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

Transformation of human bronchial epithelial cells alters responsiveness to inflammatory cytokines

BACKGROUND: Inflammation is commonly associated with lung tumors. Since inflammatory mediators, including members of the interleukin-6 (IL-6) cytokine family, suppress proliferation of normal epithelial cells, we hypothesized that epithelial cells must develop mechanisms to evade this inhibition during the tumorigenesis. This study compared the cytokine responses of normal epithelial cells to that of premalignant cells. METHODS: Short-term primary cultures of epithelial cells were established from bronchial brushings. Paired sets of brushings were obtained from areas of normal bronchial epithelium and from areas of metaplastic or dysplastic epithelium, or areas of frank endobronchial carcinoma. In 43 paired cultures, the signalling through the signal transducer and activator of transcription (STAT) and extracellular regulated kinase (ERK) pathways and growth regulation by IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), interferon-γ (IFNγ) or epidermal growth factor (EGF) were determined. Inducible expression and function of the leukemia inhibitory factor receptor was assessed by treatment with the histone deacetylase inhibitor depsipeptide. RESULTS: Normal epithelial cells respond strongly to OSM, IFNγ and EGF, and respond moderately to IL-6, and do not exhibit a detectable response to LIF. In preneoplastic cells, the aberrant signaling that was detected most frequently was an elevated activation of ERK, a reduced or increased IL-6 and EGF response, and an increased LIF response. Some of these changes in preneoplastic cell signaling approach those observed in established lung cancer cell lines. Epigenetic control of LIF receptor expression by histone acetylation can account for the gain of LIF responsiveness. OSM and macrophage-derived cytokines suppressed proliferation of normal epithelial cells, but reduced inhibition or even stimulated proliferation was noted for preneoplastic cells. These alterations likely contribute to the supporting effects that inflammation has on lung tumor progression. CONCLUSION: This study indicates that during the earliest stage of premalignant transformation, a modified response to cytokines and EGF is evident. Some of the altered cytokine responses in primary premalignant cells are comparable to those seen in established lung cancer cell lines

PRTR カガク ブッシツ ノ カクシュ カクナイ ジュヨウタイ ニタイスル ケツゴウセイ

『大阪大学大学院工学研究科環境・エネルギー工学専攻生物圏環境工学領域 研究活動報告』, (2006.4.1～2007.3.31), pp.73～78, 大阪大学大学院工学研究科環境・エネルギー工学専攻環境資源・材料学講座生物圏環境工学領域, 2007.6 に掲

Targeted Deletion of p73 in Mice Reveals Its Role in T Cell Development and Lymphomagenesis

Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia

Mild folate deficiency induces genetic and epigenetic instability and phenotype changes in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Folate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction.</p> <p>Results</p> <p>We studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion.</p> <p>Conclusions</p> <p>This study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 μM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate <it>in vivo</it>, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.</p