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Japanese Journal of Clinical Oncology Pages 383-387

Point Mutations of Ornithine Decarboxylase Gene are an Infrequent Event in Colorectal Cancer but a Missense Mutation was Found in a Replication Error Positive Patient with hMSH2 Germline Mutation
Introduction
Materials and Methods
   Subjects
   DNA Extraction, RNA Preparation and cDNA Synthesis
   PCR-SSCP Analysis and Direct Sequencing
   Qualitative Determination of Amounts of ODC mRNA by RT-PCR
Results
Discussion
Acknowledgments
References
Point Mutations of Ornithine Decarboxylase Gene are an Infrequent Event in Colorectal Cancer but a Missense Mutation was Found in a Replication Error Positive Patient with hMSH2 Germline Mutation

Point Mutations of Ornithine Decarboxylase Gene are an Infrequent Event in Colorectal Cancer but a Missense Mutation was Found in a Replication Error Positive Patient with hMSH2 Germline Mutation

Masato Maekawa1, Kokichi Sugano1,2, Hidefumi Kashiwabara1, Mineko Ushiama2, Shin Fujita3, Hisanao Ohkura2, Tadao Kakizoe4

Divisions of 1Clinical Laboratory, 2Medical Oncology, 3Surgery and 4Urology, National Cancer Center Hospital, Tokyo, Japan

Background: Ornithine decarboxylase (ODC; EC 4.1.1.17) is the first rate-limiting enzyme in the biosynthesis of polyamines. ODC protein has a characteristic amino acid sequence, the PEST sequence, which is related to the enzyme's rapid degradation. ODC cDNA prepared from human hepatoma tissues has been reported to show nonsense or missense mutations.
Methods: We examined somatic mutations of ODC cDNA by RT-PCR-SSCP analysis and mRNA expressions by RT-PCR in 50 colorectal cancer tissues to investigate the involvement of ODC gene alterations in colorectal cancers.
Results: Increased expression of the ODC gene was observed in 36 cases (86%) out of the 42 examined by RT-PCR. In one case, a missense mutation was found in the cancer tissue but not in normal mucosa. The missense mutation from Asp to Asn at codon 424, in the PEST region, possibly stabilizes the ODC protein. In colorectal cancer, replication error and a germline mutation in hMSH2 gene were observed.
Conclusions: The missense mutation at codon 424 is speculated to be a cause of stabilization and a passenger mutation owing to the mutator phenotype. Since only one of 50 colorectal cancers exhibited a missense mutation of the ODC gene, mutations in ODC gene are not frequent in colorectal cancer. The increased expression of the ODC gene was noted in 86% of colorectal cancer tissues by RT-PCR, however, it was not due to point mutations in ODC coding exons.

Key words: ornithine decarboxylase - mutation analysis - colorectal cancer - missense mutation

INTRODUCTION

Ornithine decarboxylase (ODC; EC 4.1.1.17) activity has been recognized to be a biochemical marker of cellular proliferation. ODC is a cytoplasmic enzyme, which is the first rate-limiting enzyme in the biosynthesis of polyamines, catalyzing the conversion of the polyamine ornithine to putrescine. The polyamines have essential regulatory functions in protein and nucleic acid synthesis. The elevation of polyamine biosynthesis is characteristically associated with the onset of rapid growth and cell transformation (1-3). Animal studies have shown that ODC activity increases in the early stages of tumor proliferation and in the promotion step of carcinogenesis (3). In addition, several reports have described that ODC activity is high in human neoplastic lesions, i.e. those of breast (4), liver (5), stomach (6) and colon (7).

Human ODC cDNA was isolated from a human liver cDNA library and the nucleotide sequence coding for the entire enzyme was determined. The 1825 nucleotide cDNA contained an open reading frame of 1383 nucleotides, 87 nucleotides 5[prime] from the first methionine codon, 346 nucleotides in the 3[prime]-noncoding region and a poly(A) tail of nine bases (8). Human ODC gene is divided into 12 exons and spans 8 kb (9-11). It mapped to chromosome 2pter-p23 (11,12).

Recently, certain proteins degraded rapidly in cells have been found to have a characteristic amino acid sequence, the PEST sequence (13), and ODC has been demonstrated to be one of such proteins (13,14). Tamori et al. (15) examined the nucleotide sequence of ODC cDNA prepared from human hepatoma tissues of three Japanese patients and found three point mutations, two missense and one nonsense. In the present study we searched for somatic mutations of ODC cDNA in colorectal cancer tissues using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis and semi-quantified the ODC mRNA by reverse transcriptase (RT)-PCR.

