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[Cancer Research 60, 3165-3169, June 15, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Instability of X Chromosome Methylation in Aberrant Crypt Foci of the Human Colon1

Nobuyuki Sakurazawa, Noritake Tanaka, Masahiko Onda and Hiroyasu Esumi2

Investigative Treatment Division, National Cancer Center Research Institute East, Chiba 277-8577, Japan [N. S., H. E.]; Department of Surgery, Nippon Medical School, Chiba-Hokuso Hospital, Chiba 270-1694, Japan [N. T.]; and First Department of Surgery, Nippon Medical School, Tokyo 113-8603, Japan [N. S., M. O.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant crypt foci (ACF) in the colon have long been thought to be precancerous lesions and therefore monoclonal, but this is unresolved. Eleven ACF were isolated from five female patients. From these ACF, 178 individual aberrant crypts (ACs) were obtained and assessed for clonality using a method based on X chromosome inactivation of the polymorphic X-linked human androgen receptor (HUMARA) gene. Ten ACF were found to be mixtures of monoclonal and polyclonal types. The HUMARA analysis indicated that almost all ACF were polyclonal lesions. Simultaneously, we investigated K-ras mutations in each AC. We found that seven of the ACF harbored the K-ras mutation; strikingly, this was concordant for all of the ACs from a single ACF. These results, by contrast to the results of HUMARA analysis indicate that ACF lesions are monoclonal. This discrepancy suggests that ACF are apparently polyclonal because of de novo methylation on the active X chromosome. To confirm this possibility, we investigated the methylation status of the X chromosome in male ACF using a competitive PCR assay. One hundred nineteen individual ACs were isolated from eight ACF derived from four male patients. A total of 47 of 119 (39%) of male ACs showed de novo methylation of the HUMARA gene. We found that six of the eight male ACF harbored the K-ras mutation, and this was concordant for all of the ACs from a single ACF. We conclude that X chromosome methylation is unstable in ACF and that this might be an early event in colon carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACF3 were first identified microscopically in methylene blue-stained, whole-mount preparations of colonic mucosa from carcinogen-treated rodents (1) . ACF have also been found in the colons of humans (2 , 3) , with colorectal cancer patients having a higher number of ACF per unit area of colonic mucosa than individuals without this cancer (4) . Moreover, because K-ras mutations have been detected in over 50% of ACF of the human colon, ACF have been thought to be a precancerous lesion of colon cancer (3 , 5) .

It has been widely accepted that tumors are monoclonal in origin, arising from the growth of a single cell carrying a mutation or series of mutations. In support of this, Fearon et al. (6) reported that all colonic adenomas and cancers were monoclonal. Recently, however, Novelli (7) et al. reported that 76% of microadenomas from a patient with familial adenomatous polyposis coli, were polyclonal. Although from the high incidence of K-ras mutation in ACF these lesions appear to be monoclonal, there have been no conclusive data to indicate whether ACF is derived from a single cell or not.

Many studies have investigated the clonality of various lesions using methods based on X chromosome inactivation in females (8, 9, 10, 11) . These methods are based on two assumptions (12) . First, it is assumed that one of the two X chromosomes in the female is inactivated to compensate for the difference in X-linked gene dosage between males and females. This produces an inactivated X chromosome that is heavily methylated in multiple sites. Second, it is assumed that once X chromosome inactivation is established, the methylation status of the X chromosomes in the female cell is stably inherited by the progenitor cells.

Of the X-linked polymorphic genes, the HUMARA gene has been widely used for clonality analysis. This is because the gene has a highly polymorphic trinucleotide repeat in the first exon that is 90% informative for female specimens assessed for clonality (13 , 14) .

In this study, we used HUMARA gene analysis to examine the clonality of ACF. Unexpectedly, we found that a significant fraction of the individual ACs that made up an ACF appeared to be polyclonal, although by K-ras mutation genotyping, all ACF appeared to be monoclonal.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ACF Sampling and Crypt Isolation.
Colorectal specimens were obtained from nine patients who underwent surgery for sporadic colorectal cancers at the National Cancer Center Hospital East, between September 1998 and June 1999. These patients (five females and four males) ranged in age from 48 to 82 years and had no history of familial adenomatous polyposis, ulcerative colitis, or hereditary nonpolypoid colorectal cancer. All experiments were performed according to the guidelines of the ethics committee.

