[Role of the Nucleolus in Rearrangements of the IGH Locus].

The review summarizes the results from a series of studies focusing on the role that the nucleolus plays in maturation of the IGH locus and the choice of its partner genes in leukemia-associated translocations. The role of nuclear compartmentalization and nuclear localization of translocated oncogenes in ectopic activation of their transcription is discussed.

Genetic and Epigenetic Mechanisms of ß-Globin Gene Switching.

Vertebrates have multiple forms of hemoglobin that differ in the composition of their polypeptide chains. During ontogenesis, the composition of these subunits changes. Genes encoding different α- and ß-polypeptide chains are located in two multigene clusters on different chromosomes. Each cluster contains several genes that are expressed at different stages of ontogenesis. The phenomenon of stage-specific transcription of globin genes is referred to as globin gene switching. Mechanisms of expression switching, stage-specific activation, and repression of transcription of α- and ß-globin genes are of interest from both theoretical and practical points of view. Alteration of balanced expression of globin genes, which usually occurs due to damage to adult ß-globin genes, leads to development of severe diseases - hemoglobinopathies. In most cases, reactivation of the fetal hemoglobin gene in patients with ß-thalassemia and sickle cell disease can reduce negative consequences of irreversible alterations of expression of the ß-globin genes. This review focuses on the current state of research on genetic and epigenetic mechanisms underlying stage-specific switching of ß-globin genes.

Effect of Environmental Factors on Nuclear Organization and Transformation of Human B Lymphocytes.

Chromosomal translocations have long been known for their association with malignant transformation, particularly in hematopoietic disorders such as B-cell lymphomas. In addition to the physiological process of maturation, which creates double strand breaks in immunoglobulin gene loci, environmental factors including the Epstein-Barr and human immunodeficiency viruses, malaria-causing parasites (Plasmodium falciparum), and plant components (Euphorbia tirucalli latex) can trigger a reorganization of the nuclear architecture and DNA damage that together will facilitate the occurrence of deleterious chromosomal rearrangements.

HIV Tat induces a prolonged MYC relocalization next to IGH in circulating B-cells.

With combined antiretroviral therapy (cART), the risk for HIV-infected individuals to develop a non-Hodgkin lymphoma is diminished. However, the incidence of Burkitt lymphoma (BL) remains strikingly elevated. Most BL present a t(8;14) chromosomal translocation which must take place at a time of spatial proximity between the translocation partners. The two partner genes, MYC and IGH, were found colocalized only very rarely in the nuclei of normal peripheral blood B-cells examined using 3D-FISH while circulating B-cells from HIV-infected individuals whose exhibited consistently elevated levels of MYC-IGH colocalization. In vitro, incubating normal B-cells from healthy donors with a transcriptionally active form of the HIV-encoded Tat protein rapidly activated transcription of the nuclease-encoding RAG1 gene. This created DNA damage, including in the MYC gene locus which then moved towards the center of the nucleus where it sustainably colocalized with IGH up to 10-fold more frequently than in controls. In vivo, this could be sufficient to account for the elevated risk of BL-specific chromosomal translocations which would occur following DNA double strand breaks triggered by AID in secondary lymph nodes at the final stage of immunoglobulin gene maturation. New therapeutic attitudes can be envisioned to prevent BL in this high risk group.

Dual Role of the Extracellular Domain of Human Mucin MUC1 in Metastasis.

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains-the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1-TR) on the surface of low-invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1-TR molecule (MUC1-IR-TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002-4011, 2017. © 2017 Wiley Periodicals, Inc.

The microRNA-205-5p is correlated to metastatic potential of 21T series: A breast cancer progression model.

