Isolated Cell Nuclei Research Articles

1791 PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA BASED ON INTERPHASE FISH ANALYSIS

You have accessJournal of UrologyKidney Cancer: Advanced I1 Apr 20121791 PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA BASED ON INTERPHASE FISH ANALYSIS Jimsgene Sanjmyatav, Martin Mühr, Doriana Sava, Maria Sternal, Sophie Matthes, Heiko Wunderlich, Marc-Oliver Grimm, and Kerstin Junker Jimsgene SanjmyatavJimsgene Sanjmyatav Jena, Germany More articles by this author , Martin MührMartin Mühr Jena, Germany More articles by this author , Doriana SavaDoriana Sava Jena, Germany More articles by this author , Maria SternalMaria Sternal Jena, Germany More articles by this author , Sophie MatthesSophie Matthes Jena, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Jena, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Jena, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1822AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES By precise mapping of clear cell renal cell carcinomas using array-CGH in our previous studies we identified specific chromosomal aberrations which are significantly associated both with metastasis and cancer specific survival. The aim of this study was to examine in this cohort the prognostic value of possible combinations of the 4 most prominent aberrations detected by FISH analysis in order to develop a prognostic FISH assay. METHODS FISH experiments were performed on isolated cell nuclei from 43 ccRCCs of the same cohort of previous array-CGH analysis (26 metastasized/17 non-metastasized). For each critical chromosomal region (1q21.3, 7q36.3, 9p21.3p24.1 und 20q11.21q13.32) we hybridized a commercially available FISH probes. Mean/median follow-up was 55/41 months. RESULTS All 11 possible combinations of -9p21.3p24.1 and +1q21.3, +7q36.3 and +20q11.21q13.32 were present in tumors of this cohort. ROC-analysis showed that total number of specific aberrations (TNSA) presents the best predictor of metastasis (specificity=100%, sensitivity=77.8%) and cancer specific death of patients (specificity=100%, sensitivity=94.4%) compared to single aberrations and to different combinations of these aberrations. In Kaplan-Mayer analysis TNSA demonstrated highest significance value both in organ-confined situations and all tumors while T-status and grading of ccRCCs were not significant in this cohort. CONCLUSIONS Our results show that TNSA presents a strong prognostic factor both in all ccRCCs and organ-confined tumors. It seems that aggressiveness of the tumors increases with accumulation of specific aberrations. Based on these data it seems possible to design a combined FISH assay which can be used in routine diagnostics for outcome prediction of patients with ccRCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e723 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jimsgene Sanjmyatav Jena, Germany More articles by this author Martin Mühr Jena, Germany More articles by this author Doriana Sava Jena, Germany More articles by this author Maria Sternal Jena, Germany More articles by this author Sophie Matthes Jena, Germany More articles by this author Heiko Wunderlich Jena, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Jena, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells.

526 ESTABLISHMENT OF AN INTERPHASE FISH TEST FOR PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Evaluation and Staging1 Apr 2011526 ESTABLISHMENT OF AN INTERPHASE FISH TEST FOR PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA Jimsgene Sanjmyatav, Martin Mühr, Doriana Sava, Sophie Matthes, Maria Sternal, Heiko Wunderlich, Thomas Steiner, Marc-Oliver Grimm, and Kerstin Junker Jimsgene SanjmyatavJimsgene Sanjmyatav Jena, Germany More articles by this author , Martin MührMartin Mühr Jena, Germany More articles by this author , Doriana SavaDoriana Sava Jena, Germany More articles by this author , Sophie MatthesSophie Matthes Jena, Germany More articles by this author , Maria SternalMaria Sternal Jena, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Jena, Germany More articles by this author , Thomas SteinerThomas Steiner Jena, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Jena, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1250AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES By precise mapping of genomic imbalances of 56 clear cell renal cell carcinomas using array-CGH in our previous studies we identified specific chromosomal aberrations which are significantly associated both with metastasis and cancer specific survival. In multivariate analysis gains of 1q21.3 and of 20q11.21–q13.2 as well as loss of 9p21.3–p24.1 were independent predictors for metastasis. Multivariate Cox-regression analysis revealed gains of 7q36.3 and of 20q11.21–q13.32 and a loss of 9p21.3–p24.1 as independent prognostic factors for outcome of patients. The aim of this study was to prove the prognostic value of these aberrations using FISH probes on these critical regions in order to develop a routine diagnostic tool. METHODS FISH experiments were performed on isolated cell nuclei from tumor tissues. The same cohort of previous array-CGH analysis was used for these experiments (32 metastasized and 24 non-metastasized). For each critical chromosomal region (1q21.3, 7q36.3, 9p21.3–p24.1 und 20q11.21–q13.32) we hybridized a mixture of three different commercially available FISH probes: region specific probe in red dye, centromeric probe of corresponding chromosome or probe close to centromere in green dye and centromeric probe of chromosome 2 in blue colour as control. 100 nuclei were scored in each case. RESULTS Loss of 9p21.3–p24.1 (Fisher's exact test; p=0.017) and gains of 1q21.3 (p=0.030), 7q36.3(p=0.019) and 20q11.21–q13.32 (p=0.008) displayed significant correlations with metastasis occurrence. Our analysis revealed significant correlation of gains 7q36.3 (p=0.001) and 20q11.21–q13.32 (p=0.05) as well as loss of 9p21.3-p24.1 (p=0.00002) with cancer specific death. Patients with tumours harbouring these aberrations in more than 30% of cells have a poor outcom. CONCLUSIONS FISH results confirmed our previous findings of array-CGH and showed that these regions play an important role in cancer progression. Based on these genetic targets it seems possible to design a combined FISH assay which can be used in routine diagnostics for outcome prediction of patients with ccRCC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e214 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jimsgene Sanjmyatav Jena, Germany More articles by this author Martin Mühr Jena, Germany More articles by this author Doriana Sava Jena, Germany More articles by this author Sophie Matthes Jena, Germany More articles by this author Maria Sternal Jena, Germany More articles by this author Heiko Wunderlich Jena, Germany More articles by this author Thomas Steiner Jena, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Jena, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously 14C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that 14C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

