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Abstract Background Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. Results We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. Conclusions Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved β-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro , but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue., Competing Interests: Competing interests F.O. is a shareholder of NanoTag Biotechnologies GmbH. All other authors declare no competing interests.

Our previous work demonstrated that vitamin D (VitD) reduces experimental autoimmune encephalomyelitis (EAE) disease severity in wild-type (WT) but not in T cell-specific glucocorticoid (GC) receptor (GR)-deficient (GR lck ) mice. This study aimed to investigate the interplay between the GR- and VitD receptor (VDR) signaling. In vivo, we confirmed the involvement of the GR in the VitD-induced effects in EAE using WT and GR lck mice. Furthermore, we observed that VitD-enhanced T cell apoptosis and T regulatory cell differentiation are diminished in vitro in CD3+ T cells of GR lck but not WT mice. Mechanistically, VitD does not appear to signal directly via the GR, as it does not bind to the GR, does not induce its nuclear translocation, and does not modulate the expression of two GR-induced genes. However, we observed that VitD enhances VDR protein expression in CD3+ T cells from WT but not GR lck mice in vitro, that the GR and the VDR spatially co-localize after VitD treatment, and that VitD does not modulate the expression of two VDR-induced genes in the absence of the GR. Our data suggest that a functional GR, specifically in T cells, is required for the VDR to signal appropriately to mediate the therapeutic effects of VitD.

The formation of Ca 2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction—NAADP to NAADPH—is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca 2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca 2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2 −/− but not in Duox1 −/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca 2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

NAADP-evoked Ca 2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca 2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca 2+ microdomains and delayed the onset and decreased the amplitude of global Ca 2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca 2+ release from the endoplasmic reticulum through RYR., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein β-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of β-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, β-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of β-synuclein in provoking T-cell-mediated pathology of the central nervous system.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca 2+ microdomains. Here, we identified a Ca 2+ entry component involved in Ca 2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca 2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca 2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca 2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca 2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca 2+ release through RYR1, in coordination with Ca 2+ entry through ORAI1 and STIM, rapidly increases the number of Ca 2+ microdomains, thereby initiating spread of Ca 2+ signals deeper into the cytoplasm to promote full T cell activation., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Antigenic activation is a central process in T-cell biology essential for efficient protection of the host from infections and tumors by the adaptive immune system. Furthermore, this process is of paramount importance for the initiation of autoimmunity. Many insights into the mechanisms of T-cell activation have been gained from real-time studies. The development of 2-photon microscopy transferred the focus of T cell activation to in vivo research and the analysis of the highly dynamic and complex T-cell response in the physiologic context of an animal model. In the last 15 years, real-time analysis of T-cell activation has progressed from a descriptive characterization of T-cell locomotion and visualization of T-cell contacts with putative antigen-presenting cells in ex vivo explants toward true intravital imaging using more functionally informative indicators of TCR-driven signaling to spot and quantify productive T-cell-APC interactions in situ. In this review we will briefly summarize and discuss current approaches to the real-time analysis of T-cell activation in vivo and their impact on our understanding of T cell function under homeostatic and pathological conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Laquinimod is currently being tested as a therapeutic drug in multiple sclerosis. However, its exact mechanism of action is still under investigation. Tracking of fluorescently-tagged encephalitogenic T cells during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, revealed that laquinimod significantly reduces the invasion of pathogenic effector T cells into the CNS tissue. T-cell activation, differentiation and amplification within secondary lymphoid organs after immunization with myelin antigen, their migratory capacity and re-activation within the nervous tissue were either only mildly affected or remained unchanged. Instead, laquinimod directly impacted the functionality of the CNS vasculature. The expression of tight junction proteins p120 and ZO-1 in human brain endothelial cells was up-regulated upon laquinimod treatment, resulting in a significant increase in the transendothelial electrical resistance of confluent monolayers of brain endothelial cells. Similarly, expression of the adhesion molecule activated leukocyte cell adhesion molecule (ALCAM) and inflammatory chemokines CCL2 and IP-10 was suppressed, leading to a significant reduction in the migration of memory T H 1 and T H 17 lymphocytes across the blood brain barrier (BBB). Our data indicate that laquinimod exerts its therapeutic effects by tightening the BBB and limiting parenchymal invasion of effector T cells, thereby reducing CNS damage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The p53-inducible cell cycle regulator 14-3-3σ exhibits tumor suppressive functions and is highly expressed in differentiating layers of the epidermis and hair follicles. 14-3-3σ/SFN/stratifin is frequently silenced in human epithelial cancers, and experimental down-regulation of 14-3-3σ expression immortalizes primary human keratinocytes. In the repeated-epilation (ER) mouse model, a heterozygous nonsense mutation of 14-3-3σ causes repeated hair-loss, hyper-proliferative epidermis, and spontaneous development of papillomas and squamous cell carcinomas in aging mice. Therefore, loss of 14-3-3σ function might contribute to epithelial tumor development. Here, we generated mice with loxP sites surrounding the single 14-3-3σ exon which allowed Cre-mediated deletion of the gene. 14-3-3σ-deficient mice are viable, but demonstrate a permanently disheveled fur. However, histological analyses of the skin did not reveal obvious defects in the hair follicles or the epidermis. Deletion of 14-3-3σ did not enhance spontaneous epidermal tumor development, whereas it increased the frequency and size of DMBA/TPA-induced papillomas. In conclusion, 14-3-3σ is dispensable for normal epidermal homeostasis but critical for suppression of chemically-induced skin carcinogenesis. In addition, these results suggest that the ER mutation of 14-3-3σ is not equivalent to loss of 14-3-3σ, but may represent a gain-of-function variant, which does not reflect the organismal function of wild-type 14-3-3σ., Competing Interests: The authors declare no conflict of interest.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals., (Copyright © 2015, American Association for the Advancement of Science.)

