Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot.

ND10, PML bodies or PODs have become the defining nuclear structure for a highly complex protein complement involved in cell activities such as aging, apoptosis, the cell cycle, stress response, hormone signaling, transcriptional regulation and development. ND10 are present in many but not all cell types and are not essential for cell survival. Here, we review the cellular proteins found in ND10, their few known interactions and their contribution to the ND10 structure per se and to functions elsewhere in the nucleus. The discrepancy between the functions of the ND10 proteins and the nonessential nature of the structure in which they are aggregated at their highest concentrations leads to the conclusion that the proteins function elsewhere. The regulated recruitment of specific proteins into ND10 as well as their controlled release upon external induced stress points to a regulated nuclear depot function for ND10. These nuclear depot functions seem important as nuclear defense against viral attack and other external insults.

Evidence for separate ND10-binding and homo-oligomerization domains of Sp100.

Nuclear domains called ND10 or PML nuclear bodies consist of an aggregation of several proteins, most notably PML and Sp100. PML is essential in the nucleation and formation of ND10 as well as in the recruitment of other ND10-associated proteins such as Daxx, pRb, BLM and Sp100. In cells induced to overexpress Sp100, ND10 binding of Sp100 was saturable and excess Sp100 formed new aggregation sites devoid of other ND10-associated proteins, suggesting that homo-oligomerization is the basis for aggregation. To determine whether Sp100 binds to ND10 through hetero- or oligomerization, Sp100 deletion variants fused with GFP were transfected into cells with and without endogenous Sp100, and the localization of the GFP-labeled fragments was determined relative to ND10. Amino acids 29-152 were sufficient for deposition of the GFP-labeled fragments at ND10 in the absence of endogenous Sp100 (heterologous binding) and for self-aggregation (formation of new Sp100 deposits). None of the shorter fragments was deposited at ND10 or self-aggregated. The 29-152 amino acid fragment and some larger fragments, but not the full-size Sp100, induced elongation of ND10, which at their ends contain only Sp100, probably due to self-aggregation. By fusing a peptide consisting of the p53-binding domain from hMDM2 to the Sp100(29-152) fragment, this self-aggregation could be blocked while retaining the limited ND10 binding capacity, indicating that the Sp100 self-aggregation domain and the ND10 binding domain are separate entities. This fusion peptide was used to demonstrate the potential of ND10 to recruit p53 as a protein not usually present at this site. Such deposited p53 was protected from turnover. The capacity of ND10 to recruit Sp100 may serve primarily to reduce its availability.

[Monoclonal antibodies against protein Daxx and its localization in nuclear domains 10]. / Monoklonal'nye antitela k belku Daxx i ego lokalizatsiia v iadernykh domenakh 10.

Nuclear domains 10 (ND10) were first detected occasionally using antibodies to an antigen of unknown nature (Ascoli, Maul, 1991). Further on it was shown that ND10 were sites of locality of the number of proteins (PML, Sp 100, pRB) (Sterndorf et al., 1992; Kamitani et al., 1998), the majority of which are modified with ubiquitin-like small proteins-modifiers (SUMO) (Ishov, Maul, 1996). In addition, it was shown that ND10 were sites of primary localization, transcription and replication of some DNA-viruses (SV40, virus of simple herpes 1, adenovirus 5) (Ishov, Maul, 1996; Ishov et al., 1997). Except for SV40, these viruses produce proteins able to modify ND10, or leading to degradation of ND10-associated proteins (Maul et al., 1993; Maul, Everett, 1994). This degradation is accompanied with protein desumofication and, later, with hydrolysis on the ubiquitin-proteosomal way (Everett et al., 1998, 1999). Cell incubation with interferon leads to augmentation of the number and dimension of ND10 owing to increased expression of Sp100 and PML (Lavau et al., 1995; Grotzinger et al., 1996). In all, these data make it possible to put forward a hypothesis that ND10 may represent a peculiar cell storage ("dépôt") of proteins regulated according to the "accumulation-drop" principle (Ishov et al., 1997; Maul, 1998). However, this hypothesis requires further factual grounds.

Hepatitis B virus-transfected Hep G2 cells demonstrate genetic alterations and de novo viral integration in cells replicating HBV.

