John D. Roberts

Inter-α-trypsin Inhibitor Promotes Bronchial Epithelial Repair after Injury through Vitronectin Binding

Pulmonary epithelial injury is central to the pathogenesis of many lung diseases, such as asthma, pulmonary fibrosis, and the acute respiratory distress syndrome. Regulated epithelial repair is crucial for lung homeostasis and prevents scar formation and inflammation that accompany dysregulated healing. The extracellular matrix (ECM) plays an important role in epithelial repair after injury. Vitronectin is a major ECM component that promotes epithelial repair. However, the factors that modify cell-vitronectin interactions after injury and help promote epithelial repair are not well studied. Inter-α-trypsin inhibitor (IaI) is an abundant serum protein. IaI heavy chains contain von Willebrand A domains that can bind the arginine-glycine-aspartate domain of vitronectin. We therefore hypothesized that IaI can bind vitronectin and promote vitronectin-induced epithelial repair after injury. We show that IaI binds vitronectin at the arginine-glycine-aspartate site, thereby promoting epithelial adhesion and migration in vitro. Furthermore, we show that IaI-deficient mice have a dysregulated response to epithelial injury in vivo, consisting of decreased proliferation and epithelial metaplasia. We conclude that IaI interacts not only with hyaluronan, as previously reported, but also other ECM components like vitronectin and is an important regulator of cellular repair after injury.

Plasminogen activator inhibitor 1 RNAi suppresses gastric cancer metastasis in vivo

Cancer metastasis remains the primary cause of pain, suffering, and death in cancer patients, and even the most current therapeutic strategies have not been highly successful in preventing or inhibiting metastasis. In most patients with scirrhous gastric cancer (one of the most aggressive of diffuse‐type gastric cancer), recurrence occurs even after potentially curative resection, most frequently in the form of peritoneal metastasis. Given that the occurrence of diffuse‐type gastric cancers has been increasing, the development of new strategies to combat metastasis of this disease is critically important. Plasminogen activator inhibitor‐1 (PAI‐1) is a critical factor in cancer progression; thus, PAI‐1 RNAi may be an effective therapy against cancer metastasis. In the present study, we used an RNAi technique to reduce PAI‐1 expression in an in vivo model system for gastric cancer metastasis. Ex vivo plasmid transfection and adenovirus infection were tested as mechanisms to incorporate specific PAI‐1 RNAi vectors into human gastric carcinoma cells. Both approaches significantly decreased peritoneal tumor growth and the formation of bloody ascites in the mouse model, suggesting that this approach may provide a new, effective strategy for inhibiting cancer metastasis. (Cancer Sci 2012; 103: 228–232)

15-Lipoxygenase-1 activates tumor suppressor p53 independent of enzymatic activity†

15‐LOX‐1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15‐LOX‐1 overexpression in HCT‐116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15‐LOX‐1 protein or its metabolites are responsible for phosphorylation of p53 in HCT‐116 cells, we used HCT‐116 cells that expressed a mutant 15‐LOX‐1. The mutant 15‐LOX‐1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT‐116 cells transiently transfected with either native or mutant 15‐LOX‐1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15‐LOX‐1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL‐4 increased the expression of 15‐LOX‐1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild‐type cells expressing physiological 15‐LOX‐1. Immunoprecipitation experiments revealed that 15‐LOX‐1 interacts with, and binds to, DNA‐dependent protein kinase (DNA‐PK). The binding of 15‐LOX‐1 to DNA‐PK caused an approximate 3.0‐fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA‐PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15‐LOX and DNA‐PK in the cells. We propose that the 15‐LOX‐1 protein binds to DNA‐PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells. Published 2008 Wiley‐Liss, Inc.