The ACF were isolated from the grossly normal-appearing mucosa of the surgical specimens after pathological examination. All mucosal strips were examined under a stereomicroscope after staining with 25% pyoktanin in saline. ACs were distinguished from normal crypts by their deep purple color, large size, oval shape of the luminal opening, and slight mucosal elevation (2) . All ACF specimens were divided into two pieces. One was used for histological examination, and the other was used for DNA extraction after crypt isolation (as described below).

Individual ACs were isolated from the ACF using a previously reported method (15) . Briefly, the ACF samples were incubated in 10 ml of HBSS containing 30 mM EDTA for 30 min at 37°C. Each AC was then identified by stereomicroscopic observation and isolated using fine forceps.

Immunohistochemical Staining for Vimentin and H&E Staining of Isolated Crypts.
A sagittal frozen section taken from each isolated crypt was used to assess the level of mesenchymal cell contamination. These sections were stained with H&E or with vimentin using an antivimentin (V9) monoclonal antibody (Roche Molecular Biochemicals, Mannheim, West Germany). Vimentin immunostaining was performed using an avidin-biotin-peroxidase complex method, as described previously (16) .

K-ras Mutation and Clonality Analyses.
Genomic DNA was extracted from each AC using the DNA extractor WB kit (Wako, Osaka, Japan), as described previously (17) . The DNA from each AC was then screened for K-ras codon 12 mutations by mismatched primer-mediated PCR-restriction fragment length polymorphism analysis, using primer sequences and PCR conditions described previously (18) . Clonality was analyzed using the polymorphic X-linked HUMARA gene. Briefly, approximately 0.2 ng of genomic crypt DNA in a total reaction volume of 3 µl was digested with 1.25 units of RsaI, with and without 1 unit of HpaII, for 6 h at 37°C. The digested products were subjected to PCR as described previously, using the AR1 (5'-CCGAGGAGCTTTCCAGAATC-3') and AR2 (5'-TACGATGGGCTTGGGGAGAA-3') primers and a 30-µl total reaction volume. AR2 is Cy5 labeled primer (17) . Fig. 1Citation shows the HUMARA gene and the scheme of this PCR. The PCR products were diluted with loading buffer [95% formamide, 0.003% (w/v) dextran blue] and separated through a denaturing polyacrylamide gel (5% acrylamide, 7 M urea, 0.6x TBE) at 30 W using the ALFred automated sequencer (Amersham Pharmacia Biotech, Upsala, Sweden). The results were then analyzed using the Fragment Manager software package (Amersham Pharmacia Biotech). The ratio of both alleles (allelic ratio) after HpaII digestion was obtained after normalization with respect to an undigested sample. In this study, we decided the clonality using this ratio, because we had used it previously for the criteria of the clonality by HUMARA (17) .



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  		Fig. 1. Schematic representation of HUMARA gene. There are one polymorphic CAG repeat and two HpaII sites. Repeat number of the CAG is from 16 to 29 in Asian people. Both AR1 and AR2 are primers that are set at the both end of this region.

 
Competitive PCR Analysis.
A competitive PCR assay to confirm that the HpaII digestion was complete was designed. The assay used DNA obtained from male peripheral blood leukocytes as an internal control, because the HUMARA gene on the male X chromosome is not normally methylated and can therefore be digested by HpaII. An excess amount of internal control male DNA was added to each DNA sample, and each particular control sample was selected to ensure that its CAG repeat number was different from the CAG repeat number of the sample. Apart from the addition of an internal control, the competitive PCR analyses were performed as described for the clonality analyses.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The ACF Were Apparently Polyclonal by HUMARA Analysis.
To analyze the clonality of ACF using the HUMARA assay, it is essential to exclude the possibility of interstitial tissue contamination (15) . In the present study, we adopted the crypt isolation method to minimize this contamination. Under stereomicroscopic examination, the isolated ACs showed smooth contours, suggesting they that were free from interstitial tissue contamination (Fig. 2ACitation ).



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  		Fig. 2. Morphological examination of ACF and isolated ACs. A, single ACs isolated from an ACF after EDTA treatment. B, H&E staining of an AC. C, vimentin immunostaining of an AC.

 
In a preliminary study using the HUMARA assay, we analyzed the clonality of 14 ACF taken from a female patient with colon cancer. After separating the epithelial lining of ACF from the interstitial tissue, the constituent crypts from single ACF were combined. This study showed that 3 of the 14 ACF were apparently polyclonal.