MATERIALS AND METHODS

Subjects

Colorectal tissues, cancerous tissues and normal accompanying mucosa were obtained from the National Cancer Center Hospital at surgery. The tissues were stored in liquid nitrogen or at -80°C until use.

DNA Extraction, RNA Preparation and cDNA Synthesis

DNA was extracted from tissue specimens by a previously described method (16). Total RNA was prepared from various cancer cell lines and colorectal tissues using a standard guanidium thiocyanate-phenol-chloroform-isoamyl alcohol extraction method (17). Reverse transcriptase reactions (20 ml) were carried out with 0.5-1.0 mg of total RNA as template with random hexamer as primers, using SuperScript RNase free reverse transcriptase (GIBCO, Gaithersburg, MD).

PCR-SSCP Analysis and Direct Sequencing

The cDNAs were amplified separately by PCR as seven fragments. The reaction mixture (25 ml) contained 10 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 1 g/l gelatin and 0.2 mmol/l each of four dNTPs, oligonucleotide primers (6 pmol each), 0.6 units of AmpliTaq DNA polymerase (Perkin Elmer-Cetus, Branchburg, NJ) and 0.5 ml of the above-mentioned cDNA. The primer sequences are shown in Table 1. After the first denaturation step at 94°C for 3 min, 40 cycles of reaction were performed at 94°C for 30 s, at 53-63°C for 30 s and at 72°C for 1 min, with a final extension at 72°C for 7 min. The yields of amplified DNA fragments were determined by electrophoresis on 8% polyacrylamide gels and visualized by ethidium bromide staining.

SSCP analysis of each amplified product was carried out using the Phast System (Pharmacia, Uppsala, Sweden) (18,19). The PCR products displaying conformation polymorphisms were treated with shrimp alkaline phosphatase and exonuclease I to remove excess PCR primers and nucleotides (Reagent Pack for use with Sequenase PCR Product Sequencing Kit; Amersham, Arlington Heights, IL) and then directly sequenced by the dideoxy sequencing procedure using a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham) or a Dye Terminator Cycle Sequencing FS Core kit and ABI PRISM 310 Genetic Analyzer (ABI, Foster City, CA), following the manufacturer's recommendations.

Qualitative Determination of Amounts of ODC mRNA by RT-PCR

Region 2 of the ODC cDNA was amplified with [beta]2 microglobulin ([beta]2M) as an internal control for RT-PCR (20) in a single reaction tube. The PCR conditions were the same as described above, using 58°C as the annealing temperature for 24, 27 and 30 cycles. Reaction products for ODC and [beta]2M were electrophoresed on 8% polyacrylamide gels and visualized by ethidium bromide staining. The stained gels were photographed with a Polaroid camera and then the films were fractionated by NIH image software. The intensity of ODC cDNA was compared with that of [beta]2M and classified into three groups at a point of mRNA overexpression (-, +, ++).

Table 1. Primer pairs for amplification of ODC cDNA
Region 1 (-80 to 176*, 257 bp) Forward 5[prime] CTG GAG AGT TGC CTT TGT GA 3[prime]
Reverse 5[prime] AGA GCT TTT AAC CAC CTC AG 3[prime]
Region 2 (127-386*, 260 bp) Forward 5[prime] GCA GAC CTG GGA GAC ATT CT 3[prime]
Reverse 5[prime] TGG ACT CCA TTA TTA GCA GC 3[prime]
Region 3 (313-542*, 230 bp) Forward 5[prime] CCA GAG AGG ATT ATC TAT GC 3[prime]
Reverse 5[prime] AAA AGG AGC CTG CTG GTT CT 3[prime]
Region 4 (496-763*, 268 bp) Forward 5[prime] CTC AGT GTG AAA TTC GGT GC 3[prime]
Reverse 5[prime] TTA CGC CGG TGA TCT CTT CA 3[prime]
Region 5 (714-1001*, 288 bp) Forward 5[prime] TCC TGG ATC TGA GGA TGT GA 3[prime]
Reverse 5[prime] GCG TGG TCA TAG AGT ATG CA 3[prime]
Region 6 (919-1207*, 289 bp) Forward 5[prime] GAT GAG TCG AGT GAG CAG AC 3[prime]
Reverse 5[prime] GCC TCT GGA AGC CAT TGA AC 3[prime]
Region 7 (1160-1416*, 257 bp) Forward 5[prime] GCG CTT ACA CTG TTG CTG CT 3[prime]
Reverse 5[prime] ACT TGC AGT TAA CAG CTA CC 3[prime]
*The nucleotide number with the first triplet of the first codon set to 1.