Members of the miR-34 family are induced by the tumor suppressor p53 and are known to inhibit epithelial-to-mesenchymal transition (EMT) and therefore presumably suppress the early phases of metastasis. Here, we determined that exposure of human colorectal cancer (CRC) cells to the cytokine IL-6 activates the oncogenic STAT3 transcription factor, which directly represses the MIR34A gene via a conserved STAT3-binding site in the first intron. Repression of MIR34A was required for IL-6-induced EMT and invasion. Furthermore, we identified the IL-6 receptor (IL-6R), which mediates IL-6-dependent STAT3 activation, as a conserved, direct miR-34a target. The resulting IL-6R/STAT3/miR-34a feedback loop was present in primary colorectal tumors as well as CRC, breast, and prostate cancer cell lines and associated with a mesenchymal phenotype. An active IL-6R/STAT3/miR-34a loop was necessary for EMT, invasion, and metastasis of CRC cell lines and was associated with nodal and distant metastasis in CRC patient samples. p53 activation in CRC cells interfered with IL-6-induced invasion and migration via miR-34a-dependent downregulation of IL6R expression. In Mir34a-deficient mice, colitis-associated intestinal tumors displayed upregulation of p-STAT3, IL-6R, and SNAIL and progressed to invasive carcinomas, which was not observed in WT animals. Collectively, our data indicate that p53-dependent expression of miR-34a suppresses tumor progression by inhibiting a IL-6R/STAT3/miR-34a feedback loop.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.

The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.

Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate/calcium signalling pathway is essential for the recruitment and the activation of autoaggressive effector T cells within their target organ. Interference with this signalling pathway suppresses the formation of autoimmune inflammatory lesions and thus might qualify as a novel strategy for the treatment of T cell mediated autoimmune diseases.