Individual ACs Were Not Significantly Contaminated with Mesenchymal Cells.
We reevaluated the possibility of mesenchymal cell contamination of the individual crypt preparations immunohistochemically by staining with a monoclonal antibody against vimentin, a mesenchymal cell-specific intermediate filament. The immunostaining of 36 crypts from three ACF showed an average of 3.8% vimentin positive cells (and at most, 9.8%) in the isolated ACs (Fig. 2CCitation ). Because we have shown previously that a monoclonal cell group with up to 15% stromal cells has an allelic ratio of less than 0.414 , an allelic ratio of 0.41 was taken to be indicative of polyclonality. Using this criterion, mesenchymal cell contamination could be excluded as contributing to the apparently polyclonality results obtained from the three ACF in the HUMARA assay.

Female ACs Were Found to Be Apparently Polyclonal by HUMARA Analysis but Monoclonal by K-ras Mutation Analysis.
Because the above results suggested that apparently polyclonal ACF might be composed of more than two groups of monoclonal ACs with inactivation of alternate X chromosomes, we investigated the clonality of each AC using the HUMARA assay. We collected 11 new ACF samples from five female patients with sporadic colorectal cancer. The characteristics of these samples are summarized in Table 1Citation .


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  		Table 1 Patient and ACF characteristics of female patients with colorectal cancer

 
The clonality of 178 ACs isolated from these 11 ACF samples was analyzed by the HUMARA assay, and representative results are shown in Fig. 3Citation . Surprisingly, some of the ACs clearly showed two curves after HpaII digestion and their allelic ratios were over 0.41. In total, 38% (67 of 178) of the ACs were found to be apparently polyclonal by HUMARA analysis (Fig. 4ACitation ). This was in marked contrast to the clonality of the crypts isolated from the normal colonic mucosa, whereas the HUMARA assay showed that less than 7% (8 of 124) of the crypts were polyclonal (data not shown). At the ACF level, 10 of 11 of the ACF were composed of both polyclonal and monoclonal ACs (Fig. 4ACitation ).



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  		Fig. 3. Representative clonal analysis. All samples were run as undigested [HpaII (–)] and HpaII-digested [HpaII (+)] pairs. The curve on the right are products with longer CAG repeats, and that on the left are products with shorter repeats. Single AC 1, monoclonal; single AC 2, polyclonal.

 


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  		Fig. 4. HUMARA and K-ras mutation analysis for 11 ACF taken from five female patients. A, clonality analysis by HUMARA. Each vertical column represents one ACF, and the left column shows the AC number. Each AC was named in order of isolation from the original tissue. Filled boxes, short type; light gray, long type; dark gray, mixed type. B, K-ras codon 12 mutation detection. Gray boxes, mutated; filled boxes, wild type.

 
These results prompted us to test for K-ras mutations in each AC because we expected the ACF to be mosaic for this mutation. We found that 7 of the 11 ACF harbored the K-ras mutation (Fig. 4BCitation ). Notably, the K-ras mutation was concordant for all of the ACs from a single ACF. These results clearly indicate that most ACF come from monoclonal cell expansion (Fig. 4BCitation ). However, this interpretation sharply contradicts the results we obtained from the HUMARA assay, in which six of those seven ACF were shown to be apparently polyclonal (Fig. 4ACitation ).

Complete HpaII Digestion.
To exclude the possibility that incomplete digestion of sample DNA with HpaII affected the results of the HUMARA analysis, we performed a competitive PCR assay by adding an excess amount of HpaII-digestible DNA into the reaction. As shown in Fig. 5Citation , the peak derived from the internal control (evident in the undigested sample) disappeared after HpaII digestion. Conversely, the two prominent peaks from the polyclonal crypt became more prominent after HpaII digestion (Fig. 5Citation ). This indicated that HpaII digestion was complete and suggested that 38% of the polyclonal ACs were actually heterogeneous in regard to their HUMARA allele methylation status.



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  		Fig. 5. Representative competitive PCR assay. All samples were run as undigested [HpaII (–)] and HpaII-digested [HpaII (+)] pairs. The sample DNA was derived from a single female AC. The two prominent peaks of the undigested DNA sample still had two prominent peaks after digestion.

