ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease.

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi-1 leukemic cell lines, the last from a t(8;21) AML. Biochemical analysis confirmed specificity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipitations of metabolically labeled 32P-proteins from Kasumi-1 cells show that AML1 and ETO are phosphorylated on serine and threonine. Investigations with normal bone marrow reveal AML1 and ETO are coexpressed in megakaryocytes and that each is expressed in a portion of the approximately 10-microns-diameter cells residing there. Using a CD34+ enriched population mobilized to peripheral blood, we found AML1 and, unexpectedly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic. Also, because ETO would not be exclusively expressed as part of chimeric AML1/ETO in leukemic patients, its presence cannot be used to monitor t(8;21) AML residual disease.

Nuclear envelope structure.

The past 18 months have seen significant advances in our knowledge of the constituents of the nuclear envelope, their interactions during interphase and the mechanisms involved in their mitotic dynamics. Although most of the new data are in general agreement with, and contribute detail to, our traditional image of the nuclear envelope, a few observations appear to mark the beginning of new and important directions in research.

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

Dynamic aspects of cytoskeletal and karyoskeletal intermediate filament systems during the cell cycle.

IF are major cytoskeletal and karyoskeletal components of eukaryotic cells (Steinert and Roop 1988). In numerous instances, their constituent protein subunits have been shown to be substrates for a variety of kinases such as A-kinase, C-kinase, and Ca++/calmodulin kinase (Geisler and Weber 1988; Inagaki et. 1988; Ando et al. 1991), as well as p34cdc2 (Chou et al. 1990; Peter et al. 1990; Ward and Kirschner 1990; Dessev et al. 1991). To date, all of the phosphorylation sites that have been mapped are in the non-alpha-helical amino- or carboxy-terminal domains (Steinert 1988; Ando et al. 1989, 1991; Geisler et al. 1989; Chou et al. 1991), and these secondary modifications can lead to IF reorganization and/or disassembly in vivo and in vitro (see, e.g., Iganaki et al. 1988; Lamb et al. 1989; Chou et al. 1990; Peter et al. 1990; Heald and McKeon 1990; Dessev et al. 1991). In addition, it is possible that the exchange seen between subunits and polymerized IF in interphase following the microinjection of unpolymerized protein (Vikstrom et al. 1989; Miller et al. 1991) may also be regulated in some fashion by phosphorylation/dephosphorylation reactions. In cultured fibroblasts such as BHK-21, the interphase equilibrium state that favors IF polymerization is shifted dramatically to a disassembled state in mitosis, apparently due to enhanced phosphorylation at specific sites mediated through the activity of p34cdc2. However, in other cells in mitosis, such as HeLa, the mechanisms involved in the regulation of cytoskeletal IF remain unclear. Therefore, no one common mechanism appears to be responsible for IF regulation during cell division. On the basis of the majority of data available, it appears that the regulation of IF phosphorylation plays an important role in the regulation of the supramolecular organization of IF cytoskeletal and karyoskeletal networks, especially in the remodeling events that take place as cells enter and exit mitosis. Although the functional significance of IF phosphorylation during interphase is not as obvious as it is in some mitotic cells, we are tempted to speculate that there may be a connection with mechanisms involved in signal transduction, since IF proteins appear to be targets for kinases known to be activated by second messengers such as Ca++ and cAMP.

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown.

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam.

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues. Synthesis of L67 is detectable in embryos 2h after fertilization; it reaches a maximum in 6h-old embryos and gradually declines thereafter. These results argue that the composition of the NL bears no obvious relationship to the structural and functional changes that take place during the embryonic development of this invertebrate.

Effect of Calcium on the Stability of the Vitelline Envelope of Surf Clam Oocytes.

