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At least 1,531 records · Page 85Linked to original sources
PubMed2001

Inhibition of DNA replication by tirapazamine.

Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.

Antineoplastic Agents↗
PubMed1999

Characterization of matrix attachment sites in the upstream region of a tobacco chitinase gene.

The nuclear matrix is thought to partition the genome into functional and structural loop domains, and it has been implicated in several cellular processes, such as the replication and transcription of DNA and the processing of RNA. Therefore, the analysis of scaffold/matrix-associated DNA regions (S/MARs) might enhance our understanding of the functional roles of the higher-order organization of chromatin. In this study, the upstream region between positions -3320 and -1095 of the basic class I chitinase gene, CHN50, was shown to have specific affinity for the tobacco nuclear scaffold. Detailed analysis of nuclear scaffold-DNA binding in vitro revealed that two regions (positions -3320 to -2621 and -2221 to -1371) bound specifically to the nuclear scaffold. These S/MAR elements, designated S/M I and S/M II, are A+T-rich sequences with 75% and 74% A+T residues, respectively, and may include a number of sequence motifs that have frequently been found in other S/MARs. Moreover, S/M II contains a curved DNA sequence with anomalous mobility on polyacrylamide gels. A circular permutation assay revealed that the center of this curved region was located between positions -1767 and -1759. The possible functions and structural features of the S/MAR elements in the upstream region of CHN50 are discussed.

Antigens, Nuclear↗
PubMed1986

Nuclear antigens recognized by antibodies present in liver disease sera.

Nuclear and nuclear matrix proteins of HeLa cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose. Antibodies present in sera of patients with primary biliary cirrhosis and autoimmune chronic active hepatitis reacted with some of the blotted proteins. The antibodies were mainly directed against chromatin-associated proteins and protein constituents of discrete RNP particles. In addition, antibodies found in autoimmune liver disease sera detected a hitherto undescribed nuclear protein of 54 kD, and a nuclear matrix protein of approximately 150 kD. Antibodies recognizing a nuclear 25 kD doublet apparently constituted a marker antibody for autoimmune liver disease. Those directed at the 17 kD centromere protein were associated with the primary biliary cirrhosis-related CREST syndrome, while those recognizing La antigen were related to cases of sicca syndrome associated with autoimmune liver diseases.

Antibodies, Antinuclear↗
PubMed2005

[Isolation and functional analysis of tobacco MARs].

Two new MAR segments (M14 and M17) were cloned from tobacco genome. Both of the sequences contained several typical consensus sequences of MARs, which were different from the original MAR sequence, such as 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomerase II, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT et al. To investigate the effects of these two sequences on gene expression in transgenic plants, 3 plant expression vectors were constructed with uidA gene coding beta-glucuronidase (GUS) which were flanked on one side and on both sides by the MARs we obtained. These plant expression vectors with one or two MARs were transformed into tobaccos via Agrobacterium-mediated transformation method, with the plant expression vector pCAMBIA2301 without MAR and wild type tobacco as controls. GUS histochemical staining results showed that the uidA gene expressed stably in transgenic tobaccos. Quantitative detection of GUS activity showed that the MARs could increase GUS expression levels in vivo in contrast to the controls, wherever they were flanked on one side or both sides of uidA gene. The vector ligated with MARs in the same direction on both sides of uidA could increase the GUS expression level much better than both vectors which just ligated with single MARs on one side. The former one increased the average GUS activity for 3.14 folds, but 1.56 and 2.43 folds for the latter two vectors with single MARs respectively contrasting to the pCAMBIA2301 control. But the expression differences among individual transformants were still obvious. Therefore, it was concluded that the DNA sequences we obtained in this experiment were two novel MARs and could enhance gene expression in vivo. In the meanwhile, although the numbers of the MARs typical motifs in M14 were more than in M17, especially the 90% AT box which had been considered to be the highest correlative motif with binding strength in vitro, the enhancement of gene expression was lower yet, which implied no correlation between improvement of gene expression and binding strength between MARs and nuclear matrix in vitro.

Cloning, Molecular↗
PubMed1987

Structural aspects of mammalian DNA replication: topoisomerase II.

The dynamic biology of mammalian DNA requires structural complexity. Previous studies on the structure and function of mammalian DNA have suggested that mammalian cell nuclei contain DNA organized into loop-domains by a nuclear structural subcomponent termed the nuclear matrix. The loop-domains, functioning as replicons during DNA synthesis, appear to be replicated by biosynthetic complexes located at fixed sites within the nuclear matrix milieu. Recently, we have found the mammalian DNA topoisomerase II enzyme to be associated with newly replicated DNA, and we speculate that the enzyme may be strategically positioned to untangle topologically intertwined daughter loop-domains before mitotic segregation. Further studies demonstrating growth-related elevations of DNA topoisomerase II levels in rat prostatic adenocarcinoma tissues support a role for the enzyme in cellular proliferation in vivo. The possible participation of DNA topoisomerase II in mammalian DNA replication and the proliferation-dependent appearance of the enzyme in neoplastic tissues may have important implications for therapeutic strategies directed at DNA topoisomerase II.