Table 2. Frequencies of the polymorphic alleles
Polymorphic site Allele Allelotype Allele frequency
Position* Exon Codon A B AA AB BB Allele A Allele B
4099 5 108 GTG GTA 49 1 0 0.99 0.01
7508 12 423 CCC CCT 12 16 16 0.45 0.55
3173 2 5[prime]-UT A G 13 3 2 0.85 0.15
*The position number was designated based on a previous report on human ODC gene (10). [dagger]5[prime]-Untranslated region.

Table 3. Relationship between ODC mRNA expression and cancer properties
Increased ODC expression Histology Location Dukes clinical stage
Well Moderate Poor A + C + T S + R A B C D
  2 4 0 0 6 2 1 2 1
+ 10 6 1 8 9 1 6 6 4
++ 9 9 1 5 14 1 4 10 4
A, ascending colon; C, cecum; T, transverse colon; S, sigmoid colon; R, rectum.

RESULTS

We investigated somatic mutations in ODC cDNA in 50 colorectal cancers using RT-PCR-SSCP analysis. However, only four point mutations were detected: one missense (codon 424), two silent (codons 108 and 423) and one in the 5[prime]-non-coding exon 2. A missense mutation of GAC to AAC at codon 424 was observed in one of 50 colorectal cancers. The missense mutation was detected as an extra band close to the double-stranded DNA (dsDNA) by PAGE of the amplified product following ethidium bromide staining and SSCP (Fig. 1A and B). The extra band was confirmed to be a heteroduplex because both the dsDNA and the extra band could be amplified from the DNA extracted from the band. This missense mutation resulted in the replacement of an aspartic acid by an asparagine (D424N) (Fig. 1). The mutation was identified in DNA prepared from the cancer tissue but was not observed in normal accompanying tissues. Hence the alteration was neither a polymorphism nor germline mutation. The missense mutation at codon 424 neither created nor suppressed any known restriction site in the ODC gene. We then designed mismatched primers and used mismatched PCR-restriction fragment length polymorphism (RFLP) (by use of EcoRI) to confirm the missense mutation. We also identified the remaining three point mutations by PCR-RFLP of 50 samples (details not shown) and calculated the allele frequency of the wild and mutant types (Table 2).

Figure 1. Missense mutation at codon 424. (A) PCR products for region 7 were electrophoresed on 8% polyacrylamide gel, then stained with ethidium bromide. An extra band was observed in lane 4, suggesting the presence of mutations. (B) RT-PCR-SSCP analysis. Three amplified products containing region 7 are shown: lanes 1 and 4, lanes 2 and 5, lanes 3 and 6 are the same sample. In lanes 4 and 6, ssDNA was separated into three bands, possibly owing to the presence of a genetic polymorphism at codon 423 (CCC and CCT) on 12.5% homogeneous gel. In lane 3, dsDNA was separated into two bands, possibly owing to the presence of a heteroduplex which originated from the heterozygous mutation for GAC and AAC at codon 424 on 8-25% gradient gel. (C) Direct sequencing analysis. The PCR products amplified from cDNA were analyzed. In cancer tissues, C and T at the last triplet of codon 423 and G and A at the first triplet of codon 424 were observed, whereas only C and T at the last triplet of codon 423 were observed in normal accompanying mucosa. Sequencing of DNA confirmed the results described above.

By RT-PCR the amplified ODC cDNA appeared as a single band of constant size. Increased expression of the ODC gene relative to [beta]2M was noted in 36 cases (17 moderately increased and 19 highly increased; 86%) out of the 42 examined by the RT-PCR method. Results representative of RT-PCR are shown in Fig. 2. Table 3 shows the relationship of ODC expression to histology, cancer location and Dukes classification. There were no significant differences between them by chi-squared test with Yates correction. The patient with the missense mutation at codon 424 described above was diagnosed as well differentiated ascending colon cancer (stage C) and his cancer tissues showed moderately increased expression of ODC gene.


Figure 2. Typical results for mRNA expression of ODC and [beta]2 microglobulin. PCR was performed for 24, 27 and 30 cycles. Pairs of normal mucosa and cancer tissues were analyzed. The upper bands correspond to ornithine decarboxylase (ODC) and the lower bands to [beta]2 microblobulin ([beta]2M). Nos 1 and 2 were recognized as an increase of mRNA expression, ++ and +, respectively, whereas No. 3 was recognized as no increase.