Caspase-9-dependent decrease of nuclear pore channel hydrophobicity is accompanied by nuclear envelope leakiness

Advances in nanomedicine require conceptual understanding of physiological processes. Apoptosis is a fundamental physiological process that is characterized, among other things, by an increased permeability of the nuclear envelope (NE). The latter is a tight transport barrier, known to restrict nuclear delivery rate of therapeutic nanoparticles. Therefore, an understanding of the underlying mechanism that leads to the breakdown of the barrier during apoptosis could stimulate the development of new approaches in gene therapy. We set out to elucidate this mechanism following induction of apoptosis on isolated cell nuclei. We tested the hypothesis whether caspases, mediators of apoptosis, trigger the NE leakiness at the level of the nuclear pore complexes (NPCs) using fluorescence techniques. As the permeability barrier inside the NPC channel is thought to be based on hydrophobic–hydrophobic protein interactions we further investigated the NPC channel hydrophobicity using atomic force microscopy. Caspase-9 was found to induce NE leakiness to large macromolecules. Leakiness was prevented by pretreatment of NPCs with an importin-β mutant, which irreversibly binds and thereby obstructs the NPC channel. Utilizing an ultra-sharp, hydrophobic atomic force microscope tip as a chemical nanosensor that reaches deep into the apoptotic NPC channel, a remarkable decrease of hydrophobic binding sites was detected therein. We conclude that caspase 9 gives rise to NE leakiness by perturbing the hydrophobicity-based barrier inside the NPC channel. This explains the high passive NE permeability in early apoptosis. From the Clinical Editor In this study, biological processes taking place in the nucleus during the course of apoptosis have been monitored using atomic force microscopy-based nanosensors. The conclusion was that one of the caspases, caspase 9 perturbes the hydrophobicity-based barrier inside the nuclear pore complex channel causing nuclear envelope leakiness.

Visiting the plant kingdom

way flow cytometers are usually configured is that thedimension of the flow orifice (50–100 lm) and the isotonicsheath fluid are optimized for the analysis of live human cells.Animal cells have similar sizes and osmolarities as humancells; therefore, they can be measured with the same setup andwithout substantial problems. By contrast, cytometric plantcell analysis is a challenge for several reasons. The rigid plantcell wall gives the cells a special, elongated form so that theycan reach sizes that exceed the usual orifice size of a typicalflow cytometer. Plant cell walls are highly organized fiber-laminate extracellular structures with strong anisotropy intheir shape and growth, and thus, polarization characteristicshave been widely used in revealing the anisotropic features ofthis complex cellulose-based structure (1). The removal of thewall leads to protoplasts with spherical form but the osmoticpressure of such cells is substantially higher than that ofhuman cells. Isolated cell nuclei or chloroplasts and mitochon-dria from tissue homogenates are more tolerant for osmoticchanges. DNA ploidy analysis is probably the most commonflow cytometric assay used in plant science and breeding (2).The DNA pattern analysis of diploid and tetraploid calli andhairy roots make following the transformation process in tis-sue culture possible (3). The use of bead beating was suggestedby Roberts to prepare suspensions of nuclei from fresh leaves,herbarium leaves, petals, and pollen for flow cytometry (4).Based on this technique, a relatively gentle homogenizationprocedure was developed by Cousin et al. (5) for releasingnuclei while leaving the leaf tissue largely intact to avoid pro-ducing in excess cellular debris. This is one part of the methodthat the authors developed or improved in order to assemblethem to a whole work-flow of preparation and analysis forhigh-throughput DNA cytometry.Fast and efficient autofocusing is a prerequisite for auto-mated imaging, slide-based cytometry, and high-contentscreening. Precise autofocus overcomes problems that includemechanical instability, movement of live specimens, variablethickness,drift(e.g.,duetothermalexpansion),andirregulari-ties of biological substrates. Reflective positioning using laser-based methods (6) can be faster, but finding the best focus bymeasuring the resolution of the images is more direct and canproducesharperimages,especiallywithhigherNAobjectives.Varga and colleagues (7) focused their work on an algo-rithm that selects the best focus position based on the sharp-ness value of the image. A higher sharpness value means abetter focused image. Sharpness calculation is based on pixelvalue differences. Varga et al. have implemented a specialalgorithm in the slide-based microscope system to lower noiseand cancel small artifacts (7). The image is shrunk byaveraging a square of pixels. Then, the algorithm goes throughthe image and calculates the difference between every pixeland its neighbor to the right. The fifth power of every differ-ence is calculated and added to the sharpness value of theimage. If, for example, the difference of two pixels was 10,then 10