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G(1) cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (2/6; 33.3%) and pancreatic (3/19; 15.7%) carcinoma cell lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32 samples; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53 after DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

Purpose: To investigate a potential mode of noninvasive screening for colorectal cancer, we evaluated the hypermethylation of the secreted frizzled-related protein-1 gene promoter in human stool DNA., Methods: In stool samples from 36 patients with colorectal neoplasia (7 adenoma, 29 colorectal cancer) and 17 healthy control subjects, isolated DNA was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction with primers specific for methylated or unmethylated promoter sequences of the secreted frizzled-related protein-1 gene., Results: Hypermethylation of the secreted frizzled-related protein-1 promoter was present in the stool DNA of patients with adenoma and colorectal cancer. A sensitivity of 89 percent and specificity of 86 percent were achieved in the detection of colorectal neoplasia. The difference in hypermethylation status of the secreted frizzled-related protein-1 promoter between the patients with colorectal neoplasia and the control group was statistically highly significant (P < 0.001). Adenoma and early tumor Stage I (International Union Against Cancer) displayed both unmethylated and methylated secreted frizzled-related protein-1 promoter sequences, whereas advanced tumor stages showed only methylated secreted frizzled-related protein-1 (P = 0.05)., Conclusions: The results indicate that this DNA stool test of hypermethylation of the secreted frizzled-related protein-1 promoter is a sensitive and specific method. It has the potential of a clinically useful test for the early detection of colorectal cancer.

In a genome-wide screen for microRNAs regulated by the transcription factor encoded by the p53 tumor suppressor gene we found that after p53-activation the abundance of thirty-four miRNAs was significantly increased, whereas sixteen miRNAs were suppressed. The induction of miR-34a was most pronounced among all differential regulations. Also expression of the primary miR-34a transcript was induced after p53 activation and by DNA damage in a p53-dependent manner. p53 occupied an evolutionarily conserved binding site proximal to the first non-coding exon of miR-34a. Ectopic miR-34a induced apoptosis and a cell cycle arrest in the G1-phase, thereby suppressing tumor cell proliferation. Other p53-induced miRNAs identified here may also have tumor suppressive potential as they are known to suppress the anti-apoptotic factor Bcl2 (miR-15a/16) and the oncogenes RAS and HMGA2 (let-7a). Our results for the first time directly integrate the regulation of miRNA expression into the transcriptional network regulated by p53. siRNAs corresponding to p53-induced miRNAs may have potential as cancer therapeutic agents as RNA interference based therapies are currently emerging.

Here we show that the human BubR1 and MAD2 genes, which encode inhibitors of the anaphase promoting complex (APC/C), are directly activated by the oncogenic transcription factor c-MYC via E-box sequences in their first introns. In colorectal cancer biopsies elevated expression of c-MYC correlated with increased MAD2 levels. Activation of a conditional c-MYC allele delayed progression through mitosis in pro-metaphase in a MAD2- and BubR1-dependent manner. A fraction of the daughter cells derived from extended mitotic events underwent synchronous apoptosis, which was in part mediated by BubR1. Furthermore, c-MYC activation resulted in CIN (chromosomal instability) in the diploid MIN (microsatellite instability) cell line DLD-1 and further enhanced CIN in the aneuploid CIN-line MCF7. Unexpectedly, c-MYC-induced CIN was independent of c-MYC-induced BubR1/MAD2 expression and mitotic delay. Therefore, c-MYC-induced CIN may be caused be alternative pathways. We observed that activation of c-MYC induced DNA double-strand breaks, as evidenced by formation of gamma-H2AX foci, which colocalized with foci of active DNA replication. Furthermore, c-MYC activation resulted in mitotic chromosomes exhibiting DNA damage. Therefore, oncogenic deregulation of c-MYC prevents repair of replication-stress induced DNA lesions in the G(2)-phase. We suggest that the c-MYC-mediated persistence of DNA lesions throughout mitosis leads to chromosomal missegregation and underlies c-MYC-induced CIN. The effects of deregulated c-MYC on progression through mitosis described here may have important implications for the origin of chromosomal instability in many tumor types and the sensitivity towards cancer therapeutic agents targeting DNA or the mitotic spindle.

