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From the Division of Cancer-Related Genes,*
Institute for Genetic
Medicine, the Second Department of Surgery,
and the First Department of Surgery,
Hokkaido
University School of Medicine, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The APC gene at chromosome 5q21 is transcribed into nearly 9.5 kb-sized mRNA which encodes a huge protein of 2,843 amino acids.2,8 The coding region consists of 15 exons, the last containing more than three quarters of the whole coding region. In sporadic colorectal cancers, >60% of the somatic mutations are clustered within a small region of exon 15 designated as the mutation cluster region (MCR), which accounts for <10% of the coding region. However, most familial adenomatous polyposis (FAP) patients carry truncation mutations in the N-terminal half.9 Hence, the widespread distribution of many different mutations within the very large APC gene poses problems in molecular genetic diagnosis, and detection of the APC mutations is labor-intensive when nucleic acid-based approaches are used. An alternative method is the protein truncation test which detects the protein truncating mutations in vitro10,11 or in yeast,12 because the vast majority (>95%) of the APC mutations have truncation of the APC protein because of frameshift, nonsense, or splice-site mutations.13
We present here a new, rapid, and sensitive yeast-based screening method for detection of the truncating mutation in the APC. The assay has advantages over the similar one which uses URA3 selection,14 because it does not require a replica-plating step and can easily distinguish yeast containing mutant APC as red colonies. Moreover, mutant APC plasmid can be easily recovered from red colonies and subjected to DNA sequence analysis. The assay therefore enables us to easily test a large number of samples and many clones per sample. We applied this assay to analyze the APC mutations in clinical samples of colorectal, breast, and lung cancers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six human colon cancer cell lines (DLD1, Colo201, Colo320DM, HT29, HCT116, and SW480) were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer at Tohoku University. All cell lines were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium supplemented with 10% fetal calf serum and 0.03% L-glutamine. Resected specimens of normal colonic mucosa, two polyps, 23 colorectal cancers, 70 breast cancers, and 29 non-small-cell lung carcinomas were obtained from the First Department of Surgery, Hokkaido University Hospital, Japan. They were snap-frozen in liquid nitrogen and stored at -80°C until analyzed. No procedure to enrich the samples for tumor cells was carried out in this study. The clinical stage classifications of the colorectal and breast cancers were based on the TNM classification of the International Union Against Cancer. Breast cancers were histologically typed according to the "General Rules for Clinical and Pathological Recording of Breast Cancer" issued by the Japanese Cancer Society.
RNA and DNA Preparation
Total RNA was extracted from cell lines and tumor tissues. Approximately 100 mg of tumor samples or 1 x 105 cells were homogenized in 2 ml of Trizol reagent (Life Technologies, Inc., Tokyo, Japan), mixed with 0.4 ml of chloroform, and centrifuged. The supernatant was collected and total RNA was precipitated with isopropyl alcohol. The precipitate was washed with 75% ethanol and dissolved with 40 µl of water containing 0.1% diethyl pyrocarbonate. RNA quality was verified by electrophoresis in a 1.0% agarose gel. cDNA was synthesized at 37°C for 1 hour with 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) from 3 µg of total RNA by using a random pdN6 primer (Life Technologies, Inc.). Genomic DNA was extracted from cells and tissues or from the residue in RNA extraction.
Polymerase Chain Reactions (PCRs)
Regions I and II (see Figure 3
) of the APC coding
sequence were amplified from mRNA-derived cDNA whereas regions III to V
were amplified from genomic DNA. All PCR fragments was obtained by
using proof-reading Pfu DNA polymerase (Stratagene, La
Jolla, CA), which can minimize base misincorporation. Primers for
amplification of the five regions were 5'-ACGGCCGCAAAAAA
TGGCTGCAGCTTCATATGAT-3' and 5'-TCGGCCGCCCCCCTCCCAA
TAATTCTGCAATGGCCTGTAG-3' for region I; 5'-TCGGCCGCAAAA
AATGCGCGCTTACTGTGAAACCTG-3' and
5'-TCGGCCGCCCACCTCCCCTTCCAGAGTTCAACTGCTCATC-3' for region II;
5'-AAAAGCTTAAATGAAACCCTCGATTGAATCC-3' and
5'-CGGCCGCTGCTCTGATTCTGTTTCATTCCC-3' for region III;
5'-TCGGCCGCAAAAAATGGCCACGGAAAGTACTCCAGATG-3' and
5'-TCGGCCGTTCCAATGTACTTTTCTCCCCTGG-3' for region IV;
5'-TCGGCCGAAAATGGATTGGAAAGCTATTCAGGAAG-3' and
5'-ACGGCCGCCCAACAGATGTCACAAGGTAAGACC-3' for region V. The PCR
conditions are available from the authors on request.
