- Email: [email protected]
Somatic alterations of the DPC4 gene in human colorectal cancers in vivo
GASTROENTEROLOGY 1996;111:1369–1372
RAPID COMMUNICATIONS Somatic Alterations of the DPC4 Gene in Human Colorectal Cancers In Vivo YUKIHIRO TAKAGI,* HISASHI KOHMURA,* MANABU FUTAMURA,* HISASHI KIDA,* HIROMI TANEMURA,* KUNIYASU SHIMOKAWA,‡ and SHIGETOYO SAJI* *Department of Surgery II and ‡Department of Laboratory Medicine, Gifu University School of Medicine, Gifu, Japan
See editorial on page 1387. Background & Aims: The chromosome region 18q21 has been shown to be frequently deleted in colorectal cancers, and such frequent allelic loss is a hallmark of the presence of a tumor-suppressor gene. The DPC4 gene, which is located at 18q21, has been identified as a tumor-suppressor gene from examination of pancreatic cancers. The aim of the present study was to determine if it might also be altered in colorectal cancers. Methods: Mutation analyses of the DPC4 gene were performed on complementary DNA samples from 31 primary colorectal cancer specimens using a combination of polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Results: Four missense mutations producing amino acid substitutions and a somatic 12–base pair deletion in the coding region of the DPC4 gene were detected in the 31 cancers (16%; 5 of 31). Conclusions: The DPC4 gene may play a role as a tumor-suppressor gene in a fraction of colorectal cancers; however, while allelic loss at 18q21 is very often seen in colorectal cancers, only a minority show DPC4 mutations, suggesting that there might be another tumor-suppressor gene in this chromosome region.
T
umor-suppressor genes are characterized by alterations in cancers that inactivate both alleles.1 This is often accomplished by intragenic mutations in one allele accompanied by loss of a chromosomal region containing the other allele, termed loss of heterozygosity. Loss of heterozygosity at 18q21 has been reported to be frequent in various types of human tumors such as pancreatic and colon cancers.2 – 7 The observations suggest the possibility that the same tumor-suppressor gene may be involved in the development of cancers of diverse origins. The DCC gene was cloned as a candidate tumor-suppressor gene in colon cancer,8 but because of its length and complexity, the available data are limited.9 / 5e13$$0042
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gasa
The DPC4 gene was recently identified as a candidate tumor-suppressor gene at 18q21, with inactivation playing a possible role in the genesis of pancreatic cancers.10 Based on its sequence similarity to the Drosophila melanogaster gene, Mad, which is known to be involved in the transforming growth factor b signaling pathway, it has been suggested that the DPC4 gene product might have an important functional role in mammalian cells.10 The vast majority of human epithelial and lymphoid malignant tumor cell lines seem to have escaped from transforming growth factor b–mediated growth control, which may represent an important step in tumor progression.11 The fact of frequent 18q21 deletions in colorectal cancers suggests that, analogous to the pancreatic cancer case, alterations of the DPC4 gene might also be involved in their pathogenesis. To examine this question, we examined 31 specimens of primary colorectal cancer taken directly from patients for their DPC4 gene status. Somatic in vivo mutations were indeed identified, providing support for an authentic role as a tumor-suppressor gene in colorectal cancer, but only in a minority of cases.
Materials and Methods Tumor Specimens Thirty-one primary colorectal cancer and corresponding normal tissue specimens were obtained from patients at the time of surgery at the Gifu University School of Medicine. Fourteen men and 17 women with an age range of 30–87 years (mean age, 63 years at diagnosis) were included. The tumors were diagnosed histologically as being colorectal cancers. In all cases, tissue samples were quickly frozen in liquid nitrogen and stored at 0807C until analysis.