The 14-3-3sigma gene is a direct target of the p53 tumor suppressor and its product inhibits cell cycle progression. Recently, a proteomic analysis revealed that 14-3-3sigma regulates additional cellular processes relevant to carcinogenesis, as migration and MAP-kinase signalling. The expression of 14-3-3sigma is down-regulated by CpG methylation in several types of human cancer, among them prostate, lung, breast and several types of skin cancer. The epigenetic inactivation of 14-3-3sigma occurs at an early stage of tumor development and may allow evasion from senescence and promote genomic instability. In the future the detection of CpG methylation of 14-3-3sigma may be used for diagnostic and prognostic purposes.

In many cases, silencing of gene expression by CpG methylation is causally involved in carcinogenesis. Furthermore, cancer-specific CpG methylation may serve as a tumor marker. In order to identify candidate genes for inactivation by CpG methylation in prostate cancer, the prostate cancer cell lines LNCaP, PC3, and Du-145 were treated with 5-aza-2' deoxycytidine and trichostatin A, which leads to reversion of epigenetic silencing. By microarray analysis of 18,400 individual transcripts, several hundred genes were found to be induced when compared with cells treated with trichostatin A. Fifty re-expressed genes were selected for further analysis based on their known function, which implied a possible involvement in tumor suppression. Twelve of these genes showed a significant degree of CpG methylation in their promoters. Six genes were silenced by CpG methylation in the majority of five analyzed prostate cancer cell lines, although they displayed robust mRNA expression in normal prostate epithelial cells obtained from four different donors. In primary prostate cancer samples derived from 41 patients, the frequencies of CpG methylation detected in the promoter regions of these genes were: GPX3, 93%; SFRP1, 83%; COX2, 78%; DKK3, 68%; GSTM1, 58%; and KIP2/p57, 56%. Ectopic expression of SFRP1 or DKK3 resulted in decreased proliferation. The expression of DKK3 was accompanied by attenuation of the mitogen-activated protein kinase pathway. The high frequency of CpG methylation detected in the promoters of the identified genes suggests a potential causal involvement in prostate cancer and may prove useful for diagnostic purposes.

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3sigma undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3sigma expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3sigma is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3sigma-associated proteins by a targeted proteomics approach implies that 14-3-3sigma regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3sigma expression in cancer cells.

The 14-3-3sigma inhibitor of cell cycle progression has been shown to be target of epigenetic deregulation in many forms of human cancers; however, its role in urological and gynecological cancers has not been studied. Here, we have analyzed the expression of 14-3-3sigma, wild-type p53 and mutated p53 in over 300 cases of the most common cancers occurring in the urological and gynecological tracts and its normal counterpart tissue by immunohistochemistry using the multiple tumor tissue microarrays. 14-3-3sigma expression was detected in normal epithelia from most organs with sporadic expression in renal tubules and absence in the testis. In contrast to normal tissue, 14-3-3sigma expression was lost in 40-60% of adenocarcinomas of the breast, ovary, endometrium and prostate. There was no association between 14-3-3sigma and wild-type/mutated p53 expression. By performing methylation-specific PCR, we showed a close association of 14-3-3sigma CpG island methylation and low protein expression levels of 14-3-3sigma. In addition, a direct link of 14-3-3sigma mRNA expression levels to CpG island methylation is demonstrated in two human cancer cell lines. Loss of 14-3-3sigma expression due to promoter hypermethylation may represent the most frequent molecular aberration in ovarian, endometrial and prostate adenocarcinomas.

In order to identify tumor suppressive genes silenced by CpG methylation in prostate carcinoma (PCa), we determined genome-wide expression changes after pharmacological reversal of CpG methylation-mediated transcriptional repression in three PCa cell lines using microarray analysis. Thereby, epigenetic silencing of the 14-3-3sigma gene was detected in the cell line LNCaP. 14-3-3sigma encodes a p53-regulated inhibitor of cell cycle progression. Laser microdissection was used to isolate different cell types present in diseased prostatic tissue. Subsequent methylation-specific PCR analysis showed CpG methylation of 14-3-3sigma in all 41 primary PCa samples analysed, which was accompanied by a decrease or loss of 14-3-3sigma protein expression. In contrast, normal prostate epithelial and benign prostate hyperplasia cells showed high levels of 14-3-3sigma expression. PCa-precursor lesions (prostatic intraepithelial neoplasia) also displayed decreased levels of 14-3-3sigma expression in luminal cells, which are known to contain shortened telomeres. RNA interference-mediated inactivation of 14-3-3sigma compromised a DNA damage-induced G(2)/M arrest in the PCa cell line PC3. The generality of CpG methylation and downregulation of 14-3-3sigma expression in PCa suggests that it significantly contributes to the formation of PCa, potentially by allowing the escape from a DNA damage-induced arrest elicited by telomere shortening.

