Real-time in vivo imaging of p16Ink4a gene expression: a new approach to study senescence stress signaling in living animals

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16INK4a. To date, much of our current knowledge of how human p16INK4a gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16INK4a gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16INK4a gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16INK4a gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16INK4a gene expression in vivo and its potential for tumor suppression

Generation of Aberrant Transcripts of and Free DNA Ends in Zebrafish no tail Gene

Abstract The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. In a wild-type zebrafish population, we occasionally observed adult zebrafish with a narrow or no tailfin. This led us to examine the hypothesis that the activity of ntl was somehow genetically unstable. Here we present two findings regarding the gene. First, approximately 3% of ntl transcripts were aberrant; most of them carried deletions at various positions. Second, free, DNA double-stranded ends (DSEs) were formed at an AT dinucleotide repeat in ntl. DSEs were also generated in another zebrafish gene, noggin2 (nog2). DSEs in ntl and nog2 had common characteristics, which suggested that the AT repeats in these genes elicited DSEs by blocking progression of the replication

Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity

Generally, IgM antibodies (Abs) produced in a primary immune response show lower affinity for an inducing antigen (Ag) compared with the corresponding IgG Abs that are major switched isotypes formed in the secondary response. An IgM molecule is a pentamer with 10 Ag-binding sites that will contribute to an increase of avidity for an Ag. To estimate the contribution of the pentameric structure to the avidity of an IgM Ab, we generated IgM and IgG1 monoclonal Abs (mAbs) with identical V regions that are specific for 4-hydroxy-3-nitrophenylacetyl (NP) by in vitro class switching of B cells followed by the cell fusion with a mouse myeloma cell line. Compared with an anti-NP IgG1 mAb, the corresponding IgM mAb showed much higher avidity for NP-conjugated bovine serum albumin, which was drastically reduced after being dissociated into monomers

The p16INK4a-RB pathway : molecular link between cellular senescence and tumor suppression

The p16INK4a tumor suppressor protein functions as an inhibitor ofCDK4andCDK6, the D-type cyclin-dependent kinases that initiate the phosphorylation of the retinoblastoma tumor suppressor protein, RB. Thus, p16INK4a has the capacity to arrest cells in the G1-phase of the cell cycle and its probable physiological role is in the implementation of irreversible growth arrest termed cellular senescence. Cellular senescence is a state of permanent growth arrest that can be induced by a variety of stresses such as DNA-damage and aberrant mitogenic signaling in human primary cells. In contrast to normal cells, the function of the p16INK4a gene or its downstream mediators is frequently deregulated in many types of human cancers, illustrating the importance of cellular senescence in tumor suppression. Here we discuss the molecular mechanisms that direct cellular senescence and reveal its potential for tumor suppression

Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest

Bmi-1 regulates mucin levels and mucin O-glycosylation in the submandibular gland of mice.

Mucins, the major components of salivary mucus, are large glycoproteins abundantly modified with O-glycans. Mucins present on the surface of oral tissues contribute greatly to the maintenance of oral hygiene by selectively adhering to the surfaces of microbes via mucin O-glycans. However, due to the complex physicochemical properties of mucins, there have been relatively few detailed analyses of the mechanisms controlling the expression of mucin genes and the glycosyltransferase genes involved in glycosylation. Analysis performed using supported molecular matrix electrophoresis, a methodology developed for mucin analysis, and knockout mice without the polycomb group protein Bmi-1 revealed that Bmi-1 regulates mucin levels in the submandibular gland by suppressing the expression of the mucin Smgc gene, and that Bmi-1 also regulates mucin O-glycosylation via suppression of the glycosyltransferase Gcnt3 gene in the submandibular gland

Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity

Generally, IgM antibodies (Abs) produced in a primary immune response show lower affinity for an inducing antigen (Ag) compared with the corresponding IgG Abs that are major switched isotypes formed in the secondary response. An IgM molecule is a pentamer with 10 Ag-binding sites that will contribute to an increase of avidity for an Ag. To estimate the contribution of the pentameric structure to the avidity of an IgM Ab, we generated IgM and IgG1 monoclonal Abs (mAbs) with identical V regions that are specific for 4-hydroxy-3-nitrophenylacetyl (NP) by in vitro class switching of B cells followed by the cell fusion with a mouse myeloma cell line. Compared with an anti-NP IgG1 mAb, the corresponding IgM mAb showed much higher avidity for NP-conjugated bovine serum albumin, which was drastically reduced after being dissociated into monomers

Expression of Ror2 Associated with Fibrosis of the Submandibular Gland

The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person’s quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and Ror2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, Ror2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1, TNF-α, IL-1β, and MMP-2. Increased immunostaining signal of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG