Houtsmuller, A.B.

Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development (Article)

2009-12-01T00:00:00Z

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons.

UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells (Article)

2009-06-04T00:00:00Z

Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.

Heterochromatin protein 1 is recruited to various types of DNA damage (Article)

2009-05-18T00:00:00Z

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-{alpha}, HP1-β, and HP1-{gamma} are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.

Assembly of multiprotein complexes that control genome function (Article)

2009-04-06T00:00:00Z

Live-cell imaging studies aided by mathematical modeling have provided unprecedented insight into assembly mechanisms of multiprotein complexes that control genome function. Such studies have unveiled emerging properties of chromatin-associated systems involved in DNA repair and transcription.

Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair (Article)

2009-02-01T00:00:00Z

The ubiquitin ligase RAD18 is involved in different DNA repair processes. Here, we show that in G1 phase, human RAD18 accumulates in a few relatively large spontaneous foci that contain proteins involved in double-strand break (DSB) repair. These foci persist until cells enter S phase, when numerous small foci appear. At these sites, only 20% of RAD18 colocalizes with PCNA, a known RAD18 substrate. In late G2 phase, RAD18 relocates to nucleoli. After UVC irradiation, PCNA accumulates at the damaged site, followed by RAD18, independent of the cell cycle phase. After induction of DSBs, using low-power multi-photon laser, RAD18 accumulated at the DSB sites, but no PCNA accumulation was observed. Our data show that RAD18 accumulates on DSBs independent of the cell cycle phase. DSBs marked by RAD18 and RAD51 are also positive for RPA in G1 phase, and these DSBs persist until S phase. In addition, we show that DSBs generated in G2 phase are not all repaired, and are observed again in the next G1 phase. We conclude that repair of induced and spontaneous DSBs that accumulate RAD18 and RAD51 in G1 phase cells is delayed until S phase.

Multiple object tracking in molecular bioimaging by Rao-Blackwellized marginal particle filtering (Article)

2008-12-01T00:00:00Z

Time-lapse fluorescence microscopy imaging has rapidly evolved in the past decade and has opened new avenues for studying intracellular processes in vivo. Such studies generate vast amounts of noisy image data that cannot be analyzed efficiently and reliably by means of manual processing. Many popular tracking techniques exist but often fail to yield satisfactory results in the case of high object densities, high noise levels, and complex motion patterns. Probabilistic tracking algorithms, based on Bayesian estimation, have recently been shown to offer several improvements over classical approaches, by better integration of spatial and temporal information, and the possibility to more effectively incorporate prior knowledge about object dynamics and image formation. In this paper, we extend our previous work in this area and propose an improved, fully automated particle filtering algorithm for the tracking of many subresolution objects in fluorescence microscopy image sequences. It involves a new track management procedure and allows the use of multiple dynamics models. The accuracy and reliability of the algorithm are further improved by applying marginalization concepts. Experiments on synthetic as well as real image data from three different biological applications clearly demonstrate the superiority of the algorithm compared to previous particle filtering solutions.

Erratum: Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC (Journal of Cell Science (2008) vol. 121 (2850-2859)) (Article)

2008-12-01T00:00:00Z

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage (Article)

2008-12-01T00:00:00Z

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UVactivated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions. Copyright

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases (Article)

2008-12-01T00:00:00Z

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC (Article)

2008-09-01T00:00:00Z

To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in the nucleus under genotoxic stress conditions.

Erratum: Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC (Journal of Cell Science vol. 121 (2850-2859)) (Article)

2008-09-01T00:00:00Z

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching (Article)

2008-07-01T00:00:00Z

To study protein-protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor-acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein-protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5′ endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.