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 µM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.

Class-specific Regulation of Pro-inflammatory Genes by MyD88 Pathways and IκBζ

Toll-like receptors trigger the induction of primary response genes via MyD88-mediated activation of NF-kappaB and other transcription factors. These factors then act in concert with primary response gene products to induce secondary response genes. Although the MyD88 pathway is important for the expression of both primary and secondary response genes, we show that the recruitment of NF-kappaB, RNA polymerase, and the TATA-binding protein is MyD88-dependent only at secondary response genes. This selective dependence correlates with the fact that MyD88 is required for nucleosome remodeling and histone H3K4 trimethylation at secondary response promoters, whereas rapidly induced primary response promoters are assembled into poised MyD88-independent chromatin structures. At a subset of secondary response promoters, IkappaBzeta was identified as a selective regulator of H3K4 trimethylation and preinitiation complex assembly after nucleosome remodeling. These mechanistic distinctions advance our understanding of the diverse molecular cascades that underlie the differential regulation of pro-inflammatory genes.

A Pathway Separate from the Central Channel through the Nuclear Pore Complex for Inorganic Ions and Small Macromolecules

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.

Evidence for Small Ubiquitin-like Modifier-dependent Nuclear Import of the Thymidylate Biosynthesis Pathway

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.

Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood

During terminal cell differentiation, nuclear chromatin becomes condensed and the repertoire of epigentic heterochromatin proteins responsible for chromatin condensation is dramatically changed. In order to identify the chromatin regulatory factors associated with incomplete cell differentiation and impaired chromatin condensation in hematological malignancies, we examined expression levels of major heterochromatin proteins in normal blood cells and cells derived from a number of chronic and acute myeloid leukemia patients exhibiting different degrees of differentiation. We used immunoblotting and immunofluorescence to examine the levels and localization of epigenetic heterochromatin factors in isolated cell nuclei and fractionated peripheral blood cells. While the major epigenetic heterochromatin factor, histone H3 methylated at lysine 9, is present in all cell types, its main counterparts, nonhistone proteins, heterochromatin proteins 1 (HP1) alpha, beta, and gamma, are dramatically reduced in peripheral blood leukocytes of normal donors and chronic myeloid leukemia patients, but are substantially increased in the blood of accelerated phase and blast crisis patients. In the terminally differentiated cells, nuclear chromatin accumulates a nucleocytoplasmic serpin, monocyte and neutrophil elastase inhibitor (MNEI). HP1 and MNEI levels inversely correlate in a number of normal and leukemia myeloid cells and show strikingly opposite coordinated changes during differentiation of U937 cell line induced by retinoic acid. Our results suggest that repression of HP1 and accumulation of MNEI are linked to terminal cell differentiation and that their levels may be monitored in blood cell populations to detect transitions in cell differentiation associated with leukemia progression and treatment.

In nucleo enzymatic assays for the identification and characterization of histone modifying activities

The impact of histone phosphorylation, acetylation, and methylation onto transcription and various other cellular DNA-mediated processes is now well established. Numerous histone modification marks, specific sites carrying particular post-translational modifications, have been described and analyzed in detail. Whereas, many methods for the study of histone post-translational modifications involve sophisticated equipment and techniques, we describe the revival of a very simple procedure for the analysis of histone modifications and histone modifying enzymes, which is derived from experiments first carried out several decades ago. This method is based on the isolation of cell nuclei containing intact chromatin structures that are then incubated with defined enzymatic substrates of histone modifying enzymes. We provide quick protocols for the isolation of nuclei from yeast and mammalian cells and give basic procedures for the phosphorylation, acetylation, and methylation of histones (and other proteins) using these subcellular sources that can be carried out in any laboratory. Simple methods for the analysis of histone modifications using these assays are discussed.