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The ADE2 expression vector pLF-ADE2 was constructed as follows. A fragment containing a Saccharomyces cerevisiae ADE2 coding sequence from the initiation codon to the natural termination codon was amplified by PCR using primers that contained a unique NotI site at the 5' terminus. The primers were 5'-AGCGGCCGCGTATGGATTCTAGAACAGTTGG-3' and 5'- CTCGGCCGTTAC-TTGTTTTCTAGATAAGCTTC-3'. The plasmid pLS7615 was digested with HindIII and EagI, blunt-ended by T4 DNA polymerase, and ligated with the ADE2 fragment. In this process, a unique NotI site was generated on the 5' end of the fragment, thereby facilitating the subsequent insertion of five fragments of the APC cDNA. Five overlapping fragments spanning codons 1 to 497, codons 413 to 1040, codons 979 to 1541, codons 1492 to 2177, and codons 2100 to 2843 of the APC cDNA were amplified and inserted in-frame into the NotI site of the pLF-ADE2 to produce pLF-ADE2-I, -II, -III, -IV, and -V, respectively. Some gap vectors were made by replacing the central portions of the inserted fragments with unique restriction sites: BglII (pLF-I-ADE2g), BglII and NdeI (pLF-II-ADE2g), NdeI and PstI (pLF-III-ADE2g), PstI and BclI (pLF-IV-ADE2g), and BclI (pLF-V-ADE2g). The pLF-ADE2-III was digested with PstI to delete codons 1337 to 1475 of APC cDNA and self-ligated to create an out-of-frame deletion mutant (pLF-ADE2-IIImt). The other mutant clone (pLF-ADE2-IVmt) was prepared by inserting a single adenine at codon 2050 using PCR mutagenesis. These mutant clones were used for the assessment of the yeast color assay system.
APC Yeast Color Assay
The yeast strain yIG397 (genotype; MATaade2-1 lue2-3, 112trp1-1
his3-11, 15 can1-100) was used throughout
this study. When the strain was cultured on minimal medium plus adenine
(5 µg/ml), the cell formed red colonies because of the accumulation
of an intermediate in adenine metabolism.16
The yeast was
cultured in 100 ml of yeast extract/peptone/dextrose medium
supplement with 200 µg/ml of adenine, until
OD600 reached 0.8. The cells were collected,
washed, and resuspended in an equal volume of LiOAc solution (10 mmol/L
Tris-HCl, pH 8.0, 1 mmol/L EDTA, 0.1 mol/L lithium acetate). For each
transformation, 50 µl of yeast suspension was mixed with 1 to 5 µl
of unpurified APC cDNA PCR products, 25 to 150 ng of each
linearized plasmid, 50 µg of sonicated single-stranded salmon sperm
DNA, and 300 µl of LiOAc solution containing 40% polyethylene glycol
4000 (Kanto Chemical, Tokyo, Japan). The mixture was incubated at
30°C for 30 minutes and heat-shocked at 42°C for 15 minutes. Yeast
transformants were pelleted and plated on a synthetic minimal medium
minus leucine plus adenine (5 µg/ml) and incubated for 48 hours in a
30°C humidified atmosphere. These are summarized in Figure 1
. More than 200 colonies were examined
in this assay. When the percentage of red colonies was 10 to 20%, the
assay was repeated and the mean value was presented.
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The HCT116 (wild-type APC) and DLD1 cells (mutant APC) were mixed at serial ratios, 0, 5, 10, 15, 20, 25, 30, 50, 75, and 100% (DLD1 cells/total cells). They were equally divided into two and cultured for 6 hours, and then total RNA and genomic DNA were extracted. Region III was chosen as a representative of the five regions, and RNA and genomic DNA were used as templates for reverse transcriptase-PCR and PCR amplification, respectively. The PCR products were subjected to the yeast color assay repeatedly and the mean value was presented.
