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Molecular Cloning and Characterization of Human Frizzled-4 on Chromosome 11q14-q21
Biochemical and Biophysical Research Communications 264, 955–961 (1999) Article ID bbrc.1999.1612, available online at http://www.idealibrary.com on
Molecular Cloning and Characterization of Human Frizzled-4 on Chromosome 11q14-q21 Hiroyuki Kirikoshi,* ,† Norihiko Sagara,* Jun Koike,* Katsuaki Tanaka,† Hisahiko Sekihara,† Momoki Hirai,‡ and Masaru Katoh* ,1 *Genetics Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; †3rd Department of Internal Medicine, Yokohama City University, Yokohama 236-0004, Japan; and ‡Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Hongo 7-chome, Bunkyo-ku, Tokyo 113-0033, Japan
Received September 24, 1999
The WNT receptors, encoded by the Frizzled genes, are implicated in a variety of cellular processes such as cell fate determination, cell polarity control, and malignant transformation. Human Frizzled-4 (FZD4) cDNAs have been cloned and characterized. FZD4 spans a total of 7392 nucleotides and encodes a 537amino-acid protein with the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal S/T-X-V motif. The FZD4 mRNA of 7.7 kb in size were detected almost ubiquitously in normal human tissues and larger amounts in fetal kidney, adult heart, skeletal muscle, and ovary. Among cancer cell lines, the FZD4 mRNA level was higher in HeLa S3. The FZD4 gene has been mapped to human chromosome 11q14-q21. FZD4 is homologous to FZD9 and FZD10, and overall amino acid identity is as follows: FZD4 vs FZD9, 51.6%; FZD4 vs FZD10, 51.2%; FZD9 vs FZD10, 65.7%. FZD4 consists of two exons, while FZD9 and FZD10 consist of a single exon. FZD4 might belong to rather the independent FZD subfamily than the FZD9–FZD10 subfamily. © 1999 Academic Press Key Words: WNT receptor; gastric cancer; pancreatic cancer.
The WNT family, a group of cysteine-rich glycoproteins, plays a pivotal role in a variety of cellular processes, such as cell fate determination, cell polarity control and malignant transformation (1). The Frizzled (FZD) family consists of at least 10 members of seventransmembrane-receptors with the N-terminal cysteine rich domain (2), and functions as the WNT receptors (3). The N-terminal cysteine-rich domain of FZD is The nucleotide sequence data of FZD4 will appear in the DDBJ/ EMBL/GenBank databases under Accession No. AB032417. 1 To whom correspondence and reprint request should be addressed. Fax: 181-3-3541-2685. E-mail: [email protected].
necessary and sufficient for WNT binding, and constitutes the ligand-binding domain. The interaction between WNT and FZD induces signal transduction to the nucleus through the b-catenin/TCF pathway, or through the Jun-N-terminal kinase (JNK) pathway (4, 5). The secreted frizzled-related protein (SFRP) genes encode secreted proteins that contain the signal peptide, the cysteine-rich domain related to FZDs, and the C-terminal hydrophilic region with some homology to netrins (6). SFRP3 binds membrane-immobilized Wnt-1, and co-injection of the SFRP3 mRNA blocks the secondary axis formation induced by mRNA injection of Wnt-1 or Xwnt-8 in the Xenopus axis duplication assay (7, 8). SFRPs appear to act as soluble modulators of the WNT signaling pathway by competing with FZD receptors for the binding of WNTs (9). Human FZD1, FZD2, FZD5, FZD6, FZD7, FZD9, FZD10 (2, 10 –14) as well as SFRP1, SFRP2, SFRP3, SFRP4, and SFRP5 (6, 15–17) have been isolated so far. FZDs and SFRPs are homologous in the cysteinerich domain; however, none of these is encoded by a single gene due to alternative splicing. FZDs and SFRPs are encoded by each distinct gene. In this paper, we have cloned human Frizzled-4 (FZD4) encoding a seven-transmembrane-receptor with the N-terminal cysteine-rich domain. We have investigated the expression pattern, genomic structure, and chromosomal localization of FZD4. This is the first report on cloning and characterization of human FZD4. MATERIALS AND METHODS Cell lines and poly(A) 1 RNA extraction. OKAJIMA, TMK1, MKN7, MKN28, MKN45, MKN74, and KATO-III from gastric cancer (18, 19); PANC-1, BxPC-3, AsPC-1, PSN-1, 700T, 766T, and MIA PaCa-2 are derived from pancreatic cancer (20 –24). Poly(A) 1 RNAs
