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PubMed2000

Applications of proteomics in oncology.

Genomics has expanded the field of molecular oncology, and proteomics is complementing genomics in the fields of elucidation of pathophysiology, gene function, molecular diagnosis and anticancer drug discovery. This trend is reflected in the establishment of the Human Tumour Gene Index by the National Cancer Institute (NCI), which is now followed by the Tissue Proteomics Initiative. Laser capture microdissection (LCM) provides an ideal method for extraction of cells from specimens in which the exact morphologies of both the captured cells and the surrounding tissue are preserved. Proteomic technologies can be applied for the further characterisation and analysis of proteins. LCM can also be combined with the protein chip technology. Proteomic technologies have been used for the study of cancer of various organs including the liver, prostate, breast, bladder and oesophagus. Some of the anticancer strategies are directed against proteases that facilitate several steps in cancer progression. Proteomic mapping of blood vessels in normal and malignant tissues can be used to identify tissue-specific markers on the endothelium that serve as potential targets for in vivo drug delivery. Studies of global protein expression in human tumours have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Genes that encode proteins that are overexpressed in tumours are being identified. Demonstration of tissue or cell type specific expression of some nuclear matrix proteins has led to the search for tumour specific nuclear matrix proteins. There is considerable activity in the commercial sector to develop diagnostic tests, as well as to facilitate anticancer drug discovery using proteomic technologies. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms.

Animals↗
PubMed2000

Signal transduction pathways and chromatin structure in cancer cells.

Molecular mechanisms controlling gene expression include cell shape, mechanical and chemical signal transduction pathways, chromatin remodeling, and DNA methylation. In this article, we will review the contribution of these molecular mechanisms and structural alterations in the malignant transformation of cells. The mechanical signaling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. The cytoskeleton array is a dynamic system that transmits signals from the cell exterior to nuclear DNA. The composition and function of this mechanical signaling pathway is altered in cancer cells. Chemical signaling pathways such as the Ras/mitogen-activated protein kinase (MAPK) pathway stimulate the activity of kinases that modify transcription factors, histones, and chromatin remodeling factors. Oncoproteins deregulating this signaling pathway set in motion a series of events that cumulate to chromatin remodeling and aberrant gene expression. J. Cell. Biochem. Suppl. 35:27-35, 2000.

Animals↗
PubMed1982

The specific binding of the thyroid hormones to matrix isolated from rat liver nuclei.

Specific binding sites for the thyroid hormones have been demonstrated in the liver nuclear matrix, a structural framework of the nucleus. When labelled 3,5,3'-tri-iodo-L-thyronine ([125I]T3) is injected into rats, 5% of the total nucleus bound T3 is bound to the matrix after 1 hour. However, when either isolated nuclei or isolated nuclear matrices were incubated with [125I]T3 in vitro, a 3- to 7-fold greater number of specific T3 binding sites were revealed in the nuclear matrix. The properties of the matrix-associated thyroid hormone binding sites were investigated in vitro. These binding sites showed limited capacity and high affinity for T3; the equilibrium association constant (Ka) was 1,3 x 10(9) M-1 and the binding capacity was 20,2 fmol T3 per 100 microgram matrix protein.

Animals↗
PubMed1996

Dynamics of human replication protein A subunit distribution and partitioning in the cell cycle.

Human replication protein A (RPA) is a three-sub-unit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In meta-phase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.

Cell Cycle↗
PubMed2001

The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs.

How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.

Active Transport, Cell Nucleus↗
PubMed1999

Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Cell Nucleus↗
PubMed2001

Chromatin fibers organization within the nucleus.

In order to evaluate at the ultrastructural level the three dimensional arrangement of the dispersed chromatin during the intephase, the immunogold detection of Bromodeoxyuridine (BrdU), of the DNA polymerase alpha and of the proliferating cell nuclear antigen (PCNA) was performed on human HL60 leukemia cells and nuclear matrices extracted from the same cellular model. The Field Emission In lens Scanning Electron Microscopy analysis of the ultrathin cryosectioned cells revealed the presence of a chromatin three dimensional network where the different constituents appeared repetitively assembled. Also the nuclear matrix showed a repetitive structure, on which the deprivation of the DNA corresponded to the selective loss of particular class sized fibers. The single or multiple combined immunolocalization of different structures involved in the DNA replication, where BrdU, DNA polymerase alpha and PCNA represent, respectively, the substratum, the polymerizing enzyme and a regulator of the reaction, allowed the understanding of its reciprocal spatial relationship on the dispersed interphasic chromatin and the role of the nuclear matrix in the replicative process.

Bromodeoxyuridine↗
PubMed1999

Nuclear localization of the spinocerebellar ataxia type 7 protein, ataxin-7.