DISCUSSION

The ODC activity seems to be tightly regulated at multiple levels, e.g. the rate of transcription, stability of mRNA, translational efficiency of mRNA and stability and posttranslational modifications of the enzyme protein (21,22). ODC has two PEST sequences spanning amino acid residues 292-333 and 423-449. The latter sequence was shown to be necessary for rapid turnover (23). Ghoda et al. (14) and Li et al. (24) have reported that truncation of ODC at 425 stabilized the ODC protein with full enzymatic activity. Tamori et al. (15) investigated the nucleotide sequence of ODC cDNA from human hepatoma tissues and found missense or nonsense mutations. They believe that the formation of truncated or abnormal ODC proteins due to point mutation is one reason why the ODC activity is high in human hepatoma tissue. The point mutations they found are located in the latter PEST region. Here we found one missense mutation in the latter PEST region. The missense mutation at 424 possibly stabilizes the ODC protein. Because ODC activity reflects the rate of tumor proliferation, the stabilized ODC proteins might cause tumor progression.

The amplified region 7 of one cancer specimen was found to have an abnormal electrophoretic mobility of dsDNA by electrophoresis on non-denaturing polyacrylamide gel. This was possibly due to a heteroduplex formation by a polymorphic site at codon 423 together with a missense mutation at codon 424. Nagamine et al. (25) speculated that deletions or insertions result in heteroduplexes and can be identified by analyzing the amplified products by native PAGE. We reported that heteroduplex bands were sometimes observed in heterozygotes in cases of not only deletion/insertion but also one base substitutions (18). Therefore, DNA conformation polymorphism analysis using a gradient gel can sometimes detect both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) abnormalities.

The G to A transition at codon 424 occurred in CpG dinucleotides (CG or TG originated from genetic polymorphism at codon 423). The point mutations in the CpG dinucleotides have been observed in other genes and are believed to be hotspots for mutations and to be related to methylation-mediated deamination (26). On the other hand, in eight cases out of 50, the replication error (RER) was observed at two or more microsatellites (Kashiwabara et al., in preparation). Cancers of the microsatellite mutator phenotype show exaggerated genomic instability causing the frequent accumulation of deletion and insertion mutations at simple repeat sequences (27). These mostly result from the abnormality in mismatch repair genes, i.e. hMSH2, hMLH1, etc. (28,29). Mutational analysis of hMSH2 and hMLH1 in the eight cases revealed that only one had a germline mutation, a 33 bp deletion in exon 7 of the hMSH2 gene. RT-PCR-SSCP analysis showed that normal allele was deleted by some means (Kashiwabara et al., in preparation). It was assumed that the rate of point mutations was not necessarily higher in cancer cells than in normal cells, but cancer cells go through so many divisions during their evolution that passenger mutations are likely (30). Here the passenger mutations mean the mutations which occurred randomly in any genes. The mutation in the hMSH2 gene gives mutator phenotype and possibly leads to the accumulation of genetic abnormalities in other genes including other mutator-related genes, resulting in the higher frequency of passenger mutations. In the one case with hMSH2 germline mutation, a missense mutation at codon 424 of the ODC gene was discovered. The missense mutation in the present case, therefore, might be a passenger in a mutator phenotype rather than a spontaneous mutation.

We have found three genetic polymorphisms. The polymorphism at codon 423 is novel and common among the Japanese and so will be a useful genetic marker there.

With regard to the apparent increase in expression of the ODC gene in 36 out of the 42 colorectal cancers observed by RT-PCR, the cycle number was minimized in the present study. Radford et al. (31) also reported mRNA levels were increased in colorectal tumor samples. We found only one missense mutation of ODC cDNA in 50 colorectal tissues. The increased expression of the ODC gene despite a lack of mutations in the PEST region should be investigated.

Acknowledgments

This work was supported in part by Grants-in-Aid for Cancer Research and for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, and Grants from Mitsui Life Social Welfare Foundation and Kurozumi Medical Foundation.

References

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Received December 16, 1997; accepted March 23, 1998
For reprints and all correspondence: Masato Maekawa, Division of Clinical Laboratory, National Cancer Center Hospital, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: mmaekawa{at}gan2.ncc.go.gp
Abbreviations: ODC, orinithine decarboxylase; [beta]2M, [beta]2 microglobulin; RFLP, restriction fragment length polymorphism; RER, replication error; hMSH2, human MutS homolog 2; hMLH1, human MutL homolog 1; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; RT, reverse transcriptase; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism

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