Abbreviations used in this paper: PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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Reverse-Transcription Polymerase Chain Reaction Two micrograms of total RNA, isolated and purified by standard procedures, was reverse transcribed using 0.3 mg of random primers, 20 U of ribonuclease inhibitor, 10 mmol/L deoxynucleoside triphosphate, and 20 U of reverse transcriptase (Takara Biomedicals Co., Kyoto, Japan) using the manufacturer’s suggested reaction conditions. The polymerase chain reaction (PCR) was performed in 25-mL reaction mixtures containing the following: 1 mL of the complementary DNA mix, 2.5 mL of 101 PCR buffer (50 mmol/ L KCl, 10 mmol/L Tris-HCl, pH 8.3, and 1.5 mmol/L MgCl2 ), 4.0 mmol/L deoxynucleoside triphosphate, 2 mCi [a-32P]deoxycytidine triphosphate (2000 Ci/mmol; Amersham, Buckinghamshire, England), 1.25 U of Taq polymerase (Takara), and 0.5 mmol/L of each PCR primer. Primers for DPC4 were designed to amplify the gene in 5 overlapping segments (Figure 1). The primer pairs used were as follows: S1 (sense), 5*-AGCAAGCTTGCTTCAGAAATTGGAGAC and AS1 (antisense), 5*-AGCGAATTCCCTCAAAGTCATGCACAT; S2 (sense), 5*-AGCAAGCTTCACTGCAGAGTAATGCTCCATC and AS2 (antisense), 5*-AGCGAA-TTCCAGTATACTGGCAGGCTGACTT; S3 (sense), 5*-AGCAAGCTTCCCAACATTCCTGTGGCTTC and AS3 (antisense), 5*-AGCGAATTCGAGGCTGGAATGCAAGCTCA; S4 (sense), 5*-AGCAAGCTTGGACATTACTGGCCTGTTCAC and AS4 (antisense), 5*-AGCGAATTCCCAACTGCACACCTTTGCCTA; and S5 (sense), 5*-AGCAAGCTTCATTGAGAGAGCAAGGTTGCAC and AS5 (antisense), 5*-AGCGGATCCCCATCCTGATAAGGTTAAGGGC. The reactions were programmed for thermal cycling as follows (PCR thermal cycler MP; Takara Biomedicals Co.); the initial denaturing step was for 1 minute at 947C, followed by 35 cycles of 30 seconds at 947C for denaturation, 30 seconds at 557C for annealing, and 1 minute at 727C for extension. The final extension for all PCR reactions was at 727C for 10 minutes.
One-microliter aliquots of radiolabeled PCR products were diluted with 25 mL of loading buffer (95% formamide, 20 mmol/L ethylenediaminetetraacetic acid, 0.05% bromophenol blue, and 0.05% xylene cyanol), heat denatured for 10 minutes, and chilled on ice. Three microliters of the mixture was applied to 6% nondenaturing polyacrylamide gels (acrylamide, N-N*bisacrylamide; 28:2) and electrophoretically separated at 47C and at 30-W constant power for 4 hours. The resulting bands were visualized after autoradiography with Kodak XAR films for 1 day at 0807C with intensifying screens. Abnormal samples were repeatedly tested in independent PCR reactions and with separate gel loadings to ensure reproducibility.
Sequence Analysis for Mutations After digestion with HindIII-EcoRI or HindIII-BamHI, the reverse-transcription PCR products of colorectal cancer specimens showing abnormal PCR-SSCP patterns were resolved on 0.8% agarose gels and isolated using Geneclean II (Bio 101, La Jolla, CA). After cloning into either HindIII-EcoRI or HindIII-BamHI sites of pBluescript SKII(0) (Stratagene, La Jolla, CA), plasmid DNAs prepared from pooled clones were sequenced by the dideoxy chain termination method as described previously.13 Identified mutations were confirmed by separate complementary DNA/PCR amplification and subsequent sequencing. Reverse-transcription PCR products of the corresponding normal colorectal RNAs were also subjected to PCR-SSCP and sequencing analyses.
Results The present examination of 31 colorectal cancer specimens using reverse-transcription PCR and analysis of SSCP yielded 5 colorectal cancer specimens with 5 revealed to have changed electrophoretic mobilities. We could show that the distinct DPC4 gene mobility shifts were present only in colorectal cancer specimens and not
Detection of Single-Strand Conformation Polymorphism The single-strand conformation polymorphism (SSCP) analysis was performed essentially as described by Orita et al.12
Figure 1. Schematic diagram of the strategy for PCR-SSCP analysis of DPC4 complementary DNAs. DPC4 messenger RNA is also shown. The open box represents the coding region, whereas the 5* and 3* untranslated regions are shown as shaded boxes. Restriction enzymes (SacI and PstI, respectively) were used for the S1-AS1 and S5AS5 primer sets to yield higher sensitivities due to smaller sizes.
