Abstract
BackgroundWidely 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.ResultsTo 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.ConclusionIdentifying 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.
Highlights
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
Transformation of MCF-10A cells using ICR191 treatment Mutagenesis and selection was initiated in five parallel tissue-culture plates seeded with 1 million MCF-10A cells in each plate
TP53 gene mutations identified in the transformed MCF10AαL, MCF10AβL and MCF10AγL cells derived from tumors growing in nude mice were identical to the mutations identified in the corresponding cultures derived after in vitro selection either for resistance to hypoxia or to reduced growth-factor supply, or after selection for growth in soft agar
Summary
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. It has been proposed that, even from its earliest stages, cancer development is associated with DNA replication stress, which leads to genomic instability and selective pressure for p53 inactivation [1] This hypothesis was based on the fact of frequent inactivation of the p53 pathway in human tumors and on a demonstration that human early pre-cancerous lesions show signs of DNA damage-response activation [1,2,3,4]. Similar to normal human breast epithelial cells, at confluence the MCF-10A cells form dome structures in tissue culture plates and produce mammary spheres in 3D collagen culture All of these characteristics make MCF-10A cells a model of choice for breast tumor progression studies
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