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were extracted from cancer cell lines with the FastTrack 2.0 Kit (Invitrogen, Carlsbad, CA). Computational biology and cDNA PCR. To isolate a FZD4 cDNA probe for cDNA library screening, human expressed sequence tags (ESTs) homologous to mouse frizzled-4 (Mfz4) (13) were searched by the blastn program (http://www.ncbi.nlm.nih.gov/BLAST) using Mfz4 as a query. Depending on the nucleotide sequence of ESTs with high score, PCR primers were designed: H4U (sense), 59TTCACAGTACTGACCTTCCTG-39; H4D (antisense), 59-ATGCCTGAAGTGATGCCCAC-39. The gastric cancer cDNA pool was synthesized from the mixture of poly(A) 1 RNAs extracted from the gastric cancer cell lines mentioned above with random hexamer primers with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Tokyo, Japan). Aliquots of the cDNA mixture were used for the subsequent PCR with TaqPlus Long DNA polymerase mixture (Stratagene, La Jolla, CA). PCR products were ligated to the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA). Plasmid DNAs were purified by Plasmid Mini Kit (QIAGEN, Tokyo, Japan), and aliquots were used for nucleotide sequence analyses with ABI310 Sequencer (PE Applied Biosystems, Chiba, Japan). Northern blot analyses. Two micrograms of poly(A) 1 RNA extracted from indicated sources was separated by 1.0% agarose gels containing 17.9% formaldehyde in 13 Mops buffer, and were transferred onto nitrocellulose filters, and then were fixed by baking at 80°C for 2 h in a vacuum oven. Northern blot filters were hybridized with a [a- 32P]dCTP-labeled probe at 68°C for 1 h in QuikHyb solution (Stratagene, La Jolla, CA). Filters were washed in 23 SSC buffer and 0.1% SDS at room temperature for 15 min twice, in 0.13 SSC buffer and 0.1% SDS at 60°C for 30 min, and then were exposed to XAR-5 film (Kodak, Rochester, NY). cDNA library screening. Human fetal lung cDNA library in lgt10 (Clontech, Palo Alto, CA) was screened with a FZD4 cDNA fragment. Phage plaque of human fetal lung cDNA library in lgt10 (Clontech, Palo Alto, CA) was transferred to Hybond-N1 filters (Amersham Pharmacia Biotech, Tokyo, Japan). Phage DNAs on the filters were denatured, neutralized, and then fixed by UV light. cDNA-library filters were hybridized with [a- 32P]dCTP-labeled FZGC4 as described above. Filters were washed, and then were exposed to XAR-5 film (Kodak, Rochester, NY) for 16 h. After secondary screening, phage DNAs were purified with Lambda Mini Kit (QIAGEN, Tokyo, Japan) for sequence analyses. Genomic DNA library screening. Human genomic DNA library in EMBL3 SP6/T7 (Clontech, Palo Alto, CA) was screened with FZD4 cDNA fragments. After secondary screening, phage DNAs were purified with Lambda Midi Kit (QIAGEN, Tokyo, Japan) for sequence analyses. Fluorescence in situ hybridization (FISH). Human metaphase chromosomes with replication R-bands were prepared and hybridized to a biotin-14-dATP-labeled probe, followed by washing, detection with rabbit anti-biotin (Enzo, Farmingdale, NY) and fluoresceinlabeled goat anti-rabbit IgG (Enzo, Farmingdale, NY), and counterstained with propidium iodide (25).
RESULTS Isolation of FZD4 cDNAs Two human ESTs (Accession Nos. R26355 and W35336) homologous to Mfz4 were identified by the blastn program. R26355 corresponds to the nucleotide position 701–1062 of Mfz4, and W35336 corresponds to the nucleotide position 1642–1867 of Mfz4; however, neither R26355 or W35336 spans to the entire coding region of FZD4.
FIG. 1. (A) Structure of FZD4 cDNA. FZD4 spans a total of 7392 nucleotides. The coding region and the noncoding regions are indicated by the closed box and the open box, respectively. Also shown are EcoRI site (E), the FZGC4 probe for cDNA library screening, and the HF4S probe for northern blot analyses. (B) Deduced amino-acid sequence of FZD4. Amino acids are numbered at the right. Transmembrane domains (double overline with Roman numeral), conserved cysteine residues in the N-terminal extracellular region (Arabic number above alignment), potential N-glycosylation sites in the N-terminal extracellular region (sharp), conserved cysteine residues in the second and third extracellular loops (cross), and the S/T-X-V motif in the C-terminus (@ marks) are indicated.
A FZD4 cDNA fragment spanning ESTs R26355 or W35336 was isolated by cDNA-PCR. PCR primers H4U and H4D correspond to the nucleotide position 30 –10 of EST R26355, and the nucleotide position 140 –159 of EST W35336, respectively. The FZGC4 cDNA fragment of 773 bp in length was isolated by cDNA-PCR from the human gastric cancer cDNA pool. The 59-portion of FZGC4 was identical to R26355, and the 39-portion of FZGC4 was identical to W35336. FZGC4 corresponded to the nucleotide position 1033– 1805 of Mfz4. Partial amino-acid identity between FZGC4 and Mfz4 was 99.6%. Thus, we used the FZGC4 cDNA fragment as the probe for cDNA library screening to isolate FZD4 cDNAs spanning the entire coding region. Since the amount of mRNA hybridized to the FZGC4 probe was relatively large in human fetal lung (data
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FIG. 2. Amino-acid comparison among human FZDs. FZD4 is compared with FZD1, FZD2, FZD6, FZD7, FZD9, and FZD10. Conserved amino-acids are indicated by asterisk below the alignment, and amino acid identity with FZD4 are indicated in the right. (A) Alignment in the N-terminal cysteine rich domain. Conserved cysteine residues in the N-terminal extracellular region are shown in Arabic number above alignment. (B) Alignment in the transmembrane domains. Transmembrane domains are shown as double overline with Roman numeral.