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Figure 2. PCR-SSCP analysis of the DPC4 gene in colorectal cancer specimens. Representative results of PCR-SSCP analysis using S4 and AS4 primers. PCR-SSCP analysis showing the somatic nature of the abnormality observed in cases 8, 18, and 29.
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November 1996
SOMATIC IN VIVO DPC4 ALTERATIONS 1371
Table 1. Somatic Mutations of the DPC4 Gene in Colorectal Cancers Tumor specimen Case 8 Case Case Case Case
9 16 18 29
Codon 375–378 118 350 340 330
Nucleotide substitution
Predicted effect
AGCCATTGAGAGA to A GCG to GAG GTT to GAT AAG to GAG GAA to GCA
Deletion Ala to Glu Val to Asp Lys to Glu Glu to Ala
in the corresponding normal colorectal tissues, indicating a somatic nature for all the changes (Figure 2). Furthermore, PCR-SSCP analysis showed the absence or only low expression of the wild-type allele in case 18, suggesting possible allelic loss at 18q21. Sequence analysis of both normal and colorectal cancer specimens of these 5 cases showed the presence of somatic mutations leading to changes in the predicted DPC4 gene product (Table 1). Case 8 showed a 12–base pair deletion leading to loss of 4 amino acid residues; Ala, Ile, Glu, and Arg (codons 375–378) (Figure 3). The others had missense mutations predicted to result in amino acid changes as follows: at codon 118, Ala to Glu (GCG to GAG) in case 9; at codon 350, Val to Asp (GTT to GAT) in case 16 (Figure 4A); at codon 340, Lys to Glu (AAG to GAG) in case 18 (Figure 4B); and at codon 330, Glu to Ala (GAA to GCA) in case 29. All of thease amino acid constitutions were nonconservative.
Figure 3. Sequencing analysis of the DPC4 gene in a colorectal cancer specimen. In case 8, a 12–base pair deletion was found starting at codon 374 (GAA) and ending at codon 378 (AGA) of the DPC4 gene, resulting in loss of four amino acids (Ala, Ile, Glu, and Arg) from the DPC4 gene product.
/ 5e13$$0042
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The present findings thus provide strong support for the authenticity of DPC4 as a tumor-suppressor gene involved in the oncogenesis of a proportion of colorectal cancers, although it is not yet known whether the amino acid substitutions found in the present study actually affect the function of the DPC4 protein. However, it should be noted that the frequency of alterations was relatively low at 16.7% (5 of 31 colorectal cancers) compared with the 75% of cases reported to have allelic loss at 18q21, where the DPC4 gene resides.7 Microscopic examination of H&E-stained sections from paraffin blocks of the same tumor samples confirmed that at least 70% of the tissue was occupied by tumor cells, so that the possibility of detecting mutations due to contamination with nonneoplastic cells was low. However, the difference in results could be explained if one assumes that in addition to the DPC4 gene there is another putative tumor-suppressor gene at 18q21 that has a role to play in colorectal carcinogenesis. For example, the DCC gene has been cloned as a candidate tumor-suppressor gene in colon cancer, although data on DCC mutations in the same set of tumor samples presented here are not available at present because of its length and complexity. A
Figure 4. Sequencing analysis of the DPC4 gene in colorectal cancer specimens. Normal indicates normal exon 8 sequence from normal colorectal tissue. T16 and T18 represents tumors from cases 16 and 18, respectively. Reverse-transcription PCR products of colorectal cancer specimens showing abnormal PCR-SSCP patterns were cloned into HindIII-EcoRI or HindIII-BamHI sites of pBluescript SKII(0), and the plasmid DNAs prepared from pooled clones were sequenced. (A and B ) A missense mutation at codon 350 (GTT to GAT) in case 16 and at codon 340 (AAG to GAG) in case 18, respectively.
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second possibility is that the DPC4 gene itself might be inactivated by other molecular mechanisms such as aberrant hypermethylation leading to transcriptional repression, because Nagatake et al. recently found that lung cancers, especially adenocarcinomas, frequently show tumor-specific aberrant hypermethylation at 18q21.14 In conclusion, the DPC4 gene may play a role as a tumor-suppressor gene in some subsets of colorectal cancers. The present findings, however, suggest that there might be another additional tumor-suppressor gene linked with the frequent 18q21 deletions in colorectal cancers. Further investigations of this chromosome region are clearly warranted to generate a better understanding of the molecular pathogenesis of this disease.
