Figure 5
ICR191-induced frameshift mutations identified using inhibition of NMD and microarray analysis in the p53 and other genes in the transformed MCF-10A cells. A. Top: P53 gene-coding sequence with the frameshift mutations sites in different MCF-10A cultures underlined. Bottom: Sequence analysis shows heterozygous insertions of one cytosine within different short poly-C repeats in the coding DNA of p53 gene in MCF10Aγ and MCF10Aβ cells. In the MCF10Aβ cells, both mutations can be seen in the sequencing graph of a single PCR amplified fragment. The insertion of one C in the poly-C repeat located at 460–464 nucleotides of the fragment results in the appearance of double peaks on the sequencing graph. The second insertion in the poly-C repeat at nucleotides 512–517 of the second allele results in the disappearance of double peaks at the end of the sequencing graph. B. Western blotting analysis of p53 protein expression and accumulation in response to ICR191 exposure in different MCF-10A derivative cell-lines. Only those cell-lines with the loss of p53 expression could form large, progressively growing tumors in nude mice. C. Top: Sequencing graphs show the homozygous insertion of one C into the coding poly-C of the SMTN gene. (C)4 (mutant) and (C)3 (wild type) repeats can be seen on the sequences of SMTN gene from the MCF-10A and MCF10Aβ1 cells, respectively. Bottom: Sequencing graph shows the heterozygous insertion of one C into the coding poly-C repeat, which results in the appearance of double peaks at the end of the sequenced fragment of a RASSF6 gene in the MCF10Aα4 cells.