not shown), the human fetal lung cDNA library was screened with FZGC4. Eight positive clones were isolated out of 1.0 3 10 6 clones. FZD4 cDNAs span a total of 7392 nucleotides, which contained two internal EcoRI sites (Fig. 1A). Putative Amino Acid Sequence of FZD4 FZD4 consists of the 306-nucleotide 59-noncoding region, the 1614-nucleotide coding region, and the 5472 39-noncoding region. FZD4 of 537 amino acids consists of seven transmembrane domains, a cysteine-rich domain in the N-terminal extracellular region, two cysteine residues in the second and third extracellular loops (Cys 282 and Cys377), two N-linked glycosylation sites in the extracellular region (Asn 59 and Asn 144), and the S/T-X-V motif in the C-terminus (Thr 535-Val 537) (Fig. 1B). The amino-acid sequence of human FZD were aligned in the cysteine-rich domain (Fig. 2A), as well as in the transmembrane domains (Fig. 2B). FZD4 are homologous to FZD10, FZD9, FZD1, FZD2, and FZD7; however homology was lower than those between FZD9 and FZD10 (2), or those among FZD1, FZD2, and
FZD7 (10). Overall amino-acid identity among FZD4, FZD9 and FZD10 is as follows: FZD4 vs FZD9, 51.6%; FZD4 vs FZD10, 51.2%; FZD9 vs FZD10, 65.7%. Gly 115, Gly 118, Leu122, Ala 313, Ala 370, Val 392,Ile 406, and Ala407 of FZD4 are conserved in the mouse homologue of FZD4, but are substituted to Pro, Arg, Glu, Thr, Gly, Leu, Leu, and Leu, respectively, in human FZDs except FZD4 (Fig. 2). Expression Analysis on FZD4 The FZD4 specific probes, HF4S, corresponds to the 39-noncoding region of FZD4 (nucleotide position 4808 –5328 of FZD4) (Fig. 1A). Northern blot analysis with HF4S detected the FZD4 mRNA of 7.7 kb in size (Fig. 3). Among normal human tissues, large amounts of FZD4 mRNA were detected in adult heart, skeletal muscle, ovary and fetal kidney, moderate amounts in adult liver, kidney, pancreas, spleen and fetal lung, small amounts in placenta, adult lung, prostate, testis, colon, fetal brain, and liver (Figs. 3A and 3B). Among the cancer cell lines examined, large amounts of FZD4 mRNA were detected in cervical cancer cell
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FIG. 3. Northern blot analyses on FZD4 mRNA expression. The HF4S probe (nucleotide position 4808 –5328 of FZD4) was hybridized to Multiple Tissue Northern filters (Clontech) containing 2 mg of poly(A) 1 RNA extracted from adult human tissues (A), fetal human tissues (B), and human cancer cell lines (C).
line HeLa S3, small amounts in colon cancer cell line SW480, chronic myelogenous leukemia cell line K-562, melanoma cell line G361, and pancreatic cancer cell line 700T and 766T (Figs. 3C and 4). Genomic Structure of FZD4 To determine the genomic structure of the FZD4 gene, the human genomic DNA library was screened with the HF4-14 cDNA, and three clones were isolated from 3.0 3 10 5 clones. Comparison of the nucleotide sequences of the genomic FZD4 clones and the FZD4 cDNAs revealed that the FZD4 mRNA consists of two exons, separated by a 2336-nucleotide intron 1 (Fig. 5A). The consensus sequence of splice donor and acceptor sites (26) are found in the exon-intron boundaries of the FZD4 gene (Fig. 5B).