The influence of ATP on poly(ADP-ribose) metabolism

ATP affects poly(ADP-ribose) metabolism at two distinct sites: it inhibits poly(ADP-ribose) polymerase-1 and activates the glycohydrolase directly. The inhibitory site of ATP on poly(ADP-ribose) polymerase-1 was identified by amino acid exchange mutation to be at the arginine 34 residue in the first Zn2+ finger. Mutation of 138 arginine residue of Zn2+ finger 2 had negligible influence on the inhibitory action of ATP, pinpointing arginine 34 of the first Zn2+ finger as the specific ATP site. The glycohydrolase protein was activated by ATP when the substrate was a long-chain ADP-ribose polymer, but not with a short-chain substrate. Isolated cell nuclei also responded to both inhibition of poly(ADP-ribose) polymerase by ATP and to poly(ADP-ribose) glycohydrolase activation by ATP, demonstrating that enzymological results can be extrapolated to cellular systems. The activation of poly(ADP-ribose) polymerase in nuclei by an alkylating drug was completely suppressed by ATP, demonstrating that the bioenergetic competence of cells can regulate the cytocidal action of DNA alkylating drugs. The potential significance of bioenergetic regulation of poly(ADP-ribose) metabolism is proposed.

Three-dimensional imaging of single isolated cell nuclei using optical projection tomography

A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.

Characterization and Quantification of Triple Helix Formation in Chromosomal DNA

DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.

Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei: A Sensitive Method for 1p and 19q Deletion Analysis in Paraffin-Embedded Oligodendroglial Tumor Specimens

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP—Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P = .001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.

Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection.

We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.

EGFR, Faster than STATS?

Although there have been a few reports that growth factor receptors cycle to the nucleus, there has not been much follow-up work attempting to answer why they are there. Lin et al. seem to have identified a role for epidermal growth factor receptors (EGFRs) in the nucleus: transcriptional activation. EGFR was found in the nucleus of cell lines and normal tissues that proliferate quickly, such as human buccal mucosa, as demonstrated by immunohistochemistry and isolation of cell nuclei. The majority of EGFR was phosphorylated in the nucleus and began to appear in the nucleus within 1 min of EGF treatment of cells. Nuclear fractions from EGFR-expressing cells treated with cross-linked 125 I-labeled EGF-EGFR contained the radiolabeled EGF-EGFR complexes, suggesting that the complexes relocalized from the plasma membrane to the nucleus. The authors identified a proline-rich region from the COOH-terminal portion of EGFR, purported to be similar to a transcriptional activation domain, and fused it to a Gal4 DNA binding domain. This construct greatly increased the expression of a Gal4-dependent reporter gene, whereas a construct that included the EGFR kinase domain in addition to the proline-rich domain and Gal4 DNA binding domain only slightly increased reporter gene expression. The authors concluded that this was evidence of a transactivation domain in EGFR and that the kinase domain acted to negatively regulate transactivation. However, taking specific regions away from the context of the full-length protein and evaluating them for specific activities is possibly to ascribe physiologically irrelevant functions to protein fragments. Nonetheless, Lin et al. identified an AT-rich sequence that bound to EGFR from EGF-treated cells in vitro and also found that EGFR bound to similar elements within the cyclin D1 promoter in chromatin immunoprecipitation assays. These results suggest that EGFR in the nucleus might increase cellular proliferation through the increased expression of cyclin D1. Additional work will need to be done to validate and further this work with other receptors; if these results remain robust and withstand scrutiny, then clearly, a brand new area of signaling research will have been opened. S.-Y. Lin, K. Makino, W. Xia, A. Matin, Y. Wen, K. Y. Kwong, L. Bourguignon, M.-C. Hung, Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nature Cell Biol. 3 , 802-808 (2001). [Online Journal]

A simple method for identification of S phase nuclei in Vicia faba root meristems using BrdUrd labeling and indirect immunofluorescence (comparison with 3H-thymidine incorporation)

A modified immunocytochemical method for identification of S-phase cells in root meristems is desribed. The technique combines incorporation of BrdUrd, rapid isolation of cell nuclei and indirect immunocytochemical detection of BrdUrd-labeled areas of chromatin using monoclonal anti-BrdUrd (I) and FITC-conjugated (II) antibodies. The labeling indexes calculated using 3H-thymidine autoradiography and BrdUrd-immunofluorescence in root meristems of Vicia faba subsp. minor were found comparable. However, the increased sensitivity of fluorescent staining reveals an enhanced complexity of visible structure emerging during successive periods of S-phase, allowing for discriminating not only quantitative but also qualitative aspects of nuclear labeling patterns characteristic for specific periods of DNA replication.