Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.
BACKGROUND
Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.


RESULTS
To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.


CONCLUSIONS
Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.
Zientek-Targosz
Helena
H
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. zient_targ@yahoo.com
Kunnev
Dimiter
D
Hawthorn
Lesleyann
L
Venkov
Mikhail
M
Matsui
Sei-Ichi
S
Cheney
Richard T
RT
Ionov
Yuri
Y
D000585:Aminacrine / Q000031:analogs & derivatives* / Q000633:toxicity; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D043171:Chromosomal Instability / Q000187:drug effects; D005260:Female; D016368:Frameshift Mutation / Q000187:drug effects / Q000235:genetics*; D016147:Genes, Tumor Suppressor; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D016296:Mutagenesis / Q000187:drug effects*; D009368:Neoplasm Transplantation; D009588:Nitrogen Mustard Compounds / Q000633:toxicity*; D009693:Nucleic Acid Hybridization; D020871:RNA Stability / Q000187:drug effects; D032681:Spectral Karyotyping
D009588:Nitrogen Mustard Compounds; D000585:Aminacrine; C004294:acridine half-mustard