seven-transmembrane-receptor with the N-terminal cysteine-rich domain and the C-terminal S/T-X-V motif (Fig. 1). The domain structure with the N-terminal cysteine rich domain and seven transmembrane domains are conserved among all members of the human FZD family isolated so far, while the C-terminal S/TX-V motif is conserved among some members of the human FZD family: FZD4, FZD1, FZD2, FZD5, FZD7, and FZD10. In the transmembrane domains, the homology between FZD4 and other members human FZD family is highest in FZD10 (69%), followed by FZD9 (67%), FZD2 (61%), FZD1 (61%), FZD7 (61%), FZD5 (58%), and FZD6 (42%) (Fig. 2B). As we have previously reported, FZD1, FZD2 and FZD7 constitute one subfamily, and FZD9 and FZD10 constitute another subfamily (2, 10). Homology among subfamily members in the transmembrane domains shown in Fig. 2B is much higher than the homology between FZD4 and other members of human FZD family: FZD1 vs FZD2, 92%; FZD1 vs FZD7, 91%; FZD2 vs FZD7, 91%; FZD9 vs FZD10, 79%. The FZD4 gene consists of two exons, and the coding region of FZD4 is split by intron 1 (Figs. 5A and 5B). We have previously reported that FZD10 consists of a single exon (2). We have also isolated human FZD9 genomic clones, and found that FZD9 also consists of a single exon (Koike and Katoh, unpublished data). Difference in the genomic structure as well as relatively lower homology between FZD4 and other members of human FZD family suggest that FZD4 might belong to an independent FZD subfamily. The expression of FZD4 mRNAs in normal human tissues was almost ubiquitous, while the expression of
Chromosomal Localization of FZD4 Human metaphase chromosomes with replication R-bands were prepared and hybridized to a biotin-14dATP-labeled HF4T probe corresponding to the 39noncoding region and a part of the open reading frame of the FZD4 (nucleotide position 1842– 4811). The hybridization signals were detected on human chromosome 11q14-q21 with HF4T (Fig. 5C). DISCUSSION This is the first report on molecular cloning and characterization of human FZD4, which encodes a
FIG. 4. FZD4 mRNA expression in human gastroenterological cancer. (A) Gastric cancer. (B) Pancreatic cancer.
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FIG. 5. Structure and human chromosomal localization of the FZD4 gene. (A) Structure of the FZD4 gene. Exons are indicated by boxes. The coding region and the noncoding regions are indicated by the closed box and the open box, respectively. Also shown are EcoRI site (E) and the HF4T probe for FISH. (B) Nucleotide and deduced amino-acid sequence of the FZD4 gene around the exon-intron boundaries. Exon sequence is indicated by large caps, while intron sequence is indicated by small caps. (C) Human chromosomal localization of the FZD4 gene. Human metaphase chromosomes with replication R-bands were prepared and hybridized with a biotin-14-dATP-labeled HF4T probe (nucleotide position 1842– 4811). The hybridization signals were detected on human chromosome 11q14-q21 (arrow).
FZD4 mRNAs in cancer cell lines was relatively restricted (Figs. 3 and 4). Although we could successfully establish stable transformants overexpressing the mutated FZD4 (V537L), we failed to establish stable transformants overexpressing the wild type FZD4 (Sa-
gara and Katoh, unpublished data). These results suggests that overexpression of the wild type FZD4 might be disadvantageous to the growth of cancer cells. To investigate whether overexpression of the wild type FZD4 in cancer cells induces growth inhibition or not,
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Expression of FZDs in Human Cancer Cell Lines
FZD1 FZD2 FZD4 FZD6 FZD7 FZD10
HL-60
HeLa S3
K-562
MOLT-4
Raji
SW480
A549
G361
Reference
1 2 2 1 2 2
11 11 111 1 1/2 111
1 1 1 1 2 2
11 1 2 1 2 2
1 2 2 2 2 2
11 1 1 1 1 1
11 11 1/2 1 1 2
11 1 1 1 1 2
10 10 This study 13 10 2
we are now establishing stable transformants expressing FZD4 under the inducible promoter. We have previously reported cloning and characterization of human FZD1, FZD2, FZD6, FZD7, and FZD10 (2, 10, 13). Comparison of FZD expression pattern in cancer cell lines revealed that multiple FZDs were detected in each cancer cell line, except that only FZD1 is detected in Raji (Table 1). Recently, biological and pharmacological evidence for the heterodimerization of two fully functional seventransmembrane-receptors, opioid receptor kappa and opioid receptor delta, has been reported (27). FZDs might also interact to form heterodimers. We mapped the FZD4 gene to human chromosome 11q14-q21 (Fig. 5C), where the tyrosinase gene (28), the break point in t(11;18) (q21;q21) associated with mucosal-associated lymphoid tissue (MALT) lymphomas (29), and the Pallilon–Lefevre syndrome locus (30, 31) are also mapped. Although the apoptosis inhibitor gene API2/c-IAP2/HIAP1/MIHC is identified to be involved in t(11;18) (q21;q21) of MALT lymphomas (32), another gene on 11q21 might also be implicated in t(11;18) (q21;q21). The Pallilon– Lefevre syndrome is an autosomal recessively inherited palmoplantar keratoderma of unknown etiology associated with severe periodontitis leading to loss of dentition. The causative gene for the Pallilon– Lefevre syndrome has not yet been identified. Thus, we will investigate the genetic alterations of the FZD4 gene in MALT lymphomas and the Pallilon– Lefevre syndrome. ACKNOWLEDGMENTS We thank Dr. Masaaki Terada for his encouragement. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan. H.K. and J.K. are Awardees of Research Resident Fellowships from the Foundation of Promotion for Cancer Research.