Recovery of Plasmid from Yeast and DNA Sequencing
Yeast was digested with zymolyase 100T (Seikagaku-Kogyo, Tokyo, Japan), and plasmids were recovered by an alkaline lysis method (QIAprep plasmid kit, Qiagen, Hilden, Germany) and transfected into an Escherichia coli XL1Blue strain by electroporation. Recovered plasmids were subjected to sequence analysis on an ABI 377A automated sequencer (Applied Biosynthesis, Urayasu, Japan) using Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Life Science, Tokyo, Japan) and appropriate APC-specific oligonucleotide primers. To identify clonal mutations in mutation-positive cases, plasmids rescued from three to 10 colonies were sequenced and the presence of identical mutation was confirmed.
Statistical Analysis
The relationship between ratios of cells (DLD1) with APC mutation and the percentage of red colonies was studied by a regression analysis. Correlations of the clinical parameters of the patients and their APC status were analyzed by a Yates-corrected chi-square test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We divided the wild-type APC coding sequence of 8.5 kb
into five overlapping parts, and each corresponding PCR-amplified
fragment was ligated in-frame with an ADE2 open reading
frame. The resulting constructs, pLF-ADE2-I, -II, -III, -IV, and -V,
preserved the ADE2+ phenotype (white yeast
colony) after the introduction into the yeast (Figure 1)
. On the other
hand, when plasmids containing frameshift mutations, pLF-ADE2-IIImt and
pLF-ADE2-IVmt, were transfected into yeast, the yeast showed an
ADE2- phenotype (red yeast colony) because of
the accumulation of an intermediate in the adenine metabolism (Figure 1)
.16
Thus, the APC-ADE2 reporter plasmids
correctly discriminated in-frame and out-of-frame APC
sequences. For the yeast color assay of clinical samples, we prepared
linearized gap vectors, pLF-ADE2-Ig, -IIg, -IIIg, -IVg, and -Vg, by
removing most of the APC inserts, leaving ~60–150 bp of
the flanking APC sequences to allow homologous
recombination. Because the APC expression vectors are
linearized at codons 20 and 446 (region I), codons 446 and 1,013
(region II), codons 1,013 and 1,516 (region III), codons 1,516 and
2,153 (region IV), and codons 2,153 and 2,808 (region V), the assay can
test almost the entire APC coding sequence (see Figure 3
).
To test the fidelity of the system, we amplified region III of human
APC cDNA with Pfu DNA polymerase. We cloned an
unpurified PCR product into the constitutive yeast expression vector
pLF-ADE2-IIIg by homologous recombination in vivo after
co-transfection of the PCR product and linearized gap vector into
yeast, followed by selection of recombinants in plates lacking leucine,
as described previously.17
PCR-amplified wild-type and
mutant cDNA gave 2.9 ± 0.5% and 100% of red colonies,
respectively.
Testing the Assay with Colon Cancer Cell Lines
We examined five human colon cancer cell lines (four with mutant
APC and one with wild-type APC) to assess the
validity of the yeast color assay (Table 1)
. Total RNA and genomic DNA were
extracted from these cell lines. Each region of APC cDNA
consisted of multiple exons or a single exon; ie, region I (exons 1 to
10), region II (exons 10 to 15), and regions III to V (exon 15). We
therefore used RNA for the assay of regions I and II, and genomic DNA
for the assay of regions III to V. More than 96% red colonies were
observed in one of the five regions when the assay was performed in
SW480, Colo201, and Colo320DM cells. A sequencing analysis demonstrated
mutations at codon 1,338 (CAG to TAG) in SW480, 1,554 (GAA to GAAA) in
Colo201, and 811 (TCA to TGA) in Colo320DM. The DLD1 cell gave ~100%
and 50% red colonies in regions III (mutation at codon 1,414, GGC to
GG) and V (codon 2,166, CGA to TGA), respectively. HT-29 cells gave
30% and 70% red colonies in regions II (codon 853, GAG to TAG) and IV
(codon 1,556, ACT to AACT), respectively. HCT116 cells with wild-type
APC gave low percentages of red colonies (0 to 6.4%) in all
of the regions. Because normal colonic mucosa gave 2 to 9.9% red
colonies (Table 2)
, the higher values
were thought indicative of the presence of APC mutations.
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DLD1 and HCT116 cells have been shown to express similar levels of
APC protein.18
To assess the linearity of the assay, we
mixed HCT116 (wild-type APC) and DLD1 cells (mutant
APC) at serial ratios and subjected them to the assay. We
chose region III as a representative of the five regions, and used RNA
and genomic DNA as templates for reverse transcriptase-PCR and PCR
amplification, respectively. As shown in Figure 2, a
highly linear input-output relation
was observed in both DNA (r = 0.99) and RNA
(r = 0.99) samples. RNA and DNA samples obtained
from identical cell mixtures gave almost the same percentage of red
colonies, which indicated that nonsense-mediated RNA decay was
negligible in this assay.