The p53-regulated 14-3-3sigma gene encodes an inhibitor of cell cycle progression essential for senescence and clonal evolution of keratinocytes in vitro. Here we analysed the in vivo expression of 14-3-3sigma protein in several skin diseases, which are characterized by hyperproliferative keratinocytes. Unexpectedly, the 14-3-3sigma protein was expressed at high levels in psoriasis (11 of 11 patients), condylomata acuminata (11/11), actinic keratoses (11/11) and squamous cell carcinomas (SCC) (11/11). However, keratinocytes that had undergone transformation to basal cell carcinoma (BCC) showed partial (10 of 41; 24.4%) or complete (19 of 41; 46.3%) loss of 14-3-3sigma protein expression. BCC (5/5), SCC (6/6) and actinic keratoses (7/7) concomitantly expressed the p53-homolog p63 and 14-3-3sigma at high levels, ruling out potential inhibitory effects of p63 isoforms on 14-3-3sigma transcription as the basis for loss of 14-3-3sigma expression. Of 41 BCC samples isolated by laser-capture microdissection, 28 (68.3%) showed CpG-hypermethylation of the 14-3-3sigma promoter combined with reduced or absent 14-3-3sigma protein levels in 22 cases (78.6%). Since it has been reported that BCC retain wild-type p16(INK4A) and here BCC with CpG-methylation of 14-3-3sigma did not show CpG-methylation of p16(INK4A) (0/17), silencing of 14-3-3sigma may contribute to evasion of senescence in BCC. As experimental removal of 14-3-3sigma sensitizes to DNA damage, silencing of 14-3-3sigma may explain the high efficacy of radiation therapy in the treatment of BCC.

Using microarray analysis, we have detected downregulation of several components of the cGMP signaling pathway during replicative senescence of primary human diploid fibroblasts (HDFs). Therefore, the effect of pharmacological inhibition of cGMP synthesis was analyzed in HDFs. Treatment with 6-anilino-5,8-quinolinequinone (LY83583, referred to as LY hereafter), a previously described inhibitor of guanylate cyclase, induced cellular senescence. Microarray analysis revealed that LY treatment induced the Cdk inhibitor p21(WAF1/SDI/CIP1). In colorectal cancer cells, transcription of p21 was induced by LY in a p53-independent manner. Furthermore, p21, but not p53, was required for inhibition of proliferation by LY. The lack of p53 involvement suggests that LY does not induce DNA damage. Growth inhibition was also observed in malignant melanoma and breast cancer cell lines. Functional inactivation of the retinoblastoma tumor-suppressor protein, an effector of p21-mediated cell-cycle inhibition, converted LY-induced growth arrest to apoptosis. These results suggest that LY, or derivatives, may be useful therapeutic agents for the treatment of tumors.

Background: Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP., Results: We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell., Conclusions: Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved beta-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines.

The role of alpha 2-adrenergic and imidazoline receptors in the hemodynamic response to clonidine (manifested by reduced arterial pressure, heart rate, and renal blood flow rate) was studied by injecting their antagonists with different affinity (yohimbine and idazoxan) into narcotized Sprague--Dawley rats. The local introduction (microinjection into rostral ventrolateral medulla) and systemic administration (intraperitoneal injection) of the drugs showed that the hemodynamic effect of clonidine is mediated predominantly by the central alpha 2-adrenergic receptors, rather than by the imidazoline receptors.