Hepatitis B virus (HBV) is a major etiological factor associated with hepatocarcinogenesis, but its role in the transformation process remains unclear. We previously documented the accumulation of genetic alterations in a HBV-transfected cell line. In the present study, we addressed the effect of HBV and its replication on the genome and phenotype of the host cell. Parental HBV-free Hep G2 cells and two HBV-transfected variant lines Hep G2215 and Hep G2T14. 1, which do and do not replicate HBV, respectively, were used to monitor genetic alterations in conjunction with HBV profile in vitro and in vivo. Comparison of in vitro growth rates showed that Hep G2T14.1 cells grew more rapidly, while Hep G2215 cells, replicating HBV, grew slower than parental Hep G2 cells. Molecular analysis confirmed an HBV integration site (s) in both variants, and reverse trancriptase-polymerase chain reaction (RT-PCR) amplification documented expression of transcript for the HBX protein, which has recently been implicated in the compromised efficiency of cellular DNA repair. Tumorigenisity testing indicate a comparable rate of tumor formation in nude mice of both HBV-transfected variants, giving rise to tumors in 3 weeks; parental Hep G2 cells did not form tumors in nude mice. Tumor tissue from nude mice injected with Hep G2T14.1 cells showed no change in HBV status. However, a new HBV integration site was detected in tumor tissue from Hep G2215-injected mice. Two cell lines derived from the respective tumor tissue grew in vitro at rates compatible to those observe before passage in nude mice. The Hep G2215 tumor-derived line continued to replicate HBV, while HBV status remained unchanged in the Hep G2T14.1 tumor-derived line. Unique genetic alterations were detected in both transfected cell lines, and Hep G2215 cells particularly showed cellular mosaicism and clonal selection when analyzed after the passage in nude mice. Further genetic alterations were detected in tumor-derived cell lines. Interestingly, the de novo genetic alterations in the Hep G2215 cells, which maintain the ability to replicate HBV, included a new HBV integration site, several chromosome rearrangements and loss of heterozygosity (LOH) of one p53 allele. Western analyses of p21/Waf1 protein indicate an upregulation of the protein in cells that replicate HBV. Based on the combined data, we hypothesize that the genetic alterations in the cellular genome could also be generated as a function in the presence of HBV and HBV replication. Possible mechanisms that could be implicated in cumulative mutagenetic events are discussed.

Review: properties and assembly mechanisms of ND10, PML bodies, or PODs.

Nuclear domain 10 (ND10), also referred to as PML bodies or PODs, are discrete interchromosomal accumulations of several proteins including PML and Sp100. We describe here developments in the visualization of ND10 and the mechanism of ND10 assembly made possible by the identification of proteins that are essential for this process using cell lines that lack individual ND10-associated proteins. PML is critical for the proper localization of all other ND10-associated proteins under physiological conditions. Introducing PML into a PML -/- cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10, attesting to its essential nature in ND10 formation. This recruitment includes Daxx, a protein that can bind PML and is highly enriched in condensed chromatin in the absence of PML. The segregation of Daxx from condensed chromatin to ND10 by increased accumulation of Sentrin/SUMO-1 modified PML suggests the presence of a variable equilibrium between these two nuclear sites. These findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML. Additional adapter proteins are suggested to exist by the behavior of Sp100, and Sp100 will provide the basis for their identification. Further information about the dynamic balance of proteins between ND10 and the actual site of functional activity of these proteins will establish whether ND10 function as homeostatic regulators or only in storage of excess proteins destined for turnover.

Investigation of the expression of Bin1, a putative suppressor, in human hepatoma cells.

Recent data suggest that Bin1, a novel C-MYC interacting protein, is a suppressor gene whose loss of expression is a frequent aberration associated with several malignancies. The mechanism responsible for loss of BIN1 expression is not understood. The purpose of this study is to investigate DNA profile of the BIN1 gene in human hepatoma Hep G2 cells, previously documented with lack of BIN1 expression. Chromosome and molecular analyses of Hep G2 cells were initiated to exclude the possibility of genetic alterations as a factor affecting BIN1 gene expression in these cells. We used Hep G2 cell line and its hepatitis B virus (HBV) transfected variants--Hep G2T14.1 and Hep G2215 cell lines. The cytogenetic localization of BIN1 was identified in the 2q14 region. Fluorescence in situ hybridization (FISH) with the chromosome 2 whole chromosome painting probe (WCP) demonstrated three or four intact copies of chromosome 2 in all three hepatoma cell lines studied. FISH analyses with the BIN1-specific probe of the Hep G2, Hep G2T14.1, and Hep G2215 metaphase chromosomes document no rearrangement of the BIN1 gene on any of the multiple copies of chromosome 2. FISH with the specific HBV probe did not identify the HBV integration site in Hep G2T14.1 and Hep G2215 cells within the BIN1 locus. Southern blot analyses revealed no genetic rearrangements in the BIN1 gene in any of the cell lines studied. Our RNA analyses (northern blot and RT-PCR) document lack of BIN1 message in Hep G2 cells in contrast to the presence of BIN1 in Hep G2T14.1 and Hep G2215 cells. No difference was identified in other transcripts analyzed, including c-myc. Analyses of BIN1 expression of Hep G2 cells at different passages were initiated and document low levels of BIN1 transcript in Hep G2 cells of passage < 85. Furthermore, BIN1 transcript was identified in additional seven HCC cell lines analyzed. Our data indicate that lack of Bin1 expression in HepG2 cells previously documented is a characteristic of cells of passage > 85 and is not due to genetic loss, or rearrangement within the BIN1 DNA sequence. Loss of the BIN1 transcript is not a characteristic of HCCs analyzed.