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We used the new assay to test polyps (polyp-1 and -2) from a FAP
patient. The polyp-1 gave red colonies of 50% in region III (Table 2)
,
and sequencing of plasmids rescued from multiple red colonies showed
that the patient was heterozygous for mutant APC allele
(Q1114X). Polyp-2 gave red colonies of 79% in region III and the
sequencing analysis revealed that a new somatic mutation at codon 1,309
was introduced into normal APC allele.
Identification of APC Mutations in Colon Cancers
To test the suitability of the APC yeast color assay
for analyzing tumor samples, RNA and DNA were extracted from sporadic
colorectal cancers, and subjected to the assay. The results were
summarized in Table 2
. Out of 23 cancers tested, 13 cancers (57%) gave
red colonies of >10%. Sequence analysis revealed truncating
APC mutations in these samples; 10 cancers with one mutation
and three with two mutations. Clonality was demonstrated by sequencing
plasmids rescued from multiple red colonies given by both RNA and DNA
samples. The RNA and DNA samples from the identical cancers gave almost
the same results with respect to the percentage of red colonies and
clonality of the mutations. Ten out of 16 somatic mutations (62%) were
found in the APC mutation database19
and eight
out of the 16 somatic mutations (50%) were located in the mutation
cluster region (MCR, codons 1,296 to 1,513) (Figure 3)
. Of five nonsense mutations
identified, three were C to T and two were C to A base substitution.
All of the insertion mutations were found in mononucleotide repeats of
adenine or thymine. The mutations were found more frequently in TNM
stages 0 to I (5 of 5) than stages II to IV (8 of 18). Similarly, the
majority of the mutations (7 of 10) were found in well-differentiated
adenocarcinomas.
Identification of APC Mutations in Breast Cancers
We screened 70 sporadic breast cancers by the assay and the
results are summarized in Table 3
. Out of
70 RNA samples from breast cancers, 11 samples gave >15% red colonies
in a single region (I to V) of APC cDNA and two samples gave
those in double regions. DNA sequencing demonstrated the clonality in
all these samples (Table 3)
. These mutations were confirmed by the
yeast color assay on the corresponding genomic DNA regions. However, in
the four regions, B40 (region III), B42 (IV), B44 (IV), and B59 (IV),
which gave low percentages of 11 to 12% red colonies and low clonality
(2 of 4 or 2 of 6) of mutations, sequencing of the corresponding
genomic DNA regions failed to show identical clonal mutations.
Consequently, clonal APC gene mutations were confirmed in 13
of 70 (18%) breast cancers (Table 3)
. Two tumors had heterozygous
mutations. Among the total of 15 mutations identified, two were
identical (samples B41 and B50, codon 1795 TCA
TCAA); ie, 14
different mutations were identified.
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. Furthermore, the majority of base substitutions
were G to T (4 of 5) and approximately half of the mutations (8 of 14,
57%) were not found in the APC mutation
database.19
Of 10 frameshift mutations, 50% were
single-base insertions and 50% were single-base deletions. All of the
insertion mutations were found in the sites of six runs of adenine
residues. In regard to histological typing of the tumors, the majority
of the mutations were found in solid tubular carcinomas (7 of 25) or
papillotubular carcinomas (5 of 27), whereas only one was detected in
scirrhous-type tumors (1 of 14). Histological grading of the detected
mutations were grade 1, grade 2, and grade 3 in 1 of 18 (6%), 6 of 32
(19%), and 6 of 20 (30%) samples, respectively. The mutation rate of
the APC gene was significantly higher in stages 3 and 4 (5
of 10, 50%) than in stages 1 and 2 (8 of 60, 13%; P
< 0.05). Consequently, APC mutations were frequently found
at the high-grade and advanced stage of primary breast cancers. A
highly significant association was also observed between the
APC mutation and the size of primary tumor (T category): 8
of 62 (13%) in T1 and 2 versus 5 of 8 (63%) in T3 and 4
(P < 0.01) (Table 3)
. We also screened 29 non-small-cell lung cancers (21 adenocarcinomas and eight squamous cell carcinomas) by the APC yeast color assay. All of the cancers gave background level (<10%) of red colonies and were scored as negative for the APC mutation. We confirmed no clonal mutation of the APC gene in the samples which gave around 10% red colonies; ie, no APC mutations were detected in the 29 lung cancers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although a similar yeast-based assay has been reported for detection of the APC gene mutation, the advantages of the present yeast color assay, compared with the URA3 assay,14 are that it does not require a replica-plating step and that yeast containing mutant APC can be visualized as red colonies. These permit analysis of a large number of samples and many clones per sample in a short period. We have confirmed clonal APC gene mutations in both mRNA and genomic DNA of the clinical samples that gave red colonies of >15%. This sensitivity is comparable to that of the yeast functional assay for p53 mutation which we previously reported.17 It indicates that the present assay is applicable to the clinical tumor samples which may be even contaminated with a variable amount of normal tissues, whereas the URA3 assay could not be applicable, because the assay requires a replica-plating step in the selection of yeast clones and could fail to detect a mutant of a small fraction in the tumor tissue contaminated with normal host cells.14