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Molecular Cloning and Characterization of Human Frizzled-4 on Chromosome 11q14-q21 Hiroyuki Kirikoshi,* ,† Norihiko Sagara,* Jun Koike,* Katsuaki Tanaka,† Hisahiko Sekihara,† Momoki Hirai,‡ and Masaru Katoh* ,1 *Genetics Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; †3rd Department of Internal Medicine, Yokohama City University, Yokohama 236-0004, Japan; and ‡Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Hongo 7-chome, Bunkyo-ku, Tokyo 113-0033, Japan
Received September 24, 1999
The WNT receptors, encoded by the Frizzled genes, are implicated in a variety of cellular processes such as cell fate determination, cell polarity control, and malignant transformation. Human Frizzled-4 (FZD4) cDNAs have been cloned and characterized. FZD4 spans a total of 7392 nucleotides and encodes a 537amino-acid protein with the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal S/T-X-V motif. The FZD4 mRNA of 7.7 kb in size were detected almost ubiquitously in normal human tissues and larger amounts in fetal kidney, adult heart, skeletal muscle, and ovary. Among cancer cell lines, the FZD4 mRNA level was higher in HeLa S3. The FZD4 gene has been mapped to human chromosome 11q14-q21. FZD4 is homologous to FZD9 and FZD10, and overall amino acid identity is as follows: FZD4 vs FZD9, 51.6%; FZD4 vs FZD10, 51.2%; FZD9 vs FZD10, 65.7%. FZD4 consists of two exons, while FZD9 and FZD10 consist of a single exon. FZD4 might belong to rather the independent FZD subfamily than the FZD9–FZD10 subfamily. © 1999 Academic Press Key Words: WNT receptor; gastric cancer; pancreatic cancer.
The WNT family, a group of cysteine-rich glycoproteins, plays a pivotal role in a variety of cellular processes, such as cell fate determination, cell polarity control and malignant transformation (1). The Frizzled (FZD) family consists of at least 10 members of seventransmembrane-receptors with the N-terminal cysteine rich domain (2), and functions as the WNT receptors (3). The N-terminal cysteine-rich domain of FZD is The nucleotide sequence data of FZD4 will appear in the DDBJ/ EMBL/GenBank databases under Accession No. AB032417. 1 To whom correspondence and reprint request should be addressed. Fax: 181-3-3541-2685. E-mail: [email protected].
necessary and sufficient for WNT binding, and constitutes the ligand-binding domain. The interaction between WNT and FZD induces signal transduction to the nucleus through the b-catenin/TCF pathway, or through the Jun-N-terminal kinase (JNK) pathway (4, 5). The secreted frizzled-related protein (SFRP) genes encode secreted proteins that contain the signal peptide, the cysteine-rich domain related to FZDs, and the C-terminal hydrophilic region with some homology to netrins (6). SFRP3 binds membrane-immobilized Wnt-1, and co-injection of the SFRP3 mRNA blocks the secondary axis formation induced by mRNA injection of Wnt-1 or Xwnt-8 in the Xenopus axis duplication assay (7, 8). SFRPs appear to act as soluble modulators of the WNT signaling pathway by competing with FZD receptors for the binding of WNTs (9). Human FZD1, FZD2, FZD5, FZD6, FZD7, FZD9, FZD10 (2, 10 –14) as well as SFRP1, SFRP2, SFRP3, SFRP4, and SFRP5 (6, 15–17) have been isolated so far. FZDs and SFRPs are homologous in the cysteinerich domain; however, none of these is encoded by a single gene due to alternative splicing. FZDs and SFRPs are encoded by each distinct gene. In this paper, we have cloned human Frizzled-4 (FZD4) encoding a seven-transmembrane-receptor with the N-terminal cysteine-rich domain. We have investigated the expression pattern, genomic structure, and chromosomal localization of FZD4. This is the first report on cloning and characterization of human FZD4. MATERIALS AND METHODS Cell lines and poly(A) 1 RNA extraction. OKAJIMA, TMK1, MKN7, MKN28, MKN45, MKN74, and KATO-III from gastric cancer (18, 19); PANC-1, BxPC-3, AsPC-1, PSN-1, 700T, 766T, and MIA PaCa-2 are derived from pancreatic cancer (20 –24). Poly(A) 1 RNAs
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were extracted from cancer cell lines with the FastTrack 2.0 Kit (Invitrogen, Carlsbad, CA). Computational biology and cDNA PCR. To isolate a FZD4 cDNA probe for cDNA library screening, human expressed sequence tags (ESTs) homologous to mouse frizzled-4 (Mfz4) (13) were searched by the blastn program (http://www.ncbi.nlm.nih.gov/BLAST) using Mfz4 as a query. Depending on the nucleotide sequence of ESTs with high score, PCR primers were designed: H4U (sense), 59TTCACAGTACTGACCTTCCTG-39; H4D (antisense), 59-ATGCCTGAAGTGATGCCCAC-39. The gastric cancer cDNA pool was synthesized from the mixture of poly(A) 1 RNAs extracted from the gastric cancer cell lines mentioned above with random hexamer primers with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Tokyo, Japan). Aliquots