Cystic fibrosis transmembrane conductance regulator activation is reduced in the small intestine of Na+/H+ exchanger 3 regulatory factor 1 (NHERF-1)- but not NHERF-2-deficient mice (Article)

2007-12-28T00:00:00Z

Binding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to the Na+/H+exchanger 3 regulatory factor 1 (NHERF-1) and NHERF-2 scaffolding proteins has been shown to affect its localization and activation. We have for the first time studied the physiological role of these proteins in CFTR regulation in native tissue by determining CFTR-dependent chloride current in NHERF-1- and NHERF-2-deficient mice. The cAMP- and cGMP-activated chloride current and the basal chloride current in basolaterally permeabilized jejunum were reduced by ∼30% in NHERF-1-deficient mice but not in NHERF-2-deficient mice. The duodenal bicarbonate secretion was affected in a similar way, whereas no significant differences in CFTR activity were observed in ileum. CFTR abundance as determined by Western blotting was unaltered in jejunal epithelial cells and brush border membranes of NHERF-1 and NHERF-2 mutant mice. However, semi-quantitative detection of CFTR by confocal microscopy showed that the level of apically localized CFTR in jejunal crypts was reduced by ∼35% in NHERF-1-deficient and NHERF-1/2 double deficient mice but not in NHERF-2 null mice. Together our results indicate that NHERF-1 is required for full activation of CFTR in murine duodenal and jejunal mucosa and that NHERF-1 affects the local distribution of CFTR in or near the plasma membrane. These studies provide the first evidence in native intestinal epithelium that NHERF-1 but not NHERF-2 is involved in the formation of CFTR-containing functional complexes that serve to position CFTR in the crypt apical membrane and/or to optimize its function as a cAMP- and cGMP-regulated anion channel.

Human USP3 Is a Chromatin Modifier Required for S Phase Progression and Genome Stability (Article)

2007-11-20T00:00:00Z

Protein ubiquitination is critical for numerous cellular functions, including DNA damage response pathways [1, 2]. Histones are the most abundant monoubiquitin conjugates in mammalian cells; however, the regulation and the function of monoubiquitinated H2A (uH2A) and H2B (uH2B) remain poorly understood. In particular, little is known about mammalian deubiquitinating enzymes (DUBs) that catalyze the removal of ubiquitin from uH2A/uH2B. Here we identify the ubiquitin-specific protease 3 USP3 as a deubiquitinating enzyme for uH2A and uH2B. USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2A-USP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In addition, we show that in response to ionizing radiation, (1) uH2A redistributes and colocalizes in γ-H2AX DNA repair foci and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and γ-H2AX dephosphorylation. Our studies identify USP3 as a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress, and suggest its involvement in the response to DNA double-strand breaks. Together, our results implicate USP3 as a novel chromatin modifier in the maintenance of genome integrity.

UTF1 is a chromatin-associated protein involved in ES cell differentiation (Article)

2007-09-10T00:00:00Z

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation.

Cholesterol depletion and genistein as tools to promote F508delCFTR retention at the plasma membrane (Article)

2007-09-05T00:00:00Z

Background/aims: F508delCFTR-, but not wtCFTR-, expressing fibroblasts resemble Niemann Pick type C cells in the massive intracellular accumulation of free cholesterol. The recruitment and activation of F508delCFTR by cholesterol depletion was studied. Methods: Filipin staining, forskolin-stimulated anion efflux and FITC-dextran uptake were studied in control cells and fibroblasts treated with 2-hydroxypropyl β-cyclodextrin phosphatidylcholine large unilamellar vesicles to deplete cellular free cholesterol.Results: Treatment of F508delCFTR-, but not wtCFTR-, expressing fibroblasts with 2-hydroxypropyl β-cyclodextrin resulted in a reduction in cellular cholesterol and a potentiation of the forskolin-induced anion efflux. In addition, forskolin also promoted a massive increase in the rate of endocytosis in F508delCFTR fibroblasts, which was absent in genistein- or cyclodextrin-treated cultures.Conclusion: The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis. Copyright

Activation of multiple DNA repair pathways by sub-nuclear damage induction methods (Article)

2007-08-01T00:00:00Z

Live cell studies of DNA repair mechanisms are greatly enhanced by new developments in real-time visualization of repair factors in living cells. Combined with recent advances in local sub-nuclear DNA damage induction procedures these methods have yielded detailed information on the dynamics of damage recognition and repair. Here we analyze and discuss the various types of DNA damage induced in cells by three different local damage induction methods: pulsed 800 nm laser irradiation, Hoechst 33342 treatment combined with 405 nm laser irradiation and UV-C (266 nm) laser irradiation. A wide variety of damage was detected with the first two methods, including pyrimidine dimers and single- and double-strand breaks. However, many aspects of the cellular response to presensitization by Hoechst 33342 and subsequent 405 nm irradiation were aberrant from those to every other DNA damaging method described here or in the literature. Whereas, application of low-dose 266 nm laser irradiation induced only UV-specific DNA photolesions allowing the study of the UV-C-induced DNA damage response in a user-defined area in cultured cells.

Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC (Article)

2007-08-01T00:00:00Z

Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A in vivo. The majority of DDB2 and CUL4A diffuse in the nucleus with a diffusion rate consistent with a high molecular mass complex. Essentially all DDB2 binds to UV-induced DNA damage, where each molecule resides for ∼2 minutes. After the induction of DNA damage, DDB2 is proteolytically degraded with a half-life that is two orders of magnitude larger than its residence time on a DNA lesion. This indicates that binding to damaged DNA is not the primary trigger for DDB2 breakdown. The bulk of DDB2 binds to and dissociates from DNA lesions independently of damage-recognition protein XPC. Moreover, the DDB2-containing E3 ubiquitin ligase is bound to many more damaged sites than XPC, suggesting that there is little physical interaction between the two proteins. We propose a scenario in which DDB2 prepares UV-damaged chromatin for assembly of the NER complex.

Nuclear dynamics of PCNA in DNA replication and repair. (Article)

2005-11-01T00:00:00Z

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

Antiandrogens prevent stable DNA-binding of the androgen receptor (Article)

2005-01-01T00:00:00Z

The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR ( approximately 10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for approximately 45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR.

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair. (Article)

2004-11-09T00:00:00Z

Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of approximately 30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells. AD - Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.

A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron. (Article)

2004-10-01T00:00:00Z

We characterized the specifically androgen-regulated gene (SARG), which is expressed in the androgen receptor (AR) and glucocorticoid receptor (GR) positive cell line lymph node carcinoma of the prostate-1F5 (LNCaP-1F5). SARG mRNA expression can be up-regulated by androgens, but not by glucocorticoids. SARG mRNA expression is high in prostate tissue. SARG is composed of four exons and spans a region of 14.5 kbp on chromosome 1q32.2. Transcripts of 5.5, 3.3 and 2.3 kb are the result of alternative polyadenylation. SARG mRNA splice variants lack exon 2 and vary in length of exon 1. The SARG protein has a length of 601 amino acids and is located in the cytoplasm. By screening the 18 kbp genomic sequence flanking the transcription start site we identified the imperfect direct repeat 5'-TGTGCTaacTGTTCT-3'in intron 1 as an active androgen response element (ARE-SARG+4.6). A 569 bp genomic DNA fragment containing this element functioned as an androgen-specific enhancer in transiently transfected LNCaP-1F5 cells. ARE-SARG+4.6 cooperated with flanking sequences for optimal activity. Inactivation of ARE-SARG+4.6 completely abolished the androgen response of the enhancer. Chromatin immunoprecipitation (ChIP) experiments showed chromatin structural changes of the enhancer in the presence of R1881. ARE-SARG+4.6 was able to bind to the androgen receptor, but not to the glucocorticoid receptor, correlating with its androgen-specific activity in transfections.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery. (Article)

2004-07-05T00:00:00Z

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function. (Article)

2004-06-01T00:00:00Z

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction ( approximately 73%) has binding dynamics similar to TRF1 (residence time of approximately 44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of approximately 11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery (Article)

2004-01-01T00:00:00Z

Increased vesicle recycling in response to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release. (Article)

2003-10-10T00:00:00Z

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1-43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2-3 min and lasting for approximately 10-15 min. The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H. R., and Tilly, B. C. (1999) Biochem. J. 343, 579-586), was diminished after 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment. Uptake of TRITC-dextrans, however, was not affected. Treatment of the cells with the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-specific protease Clostridium botulinum toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis. Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans. Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions (Article)

2003-01-01T00:00:00Z

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.

Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells. (Article)

2003-01-01T00:00:00Z

Increasing evidence suggests that neuropeptides play a role in the regulatory mechanisms between the neuroendocrine and immune systems. A differential expression of the five known somatostatin (SS) receptors (sst1-5) has been demonstrated in human immune cells and tissues. However, little is known concerning regulation and expression of sst1-5 and the peptide SS. Therefore, we investigated the expression and the time-dependent regulation of sst1-5, SS, and cortistatin (CST), a novel SS-like peptide, in human monocytes (MO), monocyte-derived macrophages (MP), and dendritic cells (DC) in the basal and lipopolysaccharide (LPS)-activated state. MO, MP, and DC selectively expressed sst2 mRNA. SS mRNA was not detectable, whereas all samples expressed CST mRNA. Expression levels of sst2 and CST mRNA showed marked differences and were in the rank order of MP>>DC>>>MO. LPS stimulation did not induce expression of SS or sst1,3,4,5. However, sst2 mRNA expression was upregulated significantly by stimulation with LPS. CST mRNA was upregulated as well. During differentiation of MO in MP or DC, time-dependent, significantly increasing sst2 and CST mRNA levels were found. By confocal microscopy, the presence of sst2 receptors was demonstrated on MP, but not on DC. This study demonstrates for the first time a selective and inducible expression of the recently discovered CST, as well as sst2, in human monocyte-derived cells, suggesting a role for a CST-sst2 system rather than a SS-sst2 system in these immune cell types.

The formation of vault-tubes: a dynamic interaction between vaults and vault PARP (Article)

2003-01-01T00:00:00Z

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. (Article)

2002-04-15T00:00:00Z

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.

Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells (Article)

2001-01-01T00:00:00Z

Antibody-targeted chemotherapy is a promising therapy in patients with acute myeloid leukemia (AML). In a phase II study of Mylotarg (CMA-676, gemtuzumab ozogamicin), which consists of a CD33 antibody linked to calicheamicin, saturation and internalization by leukemic and normal myeloid cells were analyzed in 122 patients with relapsed AML. Peripheral blood samples were obtained just before and 3 and 6 hours after the start of the first and second Mylotarg treatment cycles. Within 3 to 6 hours after infusion, near complete saturation of CD33 antigenic sites by Mylotarg was reached for AML blasts, monocytes, and granulocytes, whereas Mylotarg did not bind to lymphocytes. Saturation levels prior to the start of the second Mylotarg treatment cycle were significantly increased compared with background levels before the start of the first cycle. This apparently was caused by remaining circulating Mylotarg from the first treatment cycle (approximately 2 weeks earlier). On binding of Mylotarg to the CD33 antigen, Mylotarg was rapidly internalized, as determined by the decrease in maximal surface membrane Mylotarg binding. Internalization of Mylotarg was also demonstrated in myeloid cells in vitro and was confirmed by confocal laser microscopy. In vitro studies using pulse labeling with Mylotarg showed a continuous renewed membrane expression of CD33 antigens, which can significantly increase the internalization process and thereby the intracellular accumulation of the drug. Finally, Mylotarg induced dose-dependent apoptosis in myeloid cells in vitro. These data indicate that Mylotarg is rapidly and specifically targeted to CD33(+) cells, followed by internalization and subsequent induction of cell death.

Action of DNA repair endonuclease ERCC1/XPF in living cells. (Article)

1999-05-07T00:00:00Z

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 +/- 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.

Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation (Article)

1998-01-01T00:00:00Z

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.

Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture (Article)

1998-01-01T00:00:00Z

The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate (CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 +/- 0.4 micrograms/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 omega.cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 omega.cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 +/- 0.03 microgram/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal binding was observed 2 days postinjury when the wounds were already morphologically closed but TER was still low. Confocal images showed that during the repair process, crystals selectively adhered to migrating cells at the wound border and to stacked cells at sites were the wounds were closed. After the barrier integrity was restored, crystal binding decreased again to the same low levels as in undamaged controls. These results indicate that, whereas functional MDCK monolayers are largely protected against COM crystal adherence, epithelial injury and the subsequent process of wound healing lead to increased crystal binding.