References 1. Knudson AG. Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Res 1985;45:1437–1443. 2. Vogelstein B, Fearon E, Kern S, Hamilton S, Preisinger A, Nakamura Y, White R. Allelotype of colorectal carcinomas. Science 1989;244:207–211. 3. Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Keeney G, Podrats KC, Jenkins RB. Human epithelial ovarian cancer allelotype. Cancer Res 1993;53:2393–2498. 4. Brewster SF, Gingell JC, Browne S, Brown KW. Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. Br J Cancer 1994;70: 697–700. 5. Shibagaki I, Shimada Y, Wagata T, Ikenaga M, Imamura M, Ishigaki K. Allelotype analysis of esophageal squamous cell carcinoma. Cancer Res 1994;54:2996–3000. 6. Hahn SA, Seumour AB, Hoque ATMS, Schutte M, da Costa LT, Redston MS, Caldas C, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern S. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995;55:4670–4675.
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7. Vogelstein B, Fearon E, Hamilton S, Kern S, Preisinger A, Leppert M, Nakamura Y, White R, Smits A, Bos J. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319: 525–532. 8. Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 1990;247:49–56. 9. Cho KR, Oliner JD, Simons JW, Hedrick L, Fearon ER, Preisinger AC, Hedge P, Silverman GA, Vogelstein B. The DCC gene: structural analysis and mutations in colorectal carcinomas. Genomics 1994;19:525–531. 10. Hahn SA, Schutte M, Hoque ATMS, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271:350–353. 11. Fynan TM, Reiss M. Resistance to inhibition of cell growth by transforming growth factor–b and its role in oncogenesis. Crit Rev Oncogenesis 1993;4:493–540. 12. Orita M, Iwahara H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as singlestrand conformation polymorphisms. Proc Natl Acad Sci USA 1989;86:2766–2770. 13. Suzuki H, Takahashi T, Kuroishi T, Suyama M, Ariyoshi Y, Takahashi T, Ueda R. p53 mutations in non-small cell lung cancer in Japan: association between mutations and smoking. Cancer Res 1992;52:734–736. 14. Nagatake M, Osada H, Kondo M, Uchida K, Nishio M, Shimokata K, Takahashi T, Takahashi T. Aberrant hypermethylation at the bcl-2 locus at 18q21 in human lung cancers. Cancer Res 1996; 56:1886–1891.
Received June 4, 1996. Accepted August 5, 1996. Address requests for reprints to: Yukihiro Takagi, M.D., Department of Surgery II, Gifu University School of Medicine, Tsukasamachi, Gifu 500, Japan. Fax: (81) 58-265-9018. The authors thank Dr. Takashi Takahashi for advice and other members of the Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, for providing assistance.
WBS-Gastro
RAPID COMMUNICATIONS Somatic Alterations of the DPC4 Gene in Human Colorectal Cancers In Vivo YUKIHIRO TAKAGI,* HISASHI KOHMURA,* MANABU FUTAMURA,* HISASHI KIDA,* HIROMI TANEMURA,* KUNIYASU SHIMOKAWA,‡ and SHIGETOYO SAJI* *Department of Surgery II and ‡Department of Laboratory Medicine, Gifu University School of Medicine, Gifu, Japan
See editorial on page 1387. Background & Aims: The chromosome region 18q21 has been shown to be frequently deleted in colorectal cancers, and such frequent allelic loss is a hallmark of the presence of a tumor-suppressor gene. The DPC4 gene, which is located at 18q21, has been identified as a tumor-suppressor gene from examination of pancreatic cancers. The aim of the present study was to determine if it might also be altered in colorectal cancers. Methods: Mutation analyses of the DPC4 gene were performed on complementary DNA samples from 31 primary colorectal cancer specimens using a combination of polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Results: Four missense mutations producing amino acid substitutions and a somatic 12–base pair deletion in the coding region of the DPC4 gene were detected in the 31 cancers (16%; 5 of 31). Conclusions: The DPC4 gene may play a role as a tumor-suppressor gene in a fraction of colorectal cancers; however, while allelic loss at 18q21 is very often seen in colorectal cancers, only a minority show DPC4 mutations, suggesting that there might be another tumor-suppressor gene in this chromosome region.