of the cDNA mixture were used for the subsequent PCR with TaqPlus Long DNA polymerase mixture (Stratagene, La Jolla, CA). PCR products were ligated to the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA). Plasmid DNAs were purified by Plasmid Mini Kit (QIAGEN, Tokyo, Japan), and aliquots were used for nucleotide sequence analyses with ABI310 Sequencer (PE Applied Biosystems, Chiba, Japan). Northern blot analyses. Two micrograms of poly(A) 1 RNA extracted from indicated sources was separated by 1.0% agarose gels containing 17.9% formaldehyde in 13 Mops buffer, and were transferred onto nitrocellulose filters, and then were fixed by baking at 80°C for 2 h in a vacuum oven. Northern blot filters were hybridized with a [a- 32P]dCTP-labeled probe at 68°C for 1 h in QuikHyb solution (Stratagene, La Jolla, CA). Filters were washed in 23 SSC buffer and 0.1% SDS at room temperature for 15 min twice, in 0.13 SSC buffer and 0.1% SDS at 60°C for 30 min, and then were exposed to XAR-5 film (Kodak, Rochester, NY). cDNA library screening. Human fetal lung cDNA library in lgt10 (Clontech, Palo Alto, CA) was screened with a FZD4 cDNA fragment. Phage plaque of human fetal lung cDNA library in lgt10 (Clontech, Palo Alto, CA) was transferred to Hybond-N1 filters (Amersham Pharmacia Biotech, Tokyo, Japan). Phage DNAs on the filters were denatured, neutralized, and then fixed by UV light. cDNA-library filters were hybridized with [a- 32P]dCTP-labeled FZGC4 as described above. Filters were washed, and then were exposed to XAR-5 film (Kodak, Rochester, NY) for 16 h. After secondary screening, phage DNAs were purified with Lambda Mini Kit (QIAGEN, Tokyo, Japan) for sequence analyses. Genomic DNA library screening. Human genomic DNA library in EMBL3 SP6/T7 (Clontech, Palo Alto, CA) was screened with FZD4 cDNA fragments. After secondary screening, phage DNAs were purified with Lambda Midi Kit (QIAGEN, Tokyo, Japan) for sequence analyses. Fluorescence in situ hybridization (FISH). Human metaphase chromosomes with replication R-bands were prepared and hybridized to a biotin-14-dATP-labeled probe, followed by washing, detection with rabbit anti-biotin (Enzo, Farmingdale, NY) and fluoresceinlabeled goat anti-rabbit IgG (Enzo, Farmingdale, NY), and counterstained with propidium iodide (25).
RESULTS Isolation of FZD4 cDNAs Two human ESTs (Accession Nos. R26355 and W35336) homologous to Mfz4 were identified by the blastn program. R26355 corresponds to the nucleotide position 701–1062 of Mfz4, and W35336 corresponds to the nucleotide position 1642–1867 of Mfz4; however, neither R26355 or W35336 spans to the entire coding region of FZD4.
FIG. 1. (A) Structure of FZD4 cDNA. FZD4 spans a total of 7392 nucleotides. The coding region and the noncoding regions are indicated by the closed box and the open box, respectively. Also shown are EcoRI site (E), the FZGC4 probe for cDNA library screening, and the HF4S probe for northern blot analyses. (B) Deduced amino-acid sequence of FZD4. Amino acids are numbered at the right. Transmembrane domains (double overline with Roman numeral), conserved cysteine residues in the N-terminal extracellular region (Arabic number above alignment), potential N-glycosylation sites in the N-terminal extracellular region (sharp), conserved cysteine residues in the second and third extracellular loops (cross), and the S/T-X-V motif in the C-terminus (@ marks) are indicated.
A FZD4 cDNA fragment spanning ESTs R26355 or W35336 was isolated by cDNA-PCR. PCR primers H4U and H4D correspond to the nucleotide position 30 –10 of EST R26355, and the nucleotide position 140 –159 of EST W35336, respectively. The FZGC4 cDNA fragment of 773 bp in length was isolated by cDNA-PCR from the human gastric cancer cDNA pool. The 59-portion of FZGC4 was identical to R26355, and the 39-portion of FZGC4 was identical to W35336. FZGC4 corresponded to the nucleotide position 1033– 1805 of Mfz4. Partial amino-acid identity between FZGC4 and Mfz4 was 99.6%. Thus, we used the FZGC4 cDNA fragment as the probe for cDNA library screening to isolate FZD4 cDNAs spanning the entire coding region. Since the amount of mRNA hybridized to the FZGC4 probe was relatively large in human fetal lung (data
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FIG. 2. Amino-acid comparison among human FZDs. FZD4 is compared with FZD1, FZD2, FZD6, FZD7, FZD9, and FZD10. Conserved amino-acids are indicated by asterisk below the alignment, and amino acid identity with FZD4 are indicated in the right. (A) Alignment in the N-terminal cysteine rich domain. Conserved cysteine residues in the N-terminal extracellular region are shown in Arabic number above alignment. (B) Alignment in the transmembrane domains. Transmembrane domains are shown as double overline with Roman numeral.
