[Development of a detection system (APC yeast color assay) of APC mutations by color change of yeast].
The author developed a sensitive yeast-based color assay which expresses APC-ADE2 (reporter) fusion protein in yeast and can screen almost the entire coding region of the APC gene. In this assay, the wild-type APC coding sequence of 8.5 kb is divided into 5 overlapping regions which are respectively ligated in-frame with an ADE2 open reading frame. The resulting five constructs containing a part of wild-type APC gene preserve the ADE2(+) phenotype (white yeast colony) when introduced into the yeast, whereas the yeast transfected with plasmids containing frameshift mutations of the APC gene shows an ADE2(-) phenotype (red yeast colony). Six human colon cancer cell lines were analyzed by this yeast color assay. HCT116 cells with wild-type APC and normal colonic mucosa gave low percentages of red colonies (0-9.9%) in all the regions. On the other hand, more than 96% red colonies were observed in one of the five regions in SW480, Colo201 and Colo320DM cells. Sequence analysis demonstrated the clonal APC mutations at codon 1,338 in SW480, 1,554 in Colo201 and 811 in Colo320DM. Moreover, the assay detected a germline mutation of the APC gene in polyps of a familial adenomatous polyposis (FAP) patient which gave about 50% red colonies. For testing the assay for clinical utilization, 18 colon cancer tissues were subjected to the assay. Eleven cancers (61%) gave more than 10% red colonies (17-57%) and clonal mutations were detected in all these samples. The same mutations were demonstrated in both DNA and RNA samples derived from idendical tissues. These results suggest that this APC yeast color assay is powerful means for detection of APC mutations in clinical samples.
Furuuchi
K
K
Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.
D011125:Adenomatous Polyposis Coli / Q000175:diagnosis / Q000235:genetics; D025601:Adenomatous Polyposis Coli Protein; D000328:Adult; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000175:diagnosis / Q000235:genetics; D003116:Color; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D017491:Genes, APC / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D012334:RNA, Neoplasm / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D025601:Adenomatous Polyposis Coli Protein; D003598:Cytoskeletal Proteins; D004273:DNA, Neoplasm; D005656:Fungal Proteins; D012334:RNA, Neoplasm