Spinocerebellar ataxia type 7 (SCA7) belongs to a group of neurological disorders caused by a CAG repeat expansion in the coding region of the associated gene. To gain insight into the pathogenesis of SCA7 and possible functions of ataxin-7, we examined the subcellular localization of ataxin-7 in transfected COS-1 cells using SCA7 cDNA clones with different CAG repeat tract lengths. In addition to a diffuse distribution throughout the nucleus, ataxin-7 associated with the nuclear matrix and the nucleolus. The location of the putative SCA7 nuclear localization sequence (NLS) was confirmed by fusing an ataxin-7 fragment with the normally cytoplasmic protein chicken muscle pyruvate kinase. Mutation of this NLS prevented protein from entering the nucleus. Thus, expanded ataxin-7 may carry out its pathogenic effects in the nucleus by altering a matrix-associated nuclear structure and/or by disrupting nucleolar function.

Animals↗
PubMed1992

Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.

Animals↗
PubMed2002

S/MARt DB: a database on scaffold/matrix attached regions.

S/MARt DB, the S/MAR transaction database, is a relational database covering scaffold/matrix attached regions (S/MARs) and nuclear matrix proteins that are involved in the chromosomal attachment to the nuclear scaffold. The data are mainly extracted from original publications, but a World Wide Web interface for direct submissions is also available. S/MARt DB is closely linked to the TRANSFAC database on transcription factors and their binding sites. It is freely accessible through the World Wide Web (http://transfac.gbf.de/SMARtDB/) for non-profit research.

Animals↗
PubMed1987

Site and mechanism of growth inhibition by prostaglandins. III. Distribution and binding of prostaglandin A2 and delta 12-prostaglandin J2 in nuclei.

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 (9-deoxy-delta 9,12-PGD2) are taken up by cultured cells and exert growth inhibition. Within cells they are transferred to and accumulate in nuclei by a temperature-dependent process. In this study, the authors analyzed and compared subnuclear distribution and binding of these PGs. Nuclei accumulating either [3H]PGA2 or [3H] delta 12-PGJ2 were extracted successively with a hypotonic solution and with a 1% Triton X-100 solution, and the radioactivities in the two extracts (fractions of nucleoplasm and nuclear membrane, respectively) and the remaining residues (a fraction of chromatin and nuclear matrix) were determined. About three-fourths of PGA2-derived nuclear radioactivity was found in the extracts, and only 20% in the residues. Most of the radioactivity in the extracts was recovered in ethyl acetate under acidic conditions and identified as intact PGA2, suggesting that the majority of PGA2 in nuclei was present as free molecules. On the other hand, more than 70% of delta 12-PGJ2-derived nuclear radioactivity was associated with the residues and resistant to extraction with acidic ethyl acetate. This radioactivity, however, became extractable after brief alkali treatment and was identified as intact delta 12-PGJ2. In order to identify nuclear component binding of this PG, the authors digested the residues with either DNases or proteases. The radioactivity in the residues was almost completely released by digestion with proteases and clearly separated from DNA by ethanol precipitation. These results suggested that most of nuclear delta 12-PGJ2 bound covalently to protein(s) of chromatin and nuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Insulin stimulates incorporation of 32Pi into nuclear lamins A and C in quiescent BHK-21 cells.

The in vivo effect of insulin upon the incorporation of 32Pi into nuclear proteins was investigated in quiescent baby hamster kidney fibroblasts (BHK-21). Insulin has previously been shown to be mitogenic in these cells (Richman, R. A., Johnson, R. A., and Friedman, D. L. (1981) Proc. Soc. Exp. Biol. Med. 168, 196-203). Incorporation into two proteins (Mr = 62,000 and 72,000) in the 0.4 M salt-resistant nuclear fraction was enhanced 2-6-fold by insulin. The effect of insulin (20 nM) was observable within 5 min of treatment, reached a maximum at 15 min, and continued for at least 90 min. The half-maximal effect of insulin was obtained at a concentration of approximately 1 nM. Analysis of nuclear matrix preparations indicated that the two insulin-sensitive proteins were prominent nuclear matrix proteins and suggested that they were lamins A and C. This was confirmed by immunostaining with lamin antibodies and by two-dimensional electrophoresis. These studies indicate that insulin rapidly stimulates the incorporation of phosphate into nuclear lamins A and C in quiescent BHK-21 fibroblasts.

Animals↗
PubMed1992

HPV16 E7 phosphorylation in fission yeast: characterization and biological effects.

Human papillomavirus type 16 E7 protein (HPV-16 E7), synthesised in Schizosaccharomyces pombe, is both phosphorylated and targeted to the nucleus [Tommasino et al., Gene 93 (1990) 265-270] as is E7 protein synthesized in primate cells. Further analysis of E7 expression in fission yeast indicates that: (i) E7 protein synthesised in S. pombe is phosphorylated only on the Ser residues which are part of a casein kinase II consensus site, as it has been shown to be the cause in human cells, and is tightly associated with the nuclear matrix; (ii) synthesis of wild type, phosphorylated E7 is responsible for a significant increase in S. pombe doubling time; and (iii) E7 phosphorylation is not required for the tight association of the protein with the nuclear matrix, but E7 mutants, in which one (Ser31) or both phosphorylated serines (Ser31 and Ser32) have been substituted, lack any effect on cell-cycle duration.

