has been described previously (46) . The pcDNA/DCC-TB construct contains a DCC cDNA insert in which the four immunoglobulin-like and FN3-like domains 1–4 have been removed, although the DCC signal sequence is maintained. The resultant DCC polypeptide contains FN3 domains 5 and 6, as well as transmembrane and cytoplasmic domains, and it is expressed on the cell surface. The CSF-DCC construct encodes a chimeric fusion protein with the five immunoglobulin-like repeats of the human CSF-1 receptor extracellular domain fused to the transmembrane and cytoplasmic domains of DCC. The pcDNA/DCC-Fc construct, encoding the four immunoglobulin and six FN3 domains of DCC fused to an immunoglobulin Fc region, was created by fusion of a PCR-amplified human IgG1 fragment from IM-9 cells (American Type Culture Collection) immediately downstream of DCC FN3 domain 6, at a point 34 amino acids proximal to the transmembrane region of DCC.

Expression and Immunoprecipitation of DCC and Netrin-1.
Transfections of COS-1 or 293 cells (American Type Culture Collection) with pNET1-myc with or without various DCC expression vectors were performed with Lipofectamine (Life Technologies, Inc.), per the manufacturer’s protocol. Total plasmid DNA masses were normalized in the transfections by adding control pcDNA3 plasmid, when necessary. For the transfection of 293 cells with pNET1-myc and pcDNA/DCC-Fc, cells were placed in Opti-MEM serum-free medium 24 h after transfection, in the presence or absence of 2 µg/ml heparin (Sigma Chemical Co., St. Louis, MO). After the cells were cultured for 24 h in the serum-free medium, the conditioned medium was concentrated 3-fold, and the DCC-Fc protein was precipitated from the media directly by incubation on protein-A agarose beads (Pierce, Rockford, IL). For Western blot studies, protein extracts were prepared as described previously (18 , 20) . Proteins were separated by 8% SDS-PAGE and then transferred to Immobilon-P membranes (Millipore, Bedford, MA) using a semidry electroblotter (Bio-Rad, Hercules, CA). The c-myc epitope-tagged netrin-1 protein was detected with an anti-myc antibody generated from a monoclonal hybridoma line MYC1–9E10.2 (ATCC). DCC proteins were detected using the G92-13 or G97-449 monoclonal antibodies (PharMingen, San Diego, CA) against the DCC extracellular or cytoplasmic domains, respectively. A secondary goat antimouse IgG antibody coupled to horseradish peroxidase (Pierce) was used, and antibody complexes were detected by ECL (Amersham, Arlington Heights, IL) and exposure to Kodak X-OMAT film. In cross-linking studies of netrin-1 and DCC, COS-1 transfected cells were washed with HBSS and treated for 30 min at room temperature with 1 mM 3,3'-dithiobis(sulfosuccinimidyl)propionate (Pierce), a cleavable, membrane-insoluble cross-linking reagent. The reaction was terminated by the addition of Tris-HCl (pH 7.5) to a final concentration of 10 mM. Protein extracts were prepared as above, and immunoprecipitation of DCC-protein complexes was carried out using rabbit polyclonal antiserum 721, as described previously (20) . SDS-PAGE and Western blotting on the immunoprecipitates was carried out as described above.


    Acknowledgments
 
We thank Drs. Sandra Bigner and Phil Kisch for generously providing cell line and tissue samples; Dr. James Anderson and Alex Brecher for the human liver cDNA library; Dr. Bert Vogelstein for assistance with p53 and NTN1L colocalization studies; and Virginia Valentine, Charlene H. An, and Kajal Sitwala for technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grants CA70097, CA71907, and CA21765. J. A. M. was supported by a predoctoral fellowship from the Howard Hughes Medical Institute. Back

2 The first two authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Division of Molecular Medicine and Genetics, University of Michigan Medical Center, 4301 MSRB III, Box 0638, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0638. Phone: (734) 764-1549; Fax: (734) 647-7979; E-mail: efearon{at}mmg.im.med.umich.edu Back

4 The abbreviations used are: DCC, deleted in colorectal cancer; FN3, fibronectin type III; FISH, fluorescence in situ hybridization; LOH, loss of heterozygosity; ECL, enhanced chemiluminescence. Back

Received for publication 7/27/98. Revision received 10/27/98. Accepted for publication 11/ 2/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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