Animals↗
PubMed1983

Generation of an internal matrix in mature avian erythrocyte nuclei during reactivation in cytoplasts.

When fused with mouse L-cell cytoplasts, chick erythrocyte nuclei enlarge, take up proteins from the host cytoplasm, and recommence RNA synthesis. We found that during this transition the erythrocyte nuclei gain an internal nuclear matrix, thus providing a novel approach to questions concerning the nature of the salt-resistant intranuclear skeleton. A new method for preparation and examination of the nuclear matrix in situ is also described.

Animals↗
PubMed2002

Engineering gene expression and protein synthesis by modulation of nuclear shape.

The current understanding of the relationships between cell shape, intracellular forces and signaling, nuclear shape and organization, and gene expression is in its infancy. Here we introduce a method for investigating gene-specific responses in individual cells with controlled nuclear shape and projected area. The shape of the nuclei of primary osteogenic cells were controlled on microfabricated substrata with regiospecific chemistry by confining attachment and spreading of isolated cells on adhesive islands. Gene expression and protein synthesis were altered by changing nuclear shape. Collagen I synthesis correlated directly with cell shape and nuclear shape index (NSI), where intermediate values of nuclear distension (6 < NSI < 8) promoted maximum synthesis. Osteocalcin mRNA, a bone-specific differentiation marker, was observed intracellularly by using reverse transcription in situ PCR at 4 days in cells constrained by the pattern and not detected in unconstrained cells of similar projected area, but different NSI. Our data supports the concept of gene expression and protein synthesis based on optimal distortion of the nucleus, possibly altering transcription factor affinity for DNA, transport to the nucleus, or nuclear matrix organization. The combination of microfabricated surfaces, reverse transcription in situ PCR, and NSI measurement is an excellent system to study how transcription factors, the nuclear matrix, and the cytoskeleton interact to control gene expression and may be useful for studying a wide variety of other cell shape/gene expression relationships.

Active Transport, Cell Nucleus↗
PubMed1987

Polyspecific reactivity of a murine monoclonal antibody that binds to nuclear matrix-associated, chromatin-bound autoantigens.

To investigate polyspecific autoantibody interactions, we have characterized the binding of a cloned murine monoclonal IgM antibody termed (RTE-23) of strain BALB/c origin. By indirect immunofluorescence this antibody displayed a nuclear speckled and peripheral pattern in interphase cells from human and rodent cell lines. In contrast, in mitotic cells, antibody RTE-23 bound to the periphery of individual chromosomes. Immunoblot analysis of soluble and insoluble nuclear proteins from purified rat fibroblast nuclei showed that antibody RTE-23 bound to molecules of 28, 29, and 33 kDa. Furthermore, antibody RTE-23 demonstrated marked polyspecificity and reacted with cytoskeletal proteins (vimentin, keratin, actin), single-stranded DNA, specific synthetic polynucleotides, and cardiolipin. Antibody RTE-23 also showed a lupus anticoagulant-like activity. Screening of sera of autoimmune disease patients with antinuclear antibodies revealed two patients, both with SLE, whose sera blocked antibody RTE-23 binding to nuclei and recognized nuclear proteins identical to those recognized by antibody RTE-23. These results suggested that antibody RTE-23 displays a pattern of self-antigen binding that is represented as well in SLE patient sera.

Antibodies, Monoclonal↗
PubMed2001

FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent.

Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-alpha (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 approximately 5-6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.

Bacterial Proteins↗
PubMed2000

Colorectal cancer-associated nuclear antigen.

By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with M(r) of 35-40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix.

Antigens, Neoplasm↗
PubMed2001

The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions.

The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.

Cell Differentiation↗
PubMed2005

Expression of a novel biomarker, EPCA, in adenocarcinomas and precancerous lesions in the prostate.

