title: Three-dimensionally specific inhibition of DNA repair-related genes by activated KRAS in colon crypt model.
abstract: Growth and differentiation of colonic epithelium are regulated in the three-dimensional (3D) physiological architecture, colonic crypt, and deregulation of 3D interactions is involved in tumorigenesis. Cell-based 3D culture systems provide a suitable approach bridging the gap between two-dimensional (2D) culture and animal models. KRAS mutations are found at high frequencies in human colorectal cancer (CRC); however, KRAS-targeted cancer therapy has not been developed. Here, we have established a 3D cell culture model resembling the colonic crypt by use of HKe3 cells, human CRC HCT116 cells disrupted at activated KRAS. In this 3D colonic crypt model, HKe3 cells showed the features of time course-dependent transit-amplifying and terminal-differentiated stages, which are characteristic of normal colonic crypt. On the basis of the features of HCT116 cells, activated KRAS inhibited normal cell polarity and apoptosis in 3D culture. The expression of DNA repair-related tumor suppressor genes including TP53, BRCA1, BRCA2, and EXO-1 was markedly suppressed by activated KRAS in 3D culture but not in 2D culture. These results together suggest that activated KRAS plays critical roles in the accumulation of genetic alterations through inhibition of DNA repair genes and apoptosis and that this 3D culture model will provide a useful tool for investigating the molecular mechanisms of CRC development.
authors.affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Tsunoda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Takashima
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Midori
authors.initials: M
authors.lastname: Koyanagi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yoshida
authors.forename: Keiko
authors.initials: K
authors.lastname: Doi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Masahide
authors.initials: M
authors.lastname: Kuroki
authors.forename: Takehiko
authors.initials: T
authors.lastname: Sasazuki
authors.forename: Senji
authors.initials: S
authors.lastname: Shirasawa
mesh_terms: D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004260:DNA Repair / Q000235:genetics; D045643:DNA Repair Enzymes / Q000096:biosynthesis / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology*; D018613:Microscopy, Confocal; D008954:Models, Biological; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D020133:Reverse Transcriptase Polymerase Chain Reaction; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D045643:DNA Repair Enzymes
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