Most of colon cancer cell lines gave 96 to 99% of red colonies in one of five regions. Although HT-29 cells gave approximately 30% and 70% of red colonies in respective regions II and IV, this can be explained from the cytogenetic finding that karyotype of this cell line is hypertriploid (ATCC no.: HTB-38). The percentages of red colonies by colon and breast cancers, which were demonstrated to harbor the APC mutations, ranged from 15 to 57% in regions I to V. The relatively low percentage of red colonies in tumor tissues compared to those of cell lines is presumed not because of reduced amounts of mutant APC mRNA (nonsense mediated decay),23 from the almost equivalent percentages of the red colonies given by the tests on the mRNA and the corresponding genomic DNA. It is most conceivable to be because of admixture of cells containing wild-type APC alleles, ie, normal cells and tumor cell clone(s) that do not acquire APC mutation.
We screened 23 colon cancers by the yeast color assay and found 15 APC mutations in 13 cancers (57%). Eight of 16 (50%) somatic mutations were located in the mutation cluster region (MCR, codon 1286 to 1513). This finding is consistent with previous reports that >60% of the somatic mutations were clustered within the MCR.9 In contrast to the findings in colon cancers, the majority of mutations found in breast cancers were scattered on the entire APC coding region and only 4 of 15 (27%) were located in the MCR, indicating that the concept of a mutation cluster region of the APC is not applicable to breast cancer. Powell et al24 presented evidence that APC mutations occur early during colorectal tumorigenesis. Our data of colon cancers reconfirmed their findings, by demonstrating the APC gene mutation at any of the clinical stages of colon cancer. In breast cancers, however, mutations were significantly more frequent in higher, advanced stages, suggesting that the APC mutations are associated with tumor progression in breast cancers. The APC inactivation may lead to suppressed degradation of ß-catenin.18 Because Tcf-4 protein was immunohistochemically detected at a high level in restricted tissues such as the intestinal and mammary gland epithelium and carcinomas derived therefrom,25 a constitutively active Tcf-4/ß-catenin complex that is a consequence of the APC mutations may result in the uncontrolled transcription of its target genes.26,27 It is noteworthy that APC mutations were detected mainly in solid tubular (7 of 25) or papillary tubular (5 of 25) breast carcinomas but only one mutation was detected in 14 scirrhous-type carcinomas. It might be possible that APC mutation plays differential roles in different types of breast cancers. Alternatively, it is possible that the lower rate of APC mutations in the scirrhous tumors might be because of the higher percentage of normal cells that may conceal a mutant APC within the background level of the APC yeast color assay.
It has been reported that most identified base substitutions in the APC gene are changes from C to T.28 In the present study, a similar trend was observed in colon cancers, whereas G to T change was dominant in breast cancers. At present it is not clear whether the difference of mutation pattern between the colon and breast cancer reflects an intrinsic (endogenous) pattern of mutation or exposures to a particular environmental mutagen(s). Furthermore, racial and/or environmental factors cannot be excluded. The age-adjusted breast cancer incidence rate and mortality rate of Japanese women are three to four times lower than those of most Western women.29,30 To elucidate the difference, it is necessary to detect APC mutations in breast cancers from ethnically different populations; the APC yeast color assay will provide a powerful means for detection of the APC mutations in such clinical samples.
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Acknowledgements |
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Footnotes |
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Supported by a Grant-in-Aid for Scientific Research of the Ministry of Education, Japan.
Accepted for publication February 9, 2000.
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, germline
mutation in FAP patients; 
, somatic mutation in colorectal cancer;
, somatic mutation in breast cancer; 
, mutations located at
the mutation cluster region (codons 1,296 to
1,513) reported in colorectal
cancers.
, RNA samples.