Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: A model for identifying candidate breast-tumor suppressors

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA)

Mismatch Repair (MMR) is closely linked to DNA replication; however, other than the role of the replicative sliding clamp (PCNA) in various MMR functions, the linkage between DNA replication and MMR has been difficult to investigate. Here we use an in vitro DNA replication system based on simian virus 40, to investigate MMR recruitment to replicating DNA. Both DNA replication and MMR proteins are recruited to replicating DNA in an origin-dependent fashion. Primer synthesis is required for recruitment of both PCNA and MMR proteins, but not for recruitment of the single-stranded DNA-binding protein (RPA). Blocking PCNA recruitment to replicating DNA with a p21-based polypeptide blocks PCNA and MMR, but not RPA recruitment. Once PCNA and subsequent proteins required for replication are loaded onto DNA, addition of p21 leaves PCNA on the replicating DNA, but actively displaces MMR proteins. These findings indicate that the MMR machinery is recruited to replicating DNA through its interaction with PCNA, and suggests that this occurs via binding of the MMR proteins to the multi-protein interaction sites on PCNA. These studies demonstrate the utility of this system for further investigation of the role of DNA replication in MMR

Origin of Life: The Point of No Return

Origin of life research is one of the greatest scientific frontiers of mankind. Many hypotheses have been proposed to explain how life began. Although different hypotheses emphasize different initial phenomena, all of them agree around one important concept: at some point, along with the chain of events toward life, Darwinian evolution emerged. There is no consensus, however, how this occurred. Frequently, the mechanism leading to Darwinian evolution is not addressed and it is assumed that this problem could be solved later, with experimental proof of the hypothesis. Here, the author first defines the minimum components required for Darwinian evolution and then from this standpoint, analyzes some of the hypotheses for the origin of life. Distinctive features of Darwinian evolution and life rooted in the interaction between information and its corresponding structure/function are then reviewed. Due to the obligatory dependency of the information and structure subject to Darwinian evolution, these components must be locked in their origin. One of the most distinctive characteristics of Darwinian evolution in comparison with all other processes is the establishment of a fundamentally new level of matter capable of evolving and adapting. Therefore, the initiation of Darwinian evolution is the &ldquo;point of no return&rdquo; after which life begins. In summary: a definition and a mechanism for Darwinian evolution are provided together with a critical analysis of some of the hypotheses for the origin of life

Possible Emergence of Sequence Specific RNA Aminoacylation via Peptide Intermediary to Initiate Darwinian Evolution and Code through Origin of Life

One of the most intriguing questions in biological science is how life originated on Earth. A large number of hypotheses have been proposed to explain it, each putting an emphasis on different events leading to functional translation and self-sustained system. Here, we propose a set of interactions that could have taken place in the prebiotic environment. According to our hypothesis, hybridization-induced proximity of short aminoacylated RNAs led to the synthesis of peptides of random sequence. We postulate that among these emerged a type of peptide(s) capable of stimulating the interaction between specific RNAs and specific amino acids, which we call &ldquo;bridge peptide&rdquo; (BP). We conclude that translation should have emerged at the same time when the standard genetic code begun to evolve due to the stabilizing effect on RNA-peptide complexes with the help of BPs. Ribosomes, ribozymes, and the enzyme-directed RNA replication could co-evolve within the same period, as logical outcome of RNA-peptide world without the need of RNA only self-sustained step

Possible Emergence of Sequence Specific RNA Aminoacylation via Peptide Intermediary to Initiate Darwinian Evolution and Code through Origin of Life

One of the most intriguing questions in biological science is how life originated on Earth. A large number of hypotheses have been proposed to explain it, each putting an emphasis on different events leading to functional translation and self-sustained system. Here, we propose a set of interactions that could have taken place in the prebiotic environment. According to our hypothesis, hybridization-induced proximity of short aminoacylated RNAs led to the synthesis of peptides of random sequence. We postulate that among these emerged a type of peptide(s) capable of stimulating the interaction between specific RNAs and specific amino acids, which we call &ldquo;bridge peptide&rdquo; (BP). We conclude that translation should have emerged at the same time when the standard genetic code begun to evolve due to the stabilizing effect on RNA-peptide complexes with the help of BPs. Ribosomes, ribozymes, and the enzyme-directed RNA replication could co-evolve within the same period, as logical outcome of RNA-peptide world without the need of RNA only self-sustained step

Par-3 partitioning defective 3 homolog <it>(C. elegans) </it>and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells

Abstract Background Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI. Methods To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation. Results Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture. Conclusion The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.</p

Proliferating Cell Nuclear Antigen (PCNA)

through interaction wit

Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR)

Nascent strand capture and release (NSCR) is a method for isolation of short nascent strands to identify origins of DNA replication.  The protocol provided involves isolation of total DNA, denaturation, size fractionation on a sucrose gradient, 5’-biotinylation of the appropriate size nucleic acids, binding to a streptavidin coated column or magnetic beads, intensive washing, and specific release only the RNA containing chimeric nascent strand DNA using RNaseI. The method has been applied to mammalian cells derived from proliferative tissues and cell culture but could be used for any system where DNA replication is primed by a small RNA resulting in chimeric RNA-DNA molecules