Knock-in of mutant K-ras in nontumorigenic human epithelial cells as a new model for studying K-ras mediated transformation.
The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT-immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream molecules extracellular signal-regulated kinase and AKT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fraction of human cancer cells.
Konishi
Hiroyuki
H
The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Karakas
Bedri
B
Abukhdeir
Abde M
AM
Lauring
Josh
J
Gustin
John P
JP
Garay
Joseph P
JP
Konishi
Yuko
Y
Gallmeier
Eike
E
Bachman
Kurtis E
KE
Park
Ben Ho
BH
D000483:Alleles; D001940:Breast / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019098:Telomerase / Q000235:genetics; D019076:Transgenes; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases; C509186:TERT protein, human; D019098:Telomerase; D018631:ras Proteins