Cell Cycle↗
PubMed2000

HET/SAF-B overexpression causes growth arrest and multinuclearity and is associated with aneuploidy in human breast cancer.

HET/SAF-B was originally cloned as a nuclear matrix protein that bound to matrix attachment regions and as a transcriptional repressor of the small heat shock protein hsp27. In addition, we have found recently that HET/SAF-B is also a corepressor of estrogen receptor activity. Estrogen receptor has a very well-described role in breast cancer, and aberrant expression of nuclear matrix and heat shock proteins has also been implicated in breast tumorigenesis. Therefore, we asked whether HET/SAF-B itself could be important in breast cancer. Toward this goal we examined its expression in breast cancer cell lines and asked whether HET/SAF-B can affect breast cancer cell proliferation. Finally, we studied HET/SAF-B expression in clinical breast cancer samples. HET/SAF-B protein and mRNA were detected at varying levels in all of the eight breast cancer cell lines examined. Using a number of different approaches to modulate the level of HET/SAF-B protein in the cell, we found that HET/SAF-B levels are inversely correlated with cell proliferation. In addition,transfection of HET/SAF-B fused to the green fluorescent protein led to the formation of multinucleated cells not observed in cells transfected with green fluorescent protein alone, suggesting that this effect is a direct result of HET/SAF-B overexpression. Western blot analysis of HET/SAF-B in 61 human breast tumors revealed widely varying levels of HET/SAF-B expression, with some tumors (16%) lacking any detectable HET/SAF-B. Statistical analysis showed that high HET/SAF-B expression in these tumors was associated with low S-phase fraction and with aneuploidy, consistent with our results from transfection experiments in tissue culture cells. We conclude that HET/SAF-B plays an important role in breast cancer, and we discuss possible mechanisms of the involvement of HET/SAF-B in cell proliferation and division.

3T3 Cells↗
PubMed1997

An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region.

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.

Amino Acid Sequence↗
PubMed2000

Alterations in subnuclear trafficking of nuclear regulatory factors in acute leukemia.

The nuclear matrix plays an important role in the functional organization of the nucleus in part by locally concentrating regulatory factors involved in nucleic acid metabolism. A number of nuclear regulatory proteins initially identified due to their involvement in human cancer are localized to discrete nuclear matrix-attached foci and correct nuclear partitioning likely plays a role in their function. Two such examples are promyelocytic leukemia (PML) and acute myelogenous leukemia-1 (AML-1; Runx1). PML, the target of the t(15;17) in acute PML, is localized to PML nuclear bodies (also termed Nuclear Domain 10 and PML oncogenic domains), a nuclear matrix-associated body whose function appears to be quite complex, with probable roles in cancer, apoptosis, and in acute viral infections. In t(15;17)-containing leukemic cells, the PML nuclear bodies are disrupted, but reform when the leukemic cells are induced to differentiate in the presence of all-trans retinoic acid. AML1 (RUNX1) is a key regulator of hematopoietic differentiation and AML1 proteins are found in nuclear compartments that reflect their roles in transcriptional activation and repression. The t(8;21), associated with AML, results in a chimeric transcription factor, AML-1/ETO (eight twenty one), that remains attached to the nuclear matrix through targeting signals contained in the ETO protein. When co-expressed, ETO and AML-1/ETO co-localize to a nuclear compartment distinct from that of AML1 or PML nuclear bodies. Interestingly, enforced expression of ETO or AML-1/ETO changes the average number of PML nuclear bodies per cell. Thus, chromosomal translocations involving AML1 result in altered nuclear trafficking of the transcription factor as well as other changes to the nuclear architecture. J. Cell. Biochem. Suppl. 35:93-98, 2000.

Animals↗
PubMed1989

Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells.

During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP proteins whose behavior is also rapidly modified by a heat shock at 45 degrees C, whereas no effect is observed at 42 degrees C. Using a monoclonal antibody, we show that in situ the antigens are available only when the cells are heat shocked at 45 degrees C. Subcellular fractionation shows that in normal cells the antigens are associated with the bulk of hnRNP (50-200S). During heat shock, whereas the overall characteristics of the bulk of preexisting hnRNP are unchanged, these antigens rapidly switch to a subpopulation of hnRNP with larger average size (50 to less than 300S) and increased stability. Structural analysis of the associated hnRNP in normal and stressed cells shows that in both cases the antigens are associated with the nuclear matrix subcomplex of hnRNP, which in situ is part of the internal nuclear matrix. Such hnRNP antigens, which are rapidly redistributed during a heat shock at the upper temperature range of the stress response, might well be involved in splicing and/or transport control.

Antibodies, Monoclonal↗
PubMed1984

Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.

Animals↗