Use of Crypt Isolation to Determine Loss of Heterozygosity of Multiple Tumor Suppressor Genes in Colorectal Carcinoma

Sugai, Tamotsu; Habano, Wataru; Nakamura, Shinichi; Yoshida, Toru; Uesugi, Noriyuki; Sasou, Shunichi; Itoh, Chuichi; Katoh, Ryouhei

doi:10.1016/s0344-0338(00)80094-1

Cited by 16 publications

(33 citation statements)

References 19 publications

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“…Contamination by other materials such as interstitial cells was not evident in the samples that were examined, as described in previous reports. 15,16 DNA from the tumor and from corresponding normal crypts was extracted by standard sodium dodecyl sulfate proteinase K treatment.…”

Section: Dna Extractionmentioning

confidence: 99%

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Analysis of Molecular Alterations in Left- and Right-Sided Colorectal Carcinomas Reveals Distinct Pathways of Carcinogenesis

Sugai

Habano

Jiao

et al. 2006

The Journal of Molecular Diagnostics

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To clarify distinct genetic profiles of colorectal cancers based on tumor location (left-and right-sided), we evaluated the status of loss of heterozygosity (LOH), CpG islands methylation phenotype (CIMP), microsatellite instability (MSI), and mutations of p53, Ki-ras, and APC genes in 119 colorectal cancers. Statuses of LOH (at 5q, 8p, 17p, 18q, and 22q), MSI, and CIMP (MINT1, MINT2, MINT31, MLH-1, MGMT, p14, p16, and RASSF1A) were determined using microsatellite polymerase chain reaction and methylation-specific polymerase chain reaction coupled with a crypt isolation method, respectively. In addition, mutations of p53, Ki-ras, and APC genes were also examined. LOH, MSI, and CIMP status allowed us to classify samples into two groups: low or negative and high or positive. Whereas the frequency of p53 mutations in the LOH-high status was significantly higher in left-sided cancers than in right-sided cancers, CIMP-high in the LOH-high status and MSI-positive status were more frequently found in rightsided cancers compared with left-sided cancers. Finally, location-specific methylated loci were seen in colorectal cancers: type I (dominant in right-sided cancer) and type II (common in both segments of cancer). Our data confirm that distinct molecular pathways to colorectal cancer dominate in the left and right sides of the bowel. (J Mol Diagn

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Section: Dna Extractionmentioning

confidence: 99%

“…LOH was determined by calculating the ratio of the peak areas of the constitutional alleles, as described previously. 16 In this study, we defined LOH as more than a 50% difference in this ratio.…”

Section: Microsatellite Analysismentioning

confidence: 99%

Analysis of Molecular Alterations in Left- and Right-Sided Colorectal Carcinomas Reveals Distinct Pathways of Carcinogenesis

Sugai

Habano

Jiao

et al. 2006

The Journal of Molecular Diagnostics

Self Cite

124

101

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“…Contamination by other materials such as interstitial cells was not evident in the samples that were examined, as shown in previous reports. 2,3 In the present study, we termed an individual isolated tumor gland a single tumor gland. A representative example of a single tumor gland is shown in Figure 1.…”

Section: Crypt Isolation Techniquementioning

confidence: 99%

“…The crypt isolation method can be used to obtain single tumor glands from a given tumor. 2,3 Using a crypt isolation method, we can accurately analyze genetic alterations of single tumor glands and compare genetic differences among the single tumor glands that occur within the same tumor. 1 In the current model of stepwise progression of colorectal tumors, each step is thought to be initiated by the acquisition of additional genetic alterations.…”

mentioning

confidence: 99%

Analysis of allelic imbalances at multiple cancer‐related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Sugai