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

Molecular cloning and RARE cleavage mapping of human 2p, 6q, 8q, 12q, and 18q telomeres.

Large terminal fragments of human chromosomes 2p, 6p, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from II unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution.

DNA from a 50-kb yeast artificial chromosome (YAC) containing one human telomere was characterized. Cloned sequences from the centromeric end of this YAC (designated yRM2001) localized to several human chromosomes by somatic hybrid panel mapping. The telomeric end of the YAC contained both (TTAGGG)n sequences and the previously characterized TelBam3.4 subterminal repeat element. A novel low-copy repeat element (designated HC1103) mapped 19 kb from the telomeric end of the YAC. This repeat was shown by fluorescence in situ hybridization to be present in several subtelomeric regions (3q, 12p, 15q, 19p, and 20p) and at an interstitial site (2q13-->q14) in all individuals studied, but to be polymorphically distributed at several other telomeres. The YAC vector-insert EcoRI cloning site was positioned 50 kb to 70 kb from chromosome termini in human genomic DNA using RecA-assisted restriction endonuclease (RARE) cleavage analysis. Our results suggest that the DNA segment cloned in yRM2001 contains a novel block of low-copy DNA consistently present at some human telomeres, but polymorphically distributed at others.

Sequence organization of the human chromosome 2q telomere.

The terminal 240 kb of a human 2q telomere region was cloned in two overlapping yeast artificial-chromosomes (YACs). This DNA contains a region of low-copy subtelomeric repeats (within 50 kb of the 2q telomere), a segment of DNA duplicated on distal 8p23 (100 kb from the 2q telomere), and a region of single-copy DNA (230 kb from the 2q telomere). Two CpG islands are present in the DNA segment duplicated on distal 8p23. RecA-assisted restriction endonuclease cleavage of genomic DNA samples revealed a potential 55 kb chromosome length polymorphism at the 2q telomere. This work provides telomeric closure of maps for human chromosome 2q, demonstrates a novel, subtelomere-specific DNA duplication, and will permit detailed molecular and cytological studies of this human telomere region.

Physical analysis of the terminal 270 kb of DNA from human chromosome 1q.

DNA from three 1q44-derived human telomeric yeast artificial chromosome clones was analyzed using physical mapping methods. The smallest clone, yRM2004 (65 kb), corresponded exactly to the distal end of the largest clone, yRM2123 (270 kb). The third clone, yRM2192, overlapped with the proximal end of yRM2123 but not the distal end, suggesting that it is most likely a deletion artifact of a clone originally derived from a 1q telomere fragment. Data from fluorescence in situ hybridization analysis, restriction mapping, and RecA-assisted restriction enzyme cleavage experiments indicate that the molecular clone yRM2123 contains a 260-kb DNA fragment colinear with a genomic telomere-terminal fragment from 1q. yRM2123 contains low-copy subtelomeric and subterminal repeats at its distal end, single-copy DNA more centromerically, and a CG-rich region with homology to mouse DNA. Markers derived from this clone will allow telomeric closure of the physical and genetic linkage maps of human chromosome 1q.

[Analysis of the nucleotide sequence of a small colicinogenic plasmid Cold gene coding for lysis]. / Analiz nukleotidnoi posledovatel'nosti gena lizisa maloi kolitsinogennoi plazmidy Cold.

The nucleotide sequence of a DNA fragment of a small colicigonenic plasmid Cold-CA23 containing the lysis gene cdl has been defined. An open reading frame has been found within the fragment for 48 amino acid polypeptide with the molecular mass 4920 Da. The polypeptide is homologous to the lysis proteins encoded by the small colicinogenic plasmids ColE1 and CloDF13. The homology was also found for the structural and functional arrangement of the cdl gene and the plasmid CloDF13 lysis gene. The analysis of the possible secondary structures of the lysis protein encoded by cdl gene has revealed the amino acid sequences able to form the alternative structures. The described feature is supposed to be required for lysis protein translocation from cytoplasmatic to outer membrane of Escherichia coli cells.