7 Fidelity of DNA Replication

The six billion nucleotides of the diploid human genome are replicated in only a few hours while generating so few errors that the spontaneous mutation rate may be less than 1 mutation per genome per cell division (Loeb 1991). This incredible accuracy results from three major error-avoidance processes: the high selectivity of DNA polymerases, exo-nucleolytic proofreading, and postreplication mismatch repair. In this chapter, we review our current understanding of the first two of these processes. Readers interested in eukaryotic mismatch repair are referred to a recent review (Modrich 1994). We begin by describing the steps in the polymerization reaction cycle that discriminate against base substitution errors, then review studies of the substitution fidelity of the five classes of eukaryotic DNA polymerases. We then consider several ways to make errors by template-primer slippage and review what is known about eukaryotic DNA polymerase frameshift error rates. Finally, we present information on the fidelity with which the multiprotein replication machinery replicates undamaged DNA and DNA containing adducts of known carcinogens. BASE SUBSTITUTION FIDELITY Discrimination Steps in a Polymerization Cycle The error discrimination steps that operate during incorporation of a single nucleotide have been worked out primarily with prokaryotic and viral DNA polymerases. The steps (Fig. 1) include binding of the polymerase to the DNA, formation of a ternary complex with the incoming deoxyribonucleoside triphosphate (dNTP), a conformational change in this complex to position the substrates for phosphodiester bond formation, the chemical reaction step to form the bond, a second conformational change following the...

PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma

Background:PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined.Methods:The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85).Results:Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells.Conclusion:PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.

Arachidonic acid stimulates formation of a novel complex containing nucleolin and RhoA

ROCK physically interacts with Nucleolin and RHOA by pull down (View interaction)

A novel role for the T-box transcription factor Tbx1 as a negative regulator of tumor cell growth in mice

The T‐box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down‐regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1‐transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage‐independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth.

Linoleic acid enhances angiogenesis through suppression of angiostatin induced by plasminogen activator inhibitor 1

Background:The intake of dietary fatty acids is highly correlated with the risk of various cancers. Linoleic acid (LA) is the most abundant polyunsaturated fat in the western diet, but the mechanism(s) by fatty acids such as LA modulate cancer cells is unclear. In this study, we examined the role of LA in various steps in gastric cancer progression.Methods:The difference in gene expression between LA-treated and untreated OCUM-2MD3 gastric carcinoma cells was examined by mRNA differential display. The involvement of candidate genes was examined by oligo- and plasmid-mediated RNA interference. Biological functions of several of these genes were examined using in vitro assays for invasion, angiogenesis, apoptosis, cell viability, and matrix digestion. Angiogenesis in vivo was measured by CD-31 immunohistochemistry and microvessel density scoring.Results:LA enhanced the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression, which are controlled by PAI-1 mRNA-binding protein. LA-stimulated invasion depended on PAI-1. LA also enhanced angiogenesis by suppression of angiostatin, also through PAI-1. LA did not alter cell growth in culture, but increased dietary LA-enhanced tumour growth in an animal model.Conclusion:Our findings suggest that dietary LA impacts multiple steps in cancer invasion and angiogenesis, and that reducing LA in the diet may help slow cancer progression.

An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression

BackgroundThe gut microbiome, a key constituent of the colonic environment, has been implicated as an important modulator of human health. The eukaryotic epigenome is postulated to respond to environmental stimuli through alterations in chromatin features and, ultimately, gene expression. How the host mediates epigenomic responses to gut microbiota is an emerging area of interest. Here, we profile the gut microbiome and chromatin characteristics in colon epithelium from mice fed either an obesogenic or control diet, followed by an analysis of the resultant changes in gene expression.ResultsThe obesogenic diet shapes the microbiome prior to the development of obesity, leading to altered bacterial metabolite production which predisposes the host to obesity. This microbiota–diet interaction leads to changes in histone modification at active enhancers that are enriched for binding sites for signal responsive transcription factors. These alterations of histone methylation and acetylation are associated with signaling pathways integral to the development of colon cancer. The transplantation of obesogenic diet-conditioned microbiota into germ free mice, combined with an obesogenic diet, recapitulates the features of the long-term diet regimen. The diet/microbiome-dependent changes are reflected in both the composition of the recipient animals’ microbiome as well as in the set of transcription factor motifs identified at diet-influenced enhancers.ConclusionsThese findings suggest that the gut microbiome, under specific dietary exposures, stimulates a reprogramming of the enhancer landscape in the colon, with downstream effects on transcription factors. These chromatin changes may be associated with those seen during colon cancer development.

Base-Resolution Analysis of DNA Methylation Patterns Downstream of Dnmt3a in Mouse Naïve B Cells

The DNA methyltransferase, Dnmt3a, is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a+/+ and Dnmt3a−/− naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a+/+ and Dnmt3a−/− naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a−/− naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development.