 
De Novo Methylation Was Also Identified in the ACs from Male ACF.
All of the results described above indicate that de novo methylation of the HUMARA gene may occur in active X chromosomes. To examine this possibility, we isolated ACs from surgical specimens taken from four male patients with colorectal cancer. Male samples were taken specifically because the HUMARA gene on a single active X chromosome is thought to be unmethylated (12) . The characteristics of these samples are summarized in Table 2Citation .


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  		Table 2 ACF in the colons of male patients with colorectal cancer

 
In total, 119 ACs were collected from the eight male ACF. To detect de novo methylation of the HUMARA gene, we conducted the following competitive PCR assay. The DNA sample from male ACs was mixed with an excess amount of internal control DNA, which was digestible with HpaII. Before restriction digestion of the mixed DNA, a large peak, originating from the internal control, was evident (Fig. 6Citation ). HpaII digestion made the PCR product peak derived from the sample DNA more prominent in 39% (47 of 119) of male ACs, whereas it abolished the internal control peak in these reactions (Fig. 6Citation , crypt 2). Similar changes in the peak ratio between the PCR product peak and the internal control peak were not observed after digestion with the methylation-insensitive enzyme MspI. These changes in peak ratio were also not observed in any of the 106 crypts isolated from the normal colonic mucosa. On the contrary, the ratio of the peaks of the nonmethylated AC did not change after HpaII digestion (Fig. 6Citation , crypt 1). Ideally, the both peaks should be eliminated after HpaII digestion, but actually trace amount PCR products was detected by automatically expanded sensitivity of ALFred sequencer. These results clearly indicate that de novo methylation of the HUMARA gene of active X chromosomes occurs only in the ACs. Fig. 7ACitation summarizes the de novo methylation status of the male ACs.



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  		Fig. 6. Representative de novo methylation of the HUMARA gene in male ACs. All samples were run as undigested [HpaII (–)], and HpaII digested [HpaII (+)] pairs. Single AC 1, nonmethylated; single AC 2, de novo methylated HUMARA gene from a male AC.

 


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  		Fig. 7. HUMARA analysis and K-ras mutation analysis of eight ACF from four male patients. A, de novo methylation analysis by competitive PCR. Each vertical column represents an ACF, and each row represents an AC. Each AC was named in order of isolation from the original tissue. Gray boxes, de novo methylated; filled boxes, nonmethylated. B, K-ras codon 12 mutation detection. Gray boxes, mutated; filled boxes, wild type.

 
Additionally, we identified the K-ras codon 12 mutation in the male ACs. The male K-ras results were completely consistent with the results obtained for the female ACF and showed that with respect to individual ACF, the presence of the K-ras mutation was either all or none. In total, six of eight ACF harbored the K-ras mutation (Fig. 7BCitation ).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the clonality of ACF by isolating individual ACs from each ACF and found two contradictory results. K-ras mutation analysis strongly suggested that each ACF was derived from a single progenitor cell. In the present study, we examined mutations only at the codon 12 of K-ras gene by PCR-restriction fragment length polymorphism method (18) . According to Yamashita et al. (3) codon 13 mutations were detected in 12% of ACF, and codon 12 mutations were detected in 46%. Forty-six % of codon 12 mutations were GGT to GAT mutations, 44% were GGT to GTT mutations, and 12% were GGT to TGT mutations. There is a possibility that analyzed ACF might be composed of different ACs having different mutations (polyclonality). However, the presence of K-ras mutation was concordant in all 19 ACF (11 female ACF and 8 male ACF), and so it was less likely that each AC from a single ACF had different point mutation.

However, HUMARA analysis indicated that each ACF was composed of different cell populations in regard to methylation states of the HUMARA gene. These results confirm previous studies that have indicated that ACF are monoclonal lesions, and HUMARA analysis cannot be applied to clonality analyses of ACF.

X chromosome inactivation is the mechanism underlying dosage compensation in the X chromosome and is critical for cellular function (12) . As such, X chromosome inactivation is strictly regulated by complex genetic and biochemical mechanisms. However, in the present study, we found that about one-third of female ACs were apparently polyclonal when analyzed using the HUMARA assay. One ACF from a female patient (F5) was found to be composed of three types of ACs, each with distinct HUMARA patterns. The AC types in this ACF were composed of six monoclonal populations with methylation at the short CAG repeat allele (short type), three monoclonal populations with methylation at the long CAG repeat allele (long type), and 15 polyclonal populations (mixed type).