T
umor-suppressor genes are characterized by alterations in cancers that inactivate both alleles.1 This is often accomplished by intragenic mutations in one allele accompanied by loss of a chromosomal region containing the other allele, termed loss of heterozygosity. Loss of heterozygosity at 18q21 has been reported to be frequent in various types of human tumors such as pancreatic and colon cancers.2 – 7 The observations suggest the possibility that the same tumor-suppressor gene may be involved in the development of cancers of diverse origins. The DCC gene was cloned as a candidate tumor-suppressor gene in colon cancer,8 but because of its length and complexity, the available data are limited.9 / 5e13$$0042
10-11-96 21:33:41
gasa
The DPC4 gene was recently identified as a candidate tumor-suppressor gene at 18q21, with inactivation playing a possible role in the genesis of pancreatic cancers.10 Based on its sequence similarity to the Drosophila melanogaster gene, Mad, which is known to be involved in the transforming growth factor b signaling pathway, it has been suggested that the DPC4 gene product might have an important functional role in mammalian cells.10 The vast majority of human epithelial and lymphoid malignant tumor cell lines seem to have escaped from transforming growth factor b–mediated growth control, which may represent an important step in tumor progression.11 The fact of frequent 18q21 deletions in colorectal cancers suggests that, analogous to the pancreatic cancer case, alterations of the DPC4 gene might also be involved in their pathogenesis. To examine this question, we examined 31 specimens of primary colorectal cancer taken directly from patients for their DPC4 gene status. Somatic in vivo mutations were indeed identified, providing support for an authentic role as a tumor-suppressor gene in colorectal cancer, but only in a minority of cases.
Materials and Methods Tumor Specimens Thirty-one primary colorectal cancer and corresponding normal tissue specimens were obtained from patients at the time of surgery at the Gifu University School of Medicine. Fourteen men and 17 women with an age range of 30–87 years (mean age, 63 years at diagnosis) were included. The tumors were diagnosed histologically as being colorectal cancers. In all cases, tissue samples were quickly frozen in liquid nitrogen and stored at 0807C until analysis.
Abbreviations used in this paper: PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
WBS-Gastro
1370 TAKAGI ET AL.
GASTROENTEROLOGY Vol. 111, No. 5
Reverse-Transcription Polymerase Chain Reaction Two micrograms of total RNA, isolated and purified by standard procedures, was reverse transcribed using 0.3 mg of random primers, 20 U of ribonuclease inhibitor, 10 mmol/L deoxynucleoside triphosphate, and 20 U of reverse transcriptase (Takara Biomedicals Co., Kyoto, Japan) using the manufacturer’s suggested reaction conditions. The polymerase chain reaction (PCR) was performed in 25-mL reaction mixtures containing the following: 1 mL of the complementary DNA mix, 2.5 mL of 101 PCR buffer (50 mmol/ L KCl, 10 mmol/L Tris-HCl, pH 8.3, and 1.5 mmol/L MgCl2 ), 4.0 mmol/L deoxynucleoside triphosphate, 2 mCi [a-32P]deoxycytidine triphosphate (2000 Ci/mmol; Amersham, Buckinghamshire, England), 1.25 U of Taq polymerase (Takara), and 0.5 mmol/L of each PCR primer. Primers for DPC4 were designed to amplify the gene in 5 overlapping segments (Figure 1). The primer pairs used were as follows: S1 (sense), 5*-AGCAAGCTTGCTTCAGAAATTGGAGAC and AS1 (antisense), 5*-AGCGAATTCCCTCAAAGTCATGCACAT; S2 (sense), 5*-AGCAAGCTTCACTGCAGAGTAATGCTCCATC and AS2 (antisense), 5*-AGCGAA-TTCCAGTATACTGGCAGGCTGACTT; S3 (sense), 5*-AGCAAGCTTCCCAACATTCCTGTGGCTTC and AS3 (antisense), 5*-AGCGAATTCGAGGCTGGAATGCAAGCTCA; S4 (sense), 5*-AGCAAGCTTGGACATTACTGGCCTGTTCAC and AS4 (antisense), 5*-AGCGAATTCCCAACTGCACACCTTTGCCTA; and S5 (sense), 5*-AGCAAGCTTCATTGAGAGAGCAAGGTTGCAC and AS5 (antisense), 5*-AGCGGATCCCCATCCTGATAAGGTTAAGGGC. The reactions were programmed for thermal cycling as follows (PCR thermal cycler MP; Takara Biomedicals Co.); the initial denaturing step was for 1 minute at 947C, followed by 35 cycles of 30 seconds at 947C for denaturation, 30 seconds at 557C for annealing, and 1 minute at 727C for extension. The final extension for all PCR reactions was at 727C for 10 minutes.