MicroRNA is a class of noncoding RNAs able to base pair with complementary messenger RNA sequences, inhibiting their expression. These regulatory molecules play important roles in key cellular processes including cell proliferation, differentiation and response to DNA damage; changes in miRNA expression are a common feature of human cancers. To gain insights into the mechanisms involved in breast cancer progression we conducted a microRNA global expression analysis on a 21T series of cell lines obtained from the same patient during different stages of breast cancer progression. These stages are represented by cell lines derived from normal epithelial (H16N2), atypical ductal hyperplasia (21PT), primary in situ ductal carcinoma (21NT) and pleural effusion of a lung metastasis (21MT-1 and 21MT-2). In a global microRNA expression analysis, miR-205-5p was the only miRNA to display an important downregulation in the metastatic cell lines (21MT-1; 21MT-2) when compared to the non-invasive cells (21PT and 21NT). The lower amounts of miR-205-5p found also correlated with high histological grades biopsies and with higher invasion rates in a Boyden chamber assay. This work pinpoints miR-205-5p as a potential player in breast tumor invasiveness.

Antagonistic functional duality of cancer genes.

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer "gene-chameleons", which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP(+)-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically "the drivers" of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and "paradoxical" effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.

Tightly bound to DNA proteins: possible universal substrates for intranuclear processes.

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.

Chromatin remodelling and DNA replication: from nucleosomes to loop domains.

Organization of DNA into chromatin is likely to participate in the control of the timing and selection of DNA replication origins. Reorganization of the chromatin is carried out by chromatin remodelling machines, which may affect the choice of replication origins and efficiency of replication. Replication itself causes a profound rearrangement in the chromatin structure, from nucleosomes to DNA loop domains, allowing to retain or switch an epigenetic state. The present review considers the effects of chromatin remodelling on replication and vice versa.

Rearrangement of chromatin domains during development in Xenopus.

A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.

Rearrangement of chromatin domains in cancer and development.

Both the accomplishment of developmental programs and neoplastic transformation are linked to changes in the long-range organization of chromatin, in particular, DNA loop domains. The development of new methods that allow the study of interactions between the bases of DNA loops and the proteins of the nuclear matrix will help our understanding of the molecular mechanisms in such changes. These methods should also allow the establishment of a fingerprint "signature" for many cancers that may serve for diagnostic purposes. J. Cell. Biochem. Suppl. 35:54-60, 2000.

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide.

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.

DNA topoisomerase II mutations and resistance to anti-tumor drugs.

Mutations in DNA topoisomerase II are often correlated with drug-resistance in tumor cell lines. Studies of topoisomerase II-mediated drug-resistance in various model systems, as well as the sequencing of such mutations from drug-resistant tumors, have shed light on the functional domains of topoisomerase II, on how it interacts with inhibitors, and on the different mechanisms by which cells avoid the toxic effects of many clinically important anti-tumor drugs.

A transcription-dependent DNase I-hypersensitive site in a far upstream segment of the chicken alpha-globin gene domain coincides with a matrix attachment region.

A site of hypersensitivity to DNase I has been found at the 5'-side of the chicken alpha-globin gene domain, ca. 8 Kbp upstream to the first gene. The presence of this site in different types of chicken cells correlates with the transcriptional status of the domain. A matrix attachment region (MAR) has also been found in the same subfragment, suggesting that it may be involved in the control of transcription of the chicken alpha-globin genes.

Topoisomerase I is associated with the regulatory region of transcriptionally active SV 40 minichromosomes.

Proteins bound to SV 40 DNA in sarkosyl-treated nuclei have been studied. The major component is topoisomerase I, a 60-70 kDa protein which possesses a strong DNA-nicking activity in the presence of sarkosyl and camptothecin. An SV 40 DNA fraction containing sarkosyl-resistant proteins constitutes 2 to 3% of the total nuclear SV 40 DNA and is enriched in transcriptionally active DNA (as monitored by distribution of RNase-resistant in vivo pulse-labeled RNA). An SV 40 DNA fraction, which is nicked due to covalent binding of topoisomerase I upon sarkosyl treatment is also enriched in transcriptionally active DNA. Topoisomerase I cleavage sites on SV 40 DNA which arise following sarkosyl treatment of nuclei or camptothecin treatment of infected cells have been mapped. The strongest site is located at nucleotide 381 on the non-coding strand and is framed by nuclease hypersensitive sites.