How could such a mixed population ACF be formed? It is unlikely that the ACF formed by collision of more than two ACF, because a significant fraction of the ACs were of mixed type and apparently polyclonal. The possibility of cellular contamination or partial digestion by HpaII was carefully eliminated, and the results were even less likely to be affected by technical errors in this study. However, there are three possible explanations for the apparent polyclonality of the individual ACs. One possible explanation is that these "polyclonal" ACs are composed of two cell populations with different methylated alleles of the HUMARA gene. This possibility is quite interesting because this type of polyclonality could not be achieved without demethylation of the HUMARA gene of an inactive X chromosome followed by de novo methylation or the presence of some cells with two unmethylated X chromosomes. Such unmethylated genes are only found in somatic cells in the very early stages of early embryonic development (12) . The second possible explanation is the mixed population was generated by de novo methylation of the HUMARA gene of an active X chromosome. In this case, the ACF would be composed of one cell population having two methylated HUMARA genes on both X chromosomes. The third possible explanation is loss of an allele of the X chromosome (loss of heterozygosity). If this were the inactive X chromosome, the AC itself would have no methylated X chromosome. Therefore, the clonality results would only look at the remaining normal contaminating tissue.

We cannot differentiate which of the three possibilities outlined above applies to the present study. However, the following observations favor the second possibility. We analyzed the methylation status of the HUMARA gene in ACF from male patients. About 40% of the ACs were apparently methylated. Because there are two HpaII sites in the HUMARA gene region (Fig. 1Citation ), it is unlikely that two HpaII sites would simultaneously mutate. Because it is more likely that the present result was acquired from de novo methylation, we conclude that the HUMARA genes from the male ACs were de novo methylated. The MspI results strongly support this conclusion. The active HUMARA genes in the female ACs are also likely to be de novo methylated.

It has long been believed that the methylation of genes on an inactive X chromosome is strictly regulated to maintain X chromosome inactivation and gene dosage in female cells (12) . The Xist and Tsix genes are known to play a role in fine tuning this process (19 , 20) . Demethylation of inactive X chromosomes only occurs during oocyte maturation, and de novo methylation only occurs during spermatogenesis and at the very early stages of embryogenesis (12) . Although, CpG methylation of autosomal genes has been described and abnormal regulation of autosomal gene methylation has been observed in carcinogenesis (21) , methylation instability of the genes on the X chromosomal gene has not been described previously.

In the present work, we have only examined the methylation status of an X chromosome-linked gene. Therefore, it might be too early to conclude that X inactivation is unstable in preneoplastic conditions. We have, however, observed a similar phenomenon in intestinal metaplasia, where monoclonal pyloric glands apparently became polyclonal glands (15) . If these cases of methylation instability of X-linked genes are associated with instability of expression of these genes, we could speculate that this instability may have a significant impact on carcinogenesis. Issa and co-workers (22 , 23) have examined methylation of a variety of tumor suppressor gene in colon cancer and in normal colon cells during aging. They reported that aberrant methylation might affect the carcinogenesis of colon. In the present study, we also found the methylation instability in colon precancerous lesion ACF. Putative oncogene-like genes might be functionally amplified by reactivation of the X chromosome in females, and putative tumor suppressor genes on the X chromosome might be inactivated by de novo methylation in both sexes in ACF. We would like to emphasize that extremely close attention must be paid to the methylation instability of X chromosomes (particularly methylation inactivation) in the clonal analysis of precancerous lesions.


   ACKNOWLEDGMENTS
 
We are deeply indebted to Drs. Yoshiaki Hitomi, Sachiyo Nomura, and Kenji Sugiyama of Investigative Treatment Division, National Cancer Center Research Institute East, for their continued support and critical discussion of this project.


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

1 This work was supported in part by a grant from the Ministry of Health and Welfare for the Second-term 10-year Comprehensive Strategy for Cancer Control. N. S. is a recipient of the Research Resident Fellowship from the Foundation for Promotion of Cancer Research. Back

2 To whom requests for reprints should be addressed, at Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa-shi, Chiba 277-8577, Japan. Phone: 81-471-33-1111, extension 5101; Fax: 81-471-34-6859; E-mail: hesumi{at}east.ncc.go.jp Back

3 The abbreviations used are: ACF, aberrant crypt foci; AC, aberrant crypt; HUMARA, human androgen receptor. Back

4 Unpublished data. Back

Received 12/ 8/99. Accepted 4/13/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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