Nuclei Of Mouse Fibroblasts Research Articles

Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

47BOVINE OOCYTE CYTOPLASM SUPPORTS NUCLEAR REMODELING BUT NOT REPROGRAMMING OF MURINE FIBROBLASTS

Nuclear transfer (NT) is used to elucidate fundamental biological functions such as cell differentiation reversibility and interactions between the cytoplasm and nucleus. In the present study, we compared nuclear and microtubule dynamics in bovine oocytes following NT of bovine and murine fibroblast cells. To clarify the nuclear reprogramming procedures, we additionally examined the expression of development-related genes (Octamer-binding transcription factors, Oct-4; E-cadherin, E-cad) and housekeeping genes (Heat shock protein 70.1, Hsp; Bos taurus apoptosis regulator box-a, Bax; Glucose transporter 1, Glut-1) in bovine embryos that had received nuclei from bovine and murine fibroblast cells. Bovine oocytes were matured in vitro and enucleated after 22h. The oocytes reconstructed with mouse embryonic fibroblast cells or bovine somatic fibroblast cells were cultured in CR1aa media. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from murine fibroblasts did not develop beyond the 8-cell stage. Similar nuclear and microtubule dynamics were observed in oocytes reconstructed with murine and bovine fibroblast cells. A small microtubule aster-containing γ-tubulin spot was observed in association with decondensed chromatin following NT of mouse fibroblasts. Within 1h of fusion of enucleated, non-activated cytoplasm, most mouse fibroblast nuclei were transformed to premature chromosome condensation (PCC). Randomly arrayed microtubules were tightly associated with PCC and formed meiotic-like microtubular spindles in all cases. Condensed chromosomes were divided into two or three chromatin masses and developed into multiple pronuclear-like structures. Microtubule asters were observed near the pronuclear-like structures during apposition in the cytoplasm. Two poles of the γ-tubulin spot evident at the mitotic metaphase stage are involved in the formation of the astral microtubule spindle for initial mitosis. A number of housekeeping mouse genes (hsp70, bax and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells. Table 1 Relative abundance of mRNA expression (mean±SEM) in mouse and xenonuclear-transferred (X-NT) embryos

Identification of a rosette-enriched chromatin fraction from mouse fibroblast nuclei

We have characterized two components of DNA isolated from mouse L-M cell nuclei. These components, designated as HMW (high molecular weight) and VHMW (very high molecular weight) DNA, were characterized by rate zonal sedimentation, agarose gel electrophoresis, and for protein content. Our electron micrographs revealed that HMW-DNA contained mainly linear molecules with few single rosette structures, while the VHMW-DNA was enriched in rosettes, many of which were significantly larger and linked together in multimeric structural forms. The VHMW-DNA component was also enriched for residual protein, which we believe represents the core of the rosette. The characteristics of this residual protein are consistent with reported findings of the most tightly bound proteins. The rosette conformation does not appear to be an artifact of microscopy or of an aggregate nature for several reasons: (i) rosettes are preferentially found in the VHMW-DNA component; (ii) further manipulation or purification of the DNA disrupts the rosette structure and produces linear fragments; (iii) the amount of proteinaceous material at the core of the rosette is diminished when the DNA is further purified; and (iv) treatment of intact nuclei with a novel bisamine reagent putatively crosslinks DNA in vivo and minimizes the disruption of rosettes by shear. We believe this separation of chromatin is critical to establish the architectural forms of euchromatin and heterochromatin of interphase DNA in the eucaryotic system. Once established, fractionated chromatin can be used to identify specifically expressed or repressed genes with linear form DNA and rosette form DNA. We discuss rosettes as derivatives of chromosomal domains that retain structural features because of residual peptide elements.

Non-random distribution of dibenzo[a, e]fluoranthene-induced DNA adducts in DNA loops in mouse fibroblast nuclei

The three-dimensional distribution of nuclear DNA damage induced by dibenzo(a,e)fluoranthene (DBF), a potent carcinogen for mouse fibroblasts, has been examined. The intact supercoiled nuclear DNA obtained from nucleoids of mouse fibroblasts incubated with DBF was fractionated into loop DNA attached to the matrix (10%) and bulk loop DNA (90%). Preferential binding of DBF to the DNA of the extremities of loops, which are rich in regulatory sequences, was observed in all experiments. An increase of the preferential DBF binding was seen when fibroblasts were incubated with both DBF and novobiocin or hydroxyurea. The excess damage seen in loop DNA attached to the cage may be due to the kinetics of diffusion to the interior of the nucleus of hydrophobic DBF metabolites accumulated in lipid-rich nuclear membrane.

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes.

An antibody was raised against "high mobility group" nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. - Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. - It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.

Expression of a liver-specific function by mouse fibroblast nuclei transplanted into rat hepatoma cytoplasts.

Data from many somatic cell fusion experiments support the notion that the cytoplasm of eukaryotic cells contains elements which can alter gene expression and replication1. More definitive evidence is perhaps lacking because the cell hybrids studied were complex cells comprising mixed genomes, frequently undergoing chromosomal losses and other aberrations, within mixed cytoplasms. Such problems have been alleviated by the development of the techniques of cybridisation, red cell-mediated microinjection and nuclear transplantation2. Using this last technique, we showed that up to 40% of a monolayer culture of cytoplasts could be renucleated to form whole viable cells3,4. Because of the high efficiency of fusion and the purity of cytoplast and karyoplast cultures used, large quantities of true cytoplasmic–nuclear hybrid cells suitable for immediate morphological and biochemical analysis could be formed. Here we describe the application of the technique to an investigation of the effects of rat liver cytoplasm on gene expression by mouse fibroblast nuclei. We show that the synthesis of mouse tryosine amino transferase (TAT)—a liver-specific enzyme never detected in the fibroblast cell line was induced in hybrids constructed by transplantation of mouse (A9) fibroblast nuclei into rat liver (HTC) cytoplasts. Furthermore, as occurs in the rat cell line, its activity was modulated by addition of the synthetic corticosteroid, dexamethasone. Clearly, this hybrid cell system will be useful in the further characterisation and eventual isolation of the newly identified regulatory substance.

Polyoma virus transcription in vitro

A transcriptionally active viral nucleoprotcin complex has been partially purified from the nuclei of mouse fibroblasts lytically infected with polyoma virus. The partially purified material contains polyoma DNA, some of which is complexed with active, preinitiated RNA polymerase II. The active transcription complex sediments at 22 to 23 S, slightly faster than supercoiled polyoma DNA (21 S). At least 95% of the RNA synthesized in vitro is viral. Both virus DNA strands are transcribed in their entirety in vitro and the RNA produced is heterogeneous in size including molecules larger than genome length. Of the in vitro product, 86% hybridizes to the virus DNA L strand, and 14% hybridizes to the E strand. The RNA synthesized in vitro is therefore similar by several criteria to viral RNA synthesized in vivo at the time when the complex is extracted.

Identification of a glucocorticoid-sensitive histone protein from mouse fibroblast nuclei

The acid-soluble proteins from mouse fibroblast nuclei show a typical elution profile for histone proteins on Bio-Rex 70 columns. Glucocorticoid treatment of cells growing in vitro decreases labeled amino acid incorporation into one specific histone region, corresponding to a peak of a lysinerich histone. The single histone affected is present in only small amounts in the cell; it normally represents about 6% of the H 1 protein, and less than 1% of the total histones. As measured by total protein nitrogen of the affected peak, glucocorticoid inhibition of this protein is first apparent between one and two hours after the start of steroid treatment.