Fertilization and parthenogenic activation of oocytes of the surf clam, Spisula solidissima, require the presence of calcium in the extracellular medium. Here we report that the depletion of calcium causes a dramatic increase in the stability of the vitelline envelopes (VE). On the basis of this effect, we have developed a method of isolating intact VE and have studied their morphology, composition, and properties. Experiments using 45Ca2+ have revealed that isolated VE bind calcium in a weak, but specific way. These findings suggest that the function of calcium may be to maintain the oocyte surface in a fertilization-competent state, while the reactions subsequent to the initial activation event, and leading to nuclear envelope breakdown (NEBD), may not require calcium. In support of this hypothesis, we have demonstrated that hypertonic conditions induce the oocytes to undergo NEBD in the absence of extracellular calcium.

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system.

Nuclei isolated from oocytes of the surf clam Spisula solidissima are disassembled when exposed to extracts from maturing oocytes. In the course of this process the nuclear lamina undergoes a marked reduction in size and the nuclear membrane appears to be fragmented into vesicles. These events are accompanied by extensive phosphorylation of the oocyte 67-kDa lamin and its solubilization. The changes observed are similar to those which occur in vivo in activated Spisula oocytes. Nuclear envelope breakdown in vitro requires ATP and Mg2+, but not Ca2+. It is not affected by protease inhibitors and is inhibited by alkaline phosphatase.

Meiotic breakdown of nuclear envelope in oocytes of Spisula solidissima involves phosphorylation and release of nuclear lamin.

During meiotic nuclear envelope breakdown (NEBD) in maturing oocytes of the surf clam, Spisula solidissima, the 67-kDa lamin is extensively phosphorylated, concurrently with its solubilization. This is accompanied by a reduction of the nuclear diameter. Quercetin, a protein kinase inhibitor, does not affect lamin phosphorylation and release, nor NEBD per se, but specifically inhibits the early phosphorylation of a set of proteins, on which NEBD seems to depend. Our results suggest that meiotic NEBD in Spisula oocytes may be controlled by a mechanism which involves lamin phosphorylation, similar to that which is thought to operate in mitosis.

Protein kinase activity associated with the nuclear lamina.

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorylation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo.

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells.

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells.

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of single-stranded regions in the course of digestion of DNA with DNAase II and micrococcal nuclease.

In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy. Most of the ss stretches are located internally in the DNA molecules. The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.

Removal of histone H1 exposes linker DNA in chromatin to DNAse I.

After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.p. and multiples, similar to those obtained with micrococcal nuclease. Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.

Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol.

Mercaptoethanol, when present in the sample buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is responsible for the appearance of two nonprotein bands (electrophoretic mobilities corresponding to 68 and 54 kdalton) stainable with silver and with Coomassie blue. After iodination in vitro of DNA preparations isolated by alkaline phenol extraction using chloramine-T procedure, part of the radioactive label is found in these bands, provided the reaction is terminated by mercaptoethanol, whereas only a diffuse background is present in this area if the reaction is stopped by sodium metabisulfite. Similar results are obtained with highly purified total cytoplasmic RNA. The results indicate that the appearance of the 68- and 54-kdalton bands is in artifact. The relevance of these results to the proteins tightly bound to DNA is discussed.

Effect of chromatin decondensation on the intranuclear matrix.

We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA. When nuclei swollen in EDTA are digested with DNAse II in the presence of EDTA, structures devoid of internal network are obtained even without subsequent treatment with high salt. When swollen nuclei are exposed to Mg++ a specific recondensation of chromatin takes place. The residual structures from recondensed nuclei are similar to those isolated from control nuclei in the presence of Mg++. The results suggest that the integrity and stability of the intranuclear matrix are acquired in the course of the isolation procedure and this is favoured by chromatin condensation.

Interaction between lysine-rich histones and DNA.

Using a membrane filter retention technique we have studied the interaction between DNA and lysine rich histone H5 in vitro. It is found that, depending on the ionic conditions, H5 can bind DNA in a random or cooperative manner and exhibits a preference to DNA with high molecular weight and/or high A + T content, as also observed with H1. The presence of 6 M urea in the assay mixture does not impair the selectivity of H5 to A + T rich DNA but partly affects its selectivity to DNA size. In contrast to H1, H5 does not discriminate between the superhelical and relaxed forms of circular SV40 DNA.