Hydrostatic pressure induces apoptosis in the human leukaemic T-cell line Jurkat via the mitochondrial pathway.

We investigated the effect of pressure levels ranging from 80 to 500 bar on the proliferative capacity and viability of Jurkat leukaemic T cells. Pressurization at 360 bar induced apoptotic cell death as shown by apoptotic morphology after Hoechst staining, DNA fragmentation in the TdT-mediated dUTP nick end labelling-assay and cleavage of several caspase substrates. Cell death could be prevented by the general caspase inhibitor zVAD-fmk. Breakdown of the mitochondrial membrane potential and the release of cytochrome c provided strong evidence for an involvement of the mitochondrial pathway, whereas a central role of the death receptor pathway was excluded because caspase-8 was not significantly activated. Pressure incubation led to calcium influx after 5 min, and we hypothesize that calcium influx could be the primary trigger for pressure-induced apoptosis.

The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells.

The mini-chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin-dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki-67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well-defined spatio-temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki-67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth-arrested, as confirmed by the absence of the Ki-67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth-arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki-67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth.

The complete DNA sequence of the Ectocarpus siliculosus Virus EsV-1 genome.

The Ectocarpus siliculosus Virus-1, EsV-1, is the type-species of a genus of Phycodnaviridae, the phaeoviruses, infecting marine filamentous brown algae. The EsV-1 genome of 335,593 bp contains tandem and dispersed repetitive elements in addition to a large number of open reading frames of which 231 are currently counted as genes. Many genes can be assigned to functional groups involved in DNA synthesis, DNA integration, transposition, and polysaccharide metabolism. Furthermore, EsV-1 contains components of a surprisingly complex signal transduction system with six different hybrid histidine protein kinases and four putative serine/threonine protein kinases. Several other genes encode polypeptides with protein-protein interaction domains. However, 50% of the predicted genes have no counterparts in data banks. Only 28 of the 231 identified genes have significant sequence similarities to genes of the Chlorella virus PBCV-1, another phycodnavirus. To our knowledge, the EsV-1 genome is the largest viral DNA sequenced to date.

Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.

Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules.

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.

DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.

The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells.

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

The proliferation-specific human Ki-67 protein is a constituent of compact chromatin.

The human nuclear Ki-67 protein (Ki-67p) is expressed in proliferating, but not in quiescent, cells and is therefore widely used as a proliferation marker in histopathological research and practice. However, information regarding its intranuclear location is scarce and controversial. Here we describe the results of cell fractionation and nuclease digestion experiments using nuclei isolated from human HeLa cells in interphase. Ki-67p dissociates at 0.3-0.4 M NaCl from its nuclear binding sites, and gradient centrifugations indicate that the released Ki-67p is most likely a single molecular entity and not complexed to other proteins. In nuclei, prepared under physiological salt conditions, the binding sites are largely resistant against micrococcal nuclease. However, when prepared at very low ionic strengths, chromatin regions with associated Ki-67p become accessible to micococcal-nuclease-producing chromatin fragments that carry bound Ki-67p. We conclude that Ki-67p is a chromatin protein and resides at densely packed regions, probably heterochromatin. Our data provide a useful basis for further biochemical research on this human nuclear protein.

The brown algal virus EsV-1 particle contains a putative hybrid histidine kinase.

The Ectocarpus siliculosus virus, EsV-1, occurs worldwide in all populations of the filamentous marine brown alga E. siliculosus. We have screened an expression library of EsV-1 restriction fragments and identified a DNA clone with the potential to code for a 52-kDa histidine protein kinase. The derived amino acid sequence includes all homology boxes diagnostic for histidine protein kinases and, in addition, amino acid motifs that are commonly found in response regulators of bacterial two-component signal transduction proteins. Thus, the novel viral protein can be classified as a hybrid histidine protein kinase of a type that has previously been detected in fungi, slime molds, and plants. By using purified antibodies, we found that the protein with its potential kinase activity is located on the outer shell of viral particles. This is the first report on a two-component regulator-like protein in viruses and could provide the basis for speculations with regard to the evolution of EsV-1 and related viruses.

The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner.

The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology is observed with chromatin but not with naked DNA and does not involve dissociation of core histones from chromatin. Moreover, these effects require histone H2A/H2B dimers in addition to histone H3/H4. We additionally tested whether the DEK protein affects DNA-utilizing processes and found that the DEK protein substantially reduces the replication efficiency of chromatin but not of naked DNA templates.

Characterization and immunolocalization of major structural proteins in the brown algal virus EsV-1.