Habano

Jiao

et al. 2004

Intl Journal of Cancer

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Genetic changes related to colorectal carcinomas are accumulated in individual tumor glands during disease progression. Microsatellite allelic analysis of individual tumor glands from 30 colorectal carcinomas using a polymerase chain reaction (PCR) assay coupled with crypt isolation was used to detect intratumoral genetic heterogeneity, the sequence of allelic imbalances (AIs) and the microsatellite instability status of single tumor glands during neoplastic progression. In addition, the CpG islands methylated phenotype (CIMP) status was examined using a methylation-specific PCR method. The specimens were divided into 2 groups: a pooled gland sample, which was composed of more than 50 tumor glands, and a single tumor gland sample. The latter consisted of 10 single tumor glands, which were obtained from the same tumor separately. Most colorectal carcinomas (27 of 30 tumors) examined were heterogeneous for at least one genetic alteration, with from 2 to 7 genotypically different subclones detected per tumor. In 12 of the 27 heterogeneous tumors, it was possible to define the order of genetic alterations during the tumor progression. By analyzing multiple single tumor glands within the same tumor, we found that various subclonal expansions were seen within the same tumors.

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“…Ten microsatellite markers (D11S1816; D11S1817; D11S1343; D11S1819; D11S2000; D11S2179; D11S1294; D11S1818; D11S1778; D11S1347) located in the 11q22-23 region known to contain the ATM gene were used. PCR analysis (GeneAmp PCR System 9600, Perkin-Ermer Cetus, Norwalk, CT, USA) was carried out according to previously described conditions to quantitatively detect allelic imbalances at each locus (Sugai et al, 2000d;Uhrhammer et al, 1999). Additional experiments were performed to examine the relationship between allelic imbalance at the ATM and 11q24 (distal to 11q22-23) loci in colorectal carcinomas using three 11q24 microsatellite markers (D11S912, D11S1320 and D11S969) shown to be frequently a ected by LOH (Connolly et al, 1999).…”

Section: Microsatellite Dna Analysismentioning

confidence: 99%

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas

et al. 2001

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DNA multiploidy may involve speci®c DNA ploidy states with respect to genetic alterations such as oncogenes, tumor suppressor gene mutation and microsatellite instability. To clarify the role of DNA multiploidy in colorectal cancer, we analysed allelic imbalance involving the ATM gene, localized to chromosome 11q22-23 and thought to be involved in genetic stability, in a series of multiploid colorectal carcinomas. In addition, p53 gene mutation (exons 5 ± 8) and allelic imbalance at 11q24 loci distal to the ATM locus were also examined. The crypt isolation technique coupled with DNA cytometric sorting and polymerase chain reaction assay using 10 microsatellite markers tightly linked to the ATM gene were used to study ATM allelic imbalance in 55 colorectal carcinomas (15 diploid, 13 aneuploid, 27 multiploid). While allelic imbalance at the ATM locus was rarely observed in diploid and aneuploid carcinomas, multiploid carcinomas exhibited a high frequency of ATM allelic imbalance. In multiploid carcinoma samples, diploid subpopulations showed a smaller range of allelic imbalance at the loci tested compared to aneuploid subpopulations that demonstrated allelic imbalance over a relatively large region. Also, the frequency of AI at 11q24 showed a similar tendency to that at the ATM locus for each DNA ploidy state. An association between p53 gene mutation and ATM allelic imbalance in multiploid carcinoma was also observed. Our results suggest that ATM allelic imbalance and p53 gene mutations occur during the progression from diploid to aneuploid cell populations in multiploid colorectal carcinomas. Oncogene (2001) 20, 6095 ± 6101.

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Use of Crypt Isolation to Determine Loss of Heterozygosity of Multiple Tumor Suppressor Genes in Colorectal Carcinoma

Cited by 16 publications

References 19 publications

Analysis of Molecular Alterations in Left- and Right-Sided Colorectal Carcinomas Reveals Distinct Pathways of Carcinogenesis

Analysis of Molecular Alterations in Left- and Right-Sided Colorectal Carcinomas Reveals Distinct Pathways of Carcinogenesis

Analysis of allelic imbalances at multiple cancer‐related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas

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