not shown), the human fetal lung cDNA library was screened with FZGC4. Eight positive clones were isolated out of 1.0 3 10 6 clones. FZD4 cDNAs span a total of 7392 nucleotides, which contained two internal EcoRI sites (Fig. 1A). Putative Amino Acid Sequence of FZD4 FZD4 consists of the 306-nucleotide 59-noncoding region, the 1614-nucleotide coding region, and the 5472 39-noncoding region. FZD4 of 537 amino acids consists of seven transmembrane domains, a cysteine-rich domain in the N-terminal extracellular region, two cysteine residues in the second and third extracellular loops (Cys 282 and Cys377), two N-linked glycosylation sites in the extracellular region (Asn 59 and Asn 144), and the S/T-X-V motif in the C-terminus (Thr 535-Val 537) (Fig. 1B). The amino-acid sequence of human FZD were aligned in the cysteine-rich domain (Fig. 2A), as well as in the transmembrane domains (Fig. 2B). FZD4 are homologous to FZD10, FZD9, FZD1, FZD2, and FZD7; however homology was lower than those between FZD9 and FZD10 (2), or those among FZD1, FZD2, and
FZD7 (10). Overall amino-acid identity among FZD4, FZD9 and FZD10 is as follows: FZD4 vs FZD9, 51.6%; FZD4 vs FZD10, 51.2%; FZD9 vs FZD10, 65.7%. Gly 115, Gly 118, Leu122, Ala 313, Ala 370, Val 392,Ile 406, and Ala407 of FZD4 are conserved in the mouse homologue of FZD4, but are substituted to Pro, Arg, Glu, Thr, Gly, Leu, Leu, and Leu, respectively, in human FZDs except FZD4 (Fig. 2). Expression Analysis on FZD4 The FZD4 specific probes, HF4S, corresponds to the 39-noncoding region of FZD4 (nucleotide position 4808 –5328 of FZD4) (Fig. 1A). Northern blot analysis with HF4S detected the FZD4 mRNA of 7.7 kb in size (Fig. 3). Among normal human tissues, large amounts of FZD4 mRNA were detected in adult heart, skeletal muscle, ovary and fetal kidney, moderate amounts in adult liver, kidney, pancreas, spleen and fetal lung, small amounts in placenta, adult lung, prostate, testis, colon, fetal brain, and liver (Figs. 3A and 3B). Among the cancer cell lines examined, large amounts of FZD4 mRNA were detected in cervical cancer cell
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FIG. 3. Northern blot analyses on FZD4 mRNA expression. The HF4S probe (nucleotide position 4808 –5328 of FZD4) was hybridized to Multiple Tissue Northern filters (Clontech) containing 2 mg of poly(A) 1 RNA extracted from adult human tissues (A), fetal human tissues (B), and human cancer cell lines (C).
line HeLa S3, small amounts in colon cancer cell line SW480, chronic myelogenous leukemia cell line K-562, melanoma cell line G361, and pancreatic cancer cell line 700T and 766T (Figs. 3C and 4). Genomic Structure of FZD4 To determine the genomic structure of the FZD4 gene, the human genomic DNA library was screened with the HF4-14 cDNA, and three clones were isolated from 3.0 3 10 5 clones. Comparison of the nucleotide sequences of the genomic FZD4 clones and the FZD4 cDNAs revealed that the FZD4 mRNA consists of two exons, separated by a 2336-nucleotide intron 1 (Fig. 5A). The consensus sequence of splice donor and acceptor sites (26) are found in the exon-intron boundaries of the FZD4 gene (Fig. 5B).
