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In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.
The epipodophyllotoxin derivative VM 26 inhibits entry of mouse mastocytoma cells into mitosis in cell cultures at drug concentrations of 0.01--1 microgram/ml, the cells being arrested in G2 phase of the cell cycle. At higher concentrations, the compound exhibits spindle poison activity which manifests itself in a shortlasting rise in the number of cells arrested in metaphase of mitosis. These cells then disintegrate after a short period of time.
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We used peripheral blood (PB) and bone marrow (BM) smears in the development of two methods based on cytomorphology and esterase cytochemistry in combination with fluorescence in situ hybridization (FISH). The first method involves photodocumentation of May-Grünewald-Giemsa (MGG)-stained cells, followed by destaining in methanol-acetic acid, fixation in paraformaldehyde, and digestion with protease and RNAse before FISH using alpha-satellite probes that specify chromosomes X, 7, 8, and 17. On average, two hybridization signals were seen in 94.5% of disomic BM cells. The hybridization sensitivity was found to vary, however, both among morphologically defined hematopoietic cell lineages and among differentation levels within a lineage. In the second method, an esterase staining technique was followed by the same treatment as for MGG-stained cells. The esterases and FISH signals could be simultaneously visualized and the method was found suitable for rapid screening of in situ signals in cytochemically defined granulocytes and lymphocytes but not in monocytes. The combined methods proved very useful in elucidating the clinical significance of chromosomal abnormalities seen in two cases of leukemia.
Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM-a higher incidence of t(11;14) (33% vs 16%; P <.025) and of t(14;16) (13% vs 1%; P <.002) though incidences of t(4;14) were identical and a higher incidence of monosomy 13 (68% vs 42%; P =.005). Hypodiploid karyotypes and monosomy 13 may explain, at least in part, the poorer prognosis of primary PCL. In contrast, significantly longer survival was observed in patients displaying t(11;14) in comparison with those lacking this translocation (P =.001).
Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G(1) phase of the cell cycle (G(1)MTC) for which little is known. The present study shows that the G(1)MTC is intact in activated T lymphocytes from mice with the p21(waf-1) gene deleted. However, p21(waf-1) gene deletion does affect the ratio of cells that arrest at the G(1)MTC and the spindle checkpoint after MTD. The G(1)MTC arrests T lymphocytes in G(1) prior to cdc2 up-regulation and prior to G(1) arrest by p21(waf-1). Once cells have progressed past the G(1)MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G(1)MTC is also present in a human myeloid cell line deficient in p21(waf-1) gene expression. The p21-independent G(1)MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.
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We studied the use of fluorescence in situ hybridization (FISH) in CSF to enhance the diagnostic yield for the detection of malignancy on the first lumbar puncture in patients clinically suspected of having leptomeningeal metastases (LMM). Although repeated lumbar punctures were still needed in some patients, the use of FISH did speed up the diagnosis in approximately one-third of the patients clinically suspected of having LMM with atypical cells at first cytology. This eliminates the need for repeated lumbar punctures in these patients and enables an earlier start of treatment.
Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions.
In order to investigate the role of centrosome duplication in mitotic spindle morphogenesis, we designed a 1 hour pulse treatment protocol on synchronized HeLa cells with nanomolar doses of taxoids that might impair centrosome biogenesis but would allow the recovery of normal microtubule (Mt) dynamics before mitosis. We were prompted to use this approach as docetaxel (DOC; taxotereTM), a taxoid known to promote Mt polymerization, was shown to be more cytotoxic when applied during S phase. We show that pulse drug exposure is most efficient in late S and in G2 and results in a marked disorganization of the centrosome in G2, the pericentriolar material (PCM) being dissociated from centrioles. Separation of centrosomes at the G2-M transition is also impaired and mitotic spindle morphogenesis is grossly abnormal: although in most spindles chromosomes align in a metaphase plate, the two centrosomes stay most often unseparated at one pole and most of the NuMA protein accumulates at the other. Interestingly, we find that the centrosomes' ability to duplicate is not abolished as they are still able to trigger parthenogenetic development of frog eggs. Despite spindle asymmetry, the progression through mitosis is not blocked. This results in a catastrophic exit from mitosis, each mitotic cell generating several micronucleated cells linked together by multiple midbodies. Lack of mitotic block appears therefore as the prime cause of cell lethality. These experiments suggest that NuMA redistribution at the onset of mitosis depends upon the correct redistribution of PCM between centriole pairs. They also indicate that the presence of aberrant spindle poles does not alert the surveillance mechanism controlling the exit of mitosis.
The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.
Promyelocytic leukemia (PML) bodies are nuclear multi-protein domains. The observations that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery suggest that PML bodies function in transcription. We have used immuno-FISH in primary human fibroblasts to determine the 3D spatial organisation of gene-rich and gene-poor chromosomal regions relative to PML bodies. We find a highly non-random association of the gene-rich major histocompatibilty complex (MHC) on chromosome 6 with PML bodies. This association is specific for the centromeric end of the MHC and extends over a genomic region of at least 1.6 megabases. We also show that PML association is maintained when a subsection of this region is integrated into another chromosomal location. This is the first demonstration that PML bodies have specific chromosomal associations and supports a model for PML bodies as part of a functional nuclear compartment.
The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33 X 10(3) Mr) and X1 (37 X 10(3) Mr) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure.
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Nuclei of in vitro cultured bovine liver cells, deprived of the membranes by Triton X-100, were treated with 2 M-NaCl and DNase. Changes in ultrastructure and protein composition were studied at successive steps during treatment. Electron micrographs of nuclei treated with 2 M-NaCl showed a peripheral lamina and an internal system of randomly coiled filaments embedded in a mass of DNA fibres. After partial removal of the DNA the filaments could be