The Ectocarpus siliculosus virus (EsV-1) is endemic in all populations of the cosmopolitan filamentous brown alga Ectocarpus siliculosus. EsV-1 has a large circular double-stranded DNA genome of about 320 kilobase pairs, and a complex virion structure with a central nucleoprotein core surrounded by several proteinaceous layers. To investigate the protein composition of the virion, we screened an expression library of EsV-1 with antibodies raised against purified detergent-disrupted viral particles. We isolated several clones encoding novel structural proteins and investigated two of them in detail. These clones encode viral proteins vp55 and vp74. Electron microscopy reveals that vp55 is most likely a component of the surface of the viral core, whereas vp74 may be part of an inner core structure. To initiate a genetic analysis, we sequenced regions of the EsV-1 genome encoding vp55 and vp74 and found several adjacent open reading frames with the potential to code for several interesting viral proteins including a putative calcium-binding protein, a collagen-like protein, and a RING finger protein.

Initiation of genome replication: assembly and disassembly of replication-competent chromatin.

Considerable progress has been made in research on the initiation of eukaryotic genome replication. This has generated a number of recent review articles. Here, we briefly summarize the major conclusions described in these articles and also include the results of more recent primary articles. The consensus view that has emerged is that a pre-replication complex assembles during the G1 phase of the cell cycle, making chromatin competent for replication. The complex consists of Orc proteins, Cdc6p, and the family of Mcm proteins. Chromatin, thus 'licenced' for replication, is guided into the S phase by the activation of cell-cycle-regulated protein kinases. Upon entry into S phase, the pre-replication complex is partially dissolved, first by the dissociation of Cdc6p and then by the gradual release of Mcm proteins. This appears to be accompanied by a recruitment of chain elongation factors and the establishment of replication forks.

Persistent virus integration into the genome of its algal host, Ectocarpus siliculosus (Phaeophyceae).

The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA molecule of about 320 kbp. After infection, which occurs in the unicellular spores or gametes, the virus is present latently in all somatic cells of the host. Virus multiplication is restricted to cells of the reproductive organs. It has been an open question whether the latent viral DNA occurs as a free episome or becomes integrated into the host genome. PCR studies showed that viral DNA co-migrates with high molecular mass DNA in pulsed-field gel electrophoresis, which confirms that latent viral DNA is integrated into the host genome.

Phosphorylation of replication protein A middle subunit (RPA32) leads to a disassembly of the RPA heterotrimer.

Replication protein A (RPA), the major eukaryotic single-strand specific DNA binding protein, consists of three subunits, RPA70, RPA32, and RPA14. The middle subunit, RPA32, is phosphorylated in a cell cycle-dependent manner. RPA occurs in two nuclear compartments, bound to chromatin or free in the nucleosol. We show here that the chromatin-associated fraction of RPA contains the phosphorylated forms of RPA32. Treatment of chromatin with 0.4 M NaCl releases bound RPA and causes a separation of the large and the phosphorylated middle RPA subunit. Unmodified RPA in the nucleosolic fraction remains perfectly stable under identical conditions. Phosphorylation is most likely an important determinant of RPA desintegration because dialysis from 0.4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphorylating conditions. Biochemical studies with isolated Cyclin-dependent protein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/CDK2, can phosphorylate human recombinant RPA in vitro. However, only a small fraction of in vitro phosphorylated RPA desintegrated, suggesting that phosphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subunit interaction are required for the proposed role of RPA during the polymerase switch at replication forks.

Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis.

Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-binding complex of eukaryotic cells that plays an important role in DNA metabolism by stabilising single-stranded regions of DNA. The functionally important binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory function. It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage. In this study we demonstrate that phosphorylation of RPA32 is rapidly induced during apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphosphorylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis. Phosphorylation of RPA32 begins in parallel to the degradation of DNA to high molecular weight fragments, and slowly continues until late apoptosis. Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation requires the activities of DNA-dependent protein kinase and of a cyclin-dependent protein kinase. Interestingly, the hyperphosphorylated, but not the less phosphorylated forms of RPA32, sediments independently from the trimeric complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.

Ser727-dependent recruitment of MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation.

Stat1alpha is a latent cytoplasmic transcription factor activated in response to interferon-gamma (IFN-gamma). The C-terminal 38 amino acids of Stat1alpha are required to trigger transcription and therefore may possibly serve as a transcription activation domain (TAD). Here we show that the C-terminus of Stat1alpha is an independent TAD which can interact with a specific group of nuclear proteins. Mutation of the Stat1 Ser727 and Leu724 decreases its transcriptional activity and affinity for the nuclear proteins. One of the interacting proteins was identified as MCM5, a member of the mini-chromosome maintenance (MCM) family involved in DNA replication. Both in vitro and in vivo interaction of Stat1alpha and MCM5 were demonstrated. Furthermore, the in vitro interaction required Ser727 and was enhanced by its phosphorylation. Transient over-expression of MCM5 enhanced transcriptional activation by Stat1alpha in a Ser727-dependent manner. Finally, changes in the level of nuclear localized MCM5 during the cell cycle correlated with the changes in transcriptional response to IFN-gamma acting through Stat1alpha. These results strongly suggest that MCM5 is recruited through interaction with Stat1alpha in a Ser727- and Leu724-dependent manner to play a role in optimal transcriptional activation.

Chromatin association of replication protein A.

Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease.

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.

Human minichromosome maintenance proteins and human origin recognition complex 2 protein on chromatin.

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.

Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.