PURPOSE: EPCA, a nuclear matrix protein, has been identified as a novel prostate cancer marker through protein profiling analyses comparing prostate cancer and its normal counterpart. A recent study of prostate biopsy specimens revealed that EPCA stained positive in the cancer negative biopsy cores of individuals with cancer at later biopsy. We clarified the relationship between EPCA expression and clinicopathological parameters, including grade and stage. Morphological characteristics of EPCA positive cells in noncancerous tissues were also analyzed. MATERIALS AND METHODS: Prostate tissue specimens were obtained from 50 patients with localized prostate cancer and 10 with invasive bladder cancer. EPCA expression was analyzed with a polyclonal antibody. Anti-P504S/anti-p63/anti-34betaE12 monoclonal antibodies were used for adjunct diagnosis for prostatic intraepithelial neoplasia. A monoclonal antibody, CD45RO, was used to confirm inflammatory infiltrates surrounding focal atrophic glands. RESULTS: EPCA staining was positive in the prostate samples of 94% of patients with prostate cancer but it was completely negative in samples from patients with bladder cancer. There was no correlation of EPCA staining intensity with Gleason grade or pT stage. In noncancerous tissues adjacent to major cancer foci EPCA was positive in 86% of prostate cancers. Most EPCA positive glands adjacent to cancer consisted of prostatic intraepithelial neoplasia and proliferative inflammatory atrophy. CONCLUSIONS: EPCA seems to reflect nuclear matrix alterations that occur in the earlier stage of prostate carcinogenesis. Individuals in whom the prostate is influenced by this field effect may be detected with this new biomarker.

Adenocarcinoma↗
PubMed2005

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato.

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.

Arabidopsis↗
PubMedDate not supplied

[Attachment of DNA loops to an artificial matrix does not affect the origin of replication initiation in early development of African clawed frog].

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.

Animals↗
PubMed1994

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Induction of apoptosis by antisense CK2 in human prostate cancer xenograft model.

Protein serine/threonine kinase CK2 (formerly casein kinase 2) is a ubiquitous protein kinase that plays key roles in cell growth, proliferation, and survival. We have shown previously that its molecular down-regulation induces apoptosis in cancer cells in culture. Here, we have employed a xenograft model of prostate cancer to extend these studies to determine whether antisense CK2alpha evokes a similar response in vivo. A single dose of antisense CK2alpha oligodeoxynucleotide given directly into the PC3-LN4 xenograft tumor in nude mouse induced a dose- and time-dependent tumor cell death in vivo. The tumor was completely resolved at the higher tested dose of the antisense. Cell death was due to apoptosis and correlated with a potent down-regulation of the CK2alpha message and loss of CK2 from the nuclear matrix in the xenograft tissue as well as in cancer cells in culture. These observations accorded with several of the earlier studies indicating that loss of CK2 from the nuclear matrix is associated with induction of apoptosis. Comparison of the effects of antisense CK2alpha oligodeoxynucleotide on cancer versus normal or noncancer cells showed that the concentration of antisense CK2alpha that elicited extensive apoptosis in tumor cells in culture or xenograft tumors in vivo had a relatively small or minimal effect on noncancer cells in culture or on normal prostate gland subjected to orthotopic injection of antisense oligodeoxynucleotide in vivo. The basis for the difference in sensitivity of cancer versus noncancer cells to antisense CK2alpha is unknown at this time; however, this differential response under similar conditions of treatment may be significant in considering the potential feasibility of targeting the CK2 signal for induction of apoptosis in cancer cells in vivo. Although much further work will be needed to establish the feasibility of targeting CK2 for cancer therapy, to our knowledge, this is the first report to provide important new evidence as an initial "proof of principle" for the potential application of antisense CK2alpha in cancer therapy, paving the way for future detailed studies of approaches to targeting CK2 in vivo to induce cancer cell death.

Animals↗
PubMed2004

AT islands - their nature and potential for anticancer strategies.

The human genome contains a unique class of domains, referred to as AT islands, which consist typically of 200-1000 bp long tracts of up to 100% A/T DNA. The significance of AT islands as potential targets for chemotherapeutic intervention stems from two main aspects. First, AT islands are inherently unstable (expandable) minisatellites that are found in various known loci of genomic instability, such as AT-rich fragile sites. Second, AT islands are involved in the organization of the genomic DNA on the nuclear matrix by acting as scaffold/matrix attachment regions, S/MARs. DNA duplexes of AT islands are unusually flexible and prone to base unpairing, which are crucial MAR attributes. Various AT islands show high binding affinity for isolated nuclear matrices and associate with the nuclear matrix in the cell. The cellular MAR function of AT islands may differ in cancer and normal cells. The abnormally expanded AT islands in the FRA16B fragile site in leukemic CEM cells act as strong, permanent MARs, while their unexpanded counterparts in normal cells are loop localized. Given their instability and involvement in the remodeling of the nuclear architecture, AT islands may be a factor in cancerous phenotypes. AT islands are preferentially targeted by the extremely potent DNA-alkylating antitumor drugs, bizelesin and U78779. High lethality of lesions in AT islands is consistent with the critical role of MAR domains in DNA replication. The abnormal structure/function of AT islands, such as their expansion and acquired strong MAR properties, may sensitize cancer cells to AT island targeting drugs.

Adenine↗