seven-transmembrane-receptor with the N-terminal cysteine-rich domain and the C-terminal S/T-X-V motif (Fig. 1). The domain structure with the N-terminal cysteine rich domain and seven transmembrane domains are conserved among all members of the human FZD family isolated so far, while the C-terminal S/TX-V motif is conserved among some members of the human FZD family: FZD4, FZD1, FZD2, FZD5, FZD7, and FZD10. In the transmembrane domains, the homology between FZD4 and other members human FZD family is highest in FZD10 (69%), followed by FZD9 (67%), FZD2 (61%), FZD1 (61%), FZD7 (61%), FZD5 (58%), and FZD6 (42%) (Fig. 2B). As we have previously reported, FZD1, FZD2 and FZD7 constitute one subfamily, and FZD9 and FZD10 constitute another subfamily (2, 10). Homology among subfamily members in the transmembrane domains shown in Fig. 2B is much higher than the homology between FZD4 and other members of human FZD family: FZD1 vs FZD2, 92%; FZD1 vs FZD7, 91%; FZD2 vs FZD7, 91%; FZD9 vs FZD10, 79%. The FZD4 gene consists of two exons, and the coding region of FZD4 is split by intron 1 (Figs. 5A and 5B). We have previously reported that FZD10 consists of a single exon (2). We have also isolated human FZD9 genomic clones, and found that FZD9 also consists of a single exon (Koike and Katoh, unpublished data). Difference in the genomic structure as well as relatively lower homology between FZD4 and other members of human FZD family suggest that FZD4 might belong to an independent FZD subfamily. The expression of FZD4 mRNAs in normal human tissues was almost ubiquitous, while the expression of
Chromosomal Localization of FZD4 Human metaphase chromosomes with replication R-bands were prepared and hybridized to a biotin-14dATP-labeled HF4T probe corresponding to the 39noncoding region and a part of the open reading frame of the FZD4 (nucleotide position 1842– 4811). The hybridization signals were detected on human chromosome 11q14-q21 with HF4T (Fig. 5C). DISCUSSION This is the first report on molecular cloning and characterization of human FZD4, which encodes a
FIG. 4. FZD4 mRNA expression in human gastroenterological cancer. (A) Gastric cancer. (B) Pancreatic cancer.
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FIG. 5. Structure and human chromosomal localization of the FZD4 gene. (A) Structure of the FZD4 gene. Exons are indicated by boxes. The coding region and the noncoding regions are indicated by the closed box and the open box, respectively. Also shown are EcoRI site (E) and the HF4T probe for FISH. (B) Nucleotide and deduced amino-acid sequence of the FZD4 gene around the exon-intron boundaries. Exon sequence is indicated by large caps, while intron sequence is indicated by small caps. (C) Human chromosomal localization of the FZD4 gene. Human metaphase chromosomes with replication R-bands were prepared and hybridized with a biotin-14-dATP-labeled HF4T probe (nucleotide position 1842– 4811). The hybridization signals were detected on human chromosome 11q14-q21 (arrow).
FZD4 mRNAs in cancer cell lines was relatively restricted (Figs. 3 and 4). Although we could successfully establish stable transformants overexpressing the mutated FZD4 (V537L), we failed to establish stable transformants overexpressing the wild type FZD4 (Sa-
gara and Katoh, unpublished data). These results suggests that overexpression of the wild type FZD4 might be disadvantageous to the growth of cancer cells. To investigate whether overexpression of the wild type FZD4 in cancer cells induces growth inhibition or not,
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Expression of FZDs in Human Cancer Cell Lines
FZD1 FZD2 FZD4 FZD6 FZD7 FZD10
HL-60
HeLa S3
K-562
MOLT-4
Raji
SW480
A549
G361
Reference
1 2 2 1 2 2
11 11 111 1 1/2 111
1 1 1 1 2 2
11 1 2 1 2 2
1 2 2 2 2 2
11 1 1 1 1 1
11 11 1/2 1 1 2
11 1 1 1 1 2
10 10 This study 13 10 2
we are now establishing stable transformants expressing FZD4 under the inducible promoter. We have previously reported cloning and characterization of human FZD1, FZD2, FZD6, FZD7, and FZD10 (2, 10, 13). Comparison of FZD expression pattern in cancer cell lines revealed that multiple FZDs were detected in each cancer cell line, except that only FZD1 is detected in Raji (Table 1). Recently, biological and pharmacological evidence for the heterodimerization of two fully functional seventransmembrane-receptors, opioid receptor kappa and opioid receptor delta, has been reported (27). FZDs might also interact to form heterodimers. We mapped the FZD4 gene to human chromosome 11q14-q21 (Fig. 5C), where the tyrosinase gene (28), the break point in t(11;18) (q21;q21) associated with mucosal-associated lymphoid tissue (MALT) lymphomas (29), and the Pallilon–Lefevre syndrome locus (30, 31) are also mapped. Although the apoptosis inhibitor gene API2/c-IAP2/HIAP1/MIHC is identified to be involved in t(11;18) (q21;q21) of MALT lymphomas (32), another gene on 11q21 might also be implicated in t(11;18) (q21;q21). The Pallilon– Lefevre syndrome is an autosomal recessively inherited palmoplantar keratoderma of unknown etiology associated with severe periodontitis leading to loss of dentition. The causative gene for the Pallilon– Lefevre syndrome has not yet been identified. Thus, we will investigate the genetic alterations of the FZD4 gene in MALT lymphomas and the Pallilon– Lefevre syndrome. ACKNOWLEDGMENTS We thank Dr. Masaaki Terada for his encouragement. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan. H.K. and J.K. are Awardees of Research Resident Fellowships from the Foundation of Promotion for Cancer Research.
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