One-microliter aliquots of radiolabeled PCR products were diluted with 25 mL of loading buffer (95% formamide, 20 mmol/L ethylenediaminetetraacetic acid, 0.05% bromophenol blue, and 0.05% xylene cyanol), heat denatured for 10 minutes, and chilled on ice. Three microliters of the mixture was applied to 6% nondenaturing polyacrylamide gels (acrylamide, N-N*bisacrylamide; 28:2) and electrophoretically separated at 47C and at 30-W constant power for 4 hours. The resulting bands were visualized after autoradiography with Kodak XAR films for 1 day at 0807C with intensifying screens. Abnormal samples were repeatedly tested in independent PCR reactions and with separate gel loadings to ensure reproducibility.
Sequence Analysis for Mutations After digestion with HindIII-EcoRI or HindIII-BamHI, the reverse-transcription PCR products of colorectal cancer specimens showing abnormal PCR-SSCP patterns were resolved on 0.8% agarose gels and isolated using Geneclean II (Bio 101, La Jolla, CA). After cloning into either HindIII-EcoRI or HindIII-BamHI sites of pBluescript SKII(0) (Stratagene, La Jolla, CA), plasmid DNAs prepared from pooled clones were sequenced by the dideoxy chain termination method as described previously.13 Identified mutations were confirmed by separate complementary DNA/PCR amplification and subsequent sequencing. Reverse-transcription PCR products of the corresponding normal colorectal RNAs were also subjected to PCR-SSCP and sequencing analyses.
Results The present examination of 31 colorectal cancer specimens using reverse-transcription PCR and analysis of SSCP yielded 5 colorectal cancer specimens with 5 revealed to have changed electrophoretic mobilities. We could show that the distinct DPC4 gene mobility shifts were present only in colorectal cancer specimens and not
Detection of Single-Strand Conformation Polymorphism The single-strand conformation polymorphism (SSCP) analysis was performed essentially as described by Orita et al.12
Figure 1. Schematic diagram of the strategy for PCR-SSCP analysis of DPC4 complementary DNAs. DPC4 messenger RNA is also shown. The open box represents the coding region, whereas the 5* and 3* untranslated regions are shown as shaded boxes. Restriction enzymes (SacI and PstI, respectively) were used for the S1-AS1 and S5AS5 primer sets to yield higher sensitivities due to smaller sizes.
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Figure 2. PCR-SSCP analysis of the DPC4 gene in colorectal cancer specimens. Representative results of PCR-SSCP analysis using S4 and AS4 primers. PCR-SSCP analysis showing the somatic nature of the abnormality observed in cases 8, 18, and 29.
WBS-Gastro
November 1996
SOMATIC IN VIVO DPC4 ALTERATIONS 1371
Table 1. Somatic Mutations of the DPC4 Gene in Colorectal Cancers Tumor specimen Case 8 Case Case Case Case
9 16 18 29
Codon 375–378 118 350 340 330
Nucleotide substitution
Predicted effect
AGCCATTGAGAGA to A GCG to GAG GTT to GAT AAG to GAG GAA to GCA
Deletion Ala to Glu Val to Asp Lys to Glu Glu to Ala
in the corresponding normal colorectal tissues, indicating a somatic nature for all the changes (Figure 2). Furthermore, PCR-SSCP analysis showed the absence or only low expression of the wild-type allele in case 18, suggesting possible allelic loss at 18q21. Sequence analysis of both normal and colorectal cancer specimens of these 5 cases showed the presence of somatic mutations leading to changes in the predicted DPC4 gene product (Table 1). Case 8 showed a 12–base pair deletion leading to loss of 4 amino acid residues; Ala, Ile, Glu, and Arg (codons 375–378) (Figure 3). The others had missense mutations predicted to result in amino acid changes as follows: at codon 118, Ala to Glu (GCG to GAG) in case 9; at codon 350, Val to Asp (GTT to GAT) in case 16 (Figure 4A); at codon 340, Lys to Glu (AAG to GAG) in case 18 (Figure 4B); and at codon 330, Glu to Ala (GAA to GCA) in case 29. All of thease amino acid constitutions were nonconservative.
Figure 3. Sequencing analysis of the DPC4 gene in a colorectal cancer specimen. In case 8, a 12–base pair deletion was found starting at codon 374 (GAA) and ending at codon 378 (AGA) of the DPC4 gene, resulting in loss of four amino acids (Ala, Ile, Glu, and Arg) from the DPC4 gene product.
/ 5e13$$0042
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The present findings thus provide strong support for the authenticity of DPC4 as a tumor-suppressor gene involved in the oncogenesis of a proportion of colorectal cancers, although it is not yet known whether the amino acid substitutions found in the present study actually affect the function of the DPC4 protein. However, it should be noted that the frequency of alterations was relatively low at 16.7% (5 of 31 colorectal cancers) compared with the 75% of cases reported to have allelic loss at 18q21, where the DPC4 gene resides.7 Microscopic examination of H&E-stained sections from paraffin blocks of the same tumor samples confirmed that at least 70% of the tissue was occupied by tumor cells, so that the possibility of detecting mutations due to contamination with nonneoplastic cells was low. However, the difference in results could be explained if one assumes that in addition to the DPC4 gene there is another putative tumor-suppressor gene at 18q21 that has a role to play in colorectal carcinogenesis. For example, the DCC gene has been cloned as a candidate tumor-suppressor gene in colon cancer, although data on DCC mutations in the same set of tumor samples presented here are not available at present because of its length and complexity. A
Figure 4. Sequencing analysis of the DPC4 gene in colorectal cancer specimens. Normal indicates normal exon 8 sequence from normal colorectal tissue. T16 and T18 represents tumors from cases 16 and 18, respectively. Reverse-transcription PCR products of colorectal cancer specimens showing abnormal PCR-SSCP patterns were cloned into HindIII-EcoRI or HindIII-BamHI sites of pBluescript SKII(0), and the plasmid DNAs prepared from pooled clones were sequenced. (A and B ) A missense mutation at codon 350 (GTT to GAT) in case 16 and at codon 340 (AAG to GAG) in case 18, respectively.
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second possibility is that the DPC4 gene itself might be inactivated by other molecular mechanisms such as aberrant hypermethylation leading to transcriptional repression, because Nagatake et al. recently found that lung cancers, especially adenocarcinomas, frequently show tumor-specific aberrant hypermethylation at 18q21.14 In conclusion, the DPC4 gene may play a role as a tumor-suppressor gene in some subsets of colorectal cancers. The present findings, however, suggest that there might be another additional tumor-suppressor gene linked with the frequent 18q21 deletions in colorectal cancers. Further investigations of this chromosome region are clearly warranted to generate a better understanding of the molecular pathogenesis of this disease.
References 1. Knudson AG. Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Res 1985;45:1437–1443. 2. Vogelstein B, Fearon E, Kern S, Hamilton S, Preisinger A, Nakamura Y, White R. Allelotype of colorectal carcinomas. Science 1989;244:207–211. 3. Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Keeney G, Podrats KC, Jenkins RB. Human epithelial ovarian cancer allelotype. Cancer Res 1993;53:2393–2498. 4. Brewster SF, Gingell JC, Browne S, Brown KW. Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. Br J Cancer 1994;70: 697–700. 5. Shibagaki I, Shimada Y, Wagata T, Ikenaga M, Imamura M, Ishigaki K. Allelotype analysis of esophageal squamous cell carcinoma. Cancer Res 1994;54:2996–3000. 6. Hahn SA, Seumour AB, Hoque ATMS, Schutte M, da Costa LT, Redston MS, Caldas C, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern S. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995;55:4670–4675.
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Received June 4, 1996. Accepted August 5, 1996. Address requests for reprints to: Yukihiro Takagi, M.D., Department of Surgery II, Gifu University School of Medicine, Tsukasamachi, Gifu 500, Japan. Fax: (81) 58-265-9018. The authors thank Dr. Takashi Takahashi for advice and other members of the Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, for providing assistance.
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