Detection of K-ras mutations in azoxymethane-induced aberrant crypt foci in mice using LNA-mediated real-time PCR clamping and mutant-specific probes.
Azoxymethane, a rodent colon-specific carcinogen, induce DNA damage, and causes proto-oncogene K-ras point mutations and subsequent tumor formation if DNA damage is not repaired or removed. The present study was designed to detect and characterize K-ras mutations in azoxymethane (AOM)-induced aberrant crypt foci (ACF) in mice, and determine whether dietary supplementation of selenium influences K-ras mutations frequency in ACF using a new PCR technique of locked nucleic acid-mediated real-time PCR clamping combined with mutant-specific probes. K-ras mutations were identified in 33% of AOM-induced ACF. In addition to G to A transition mutation, specific G to T transversion mutation was also identified for the first time in mouse ACF. Furthermore, selenium intake was associated with reduced ACF formation and reduced K-ras mutations rate, respectively, from 112 and 37% in mice fed control diet to 65 and 14% in mice fed selenium-containing diet (p < 0.05). This is the first report of the use of one-step LNA-mediated real-time PCR clamping to detect K-ras mutations in AOM-induced colon cancer model. It is highly sensitive and can be applied to the detection of early genetic alterations in carcinogen-based animal models.
Hu
Ying
Y
Department of Medicine, Flinders Centre for Cancer Prevention and Control, Flinders University of South Australia, Adelaide, Australia. ying.hu@flinders.edu.au
Le Leu
Richard K
RK
Young
Graeme P
GP
D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001397:Azoxymethane; D002273:Carcinogens; D003106:Colon / Q000187:drug effects; D003110:Colonic Neoplasms / Q000139:chemically induced* / Q000235:genetics*; D015342:DNA Probes; D004032:Diet; D011905:Genes, ras / Q000187:drug effects*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009153:Mutagens; D016133:Polymerase Chain Reaction / Q000379:methods*; D011230:Precancerous Conditions / Q000139:chemically induced* / Q000235:genetics / Q000517:prevention & control; D012643:Selenium / Q000494:pharmacology*; D012680:Sensitivity and Specificity
D016588:Anticarcinogenic Agents; D002273:Carcinogens; D015342:DNA Probes; D009153:Mutagens; D012643:Selenium; D001397:Azoxymethane