Three-dimensionally specific inhibition of DNA repair-related genes by activated KRAS in colon crypt model.
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.
Tsunoda
Toshiyuki
T
Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
Takashima
Yasuo
Y
Fujimoto
Takahiro
T
Koyanagi
Midori
M
Yoshida
Yasuhiro
Y
Doi
Keiko
K
Tanaka
Yoko
Y
Kuroki
Masahide
M
Sasazuki
Takehiko
T
Shirasawa
Senji
S
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*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D045643:DNA Repair Enzymes
Toxicol Lett. 2005 Dec 15;159(3):212-8
15979256
Nat Rev Mol Cell Biol. 2006 Mar;7(3):211-24
16496023
Nature. 2004 Nov 18;432(7015):316-23
15549093
Nat Rev Mol Cell Biol. 2002 Jul;3(7):531-7
12094219
Cell. 2007 Aug 24;130(4):601-10
17719539
Cell. 1992 Aug 21;70(4):523-6
1505019
Cancer Cell. 2005 Jan;7(1):17-23
15652746
Exp Biol Med (Maywood). 2005 Jul;230(7):464-71
15985621
Cancer Res. 2000 Dec 15;60(24):6886-9
11156386
Prostate. 2009 Sep 15;69(13):1398-403
19479898
BMC Biotechnol. 2004 Jul 13;4:14
15251044
PLoS One. 2009 Oct 02;4(10):e7314
19798417
Oncogene. 2007 Sep 20;26(43):6297-306
17452984
Cancer. 1992 Sep 15;70(6 Suppl):1727-31
1516027
Nature. 2008 Oct 9;455(7214):770-4
18806779
Science. 1989 Apr 14;244(4901):217-21
2649981
Neoplasia. 2009 Mar;11(3):260-8, 3p following 268
19242607
Oncogene. 2000 Dec 11;19(53):6159-75
11156530
Gastroenterology. 2009 May;136(5):1680-8.e7
19208363
Nat Cell Biol. 2007 Sep;9(9):1016-24
17762893
N Engl J Med. 2008 Oct 23;359(17):1757-65
18946061
PLoS One. 2009;4(1):e4240
19156225
Methods. 2003 Jul;30(3):256-68
12798140
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3787-92
17360431
Nat Rev Mol Cell Biol. 2006 Feb;7(2):131-42
16493418
Nat Med. 2004 Aug;10(8):789-99
15286780
PLoS One. 2008 Jun 11;3(6):e2336
18545657
Science. 1993 Apr 2;260(5104):85-8
8465203
EMBO J. 2001 Nov 1;20(21):5929-39
11689433
Blood. 2005 Feb 15;105(4):1734-41
15494427
Nature. 1995 Dec 21-28;378(6559):789-92
8524414
Semin Cancer Biol. 2005 Oct;15(5):405-12
16055341
Genomics. 2008 May;91(5):451-7
18329245
Cancer Res. 1998 Oct 15;58(20):4537-42
9788596
Neoplasia. 2009 Jul;11(7):683-91
19568413
Nat Clin Pract Gastroenterol Hepatol. 2006 May;3(5):267-74
16673006
Curr Opin Cell Biol. 2005 Oct;17(5):548-58
16098727
Science. 2008 Mar 7;319(5868):1352-5
18323444
Neoplasia. 2009 Oct;11(10):1084-92
19794967
Cancer. 1997 Feb 1;79(3):462-7
9028355
Methods. 2001 Dec;25(4):402-8
11846609
Cell. 2000 Jan 7;100(1):57-70
10647931
Science. 1994 Oct 7;266(5182):66-71
7545954
Clin Cancer Res. 2009 Oct 15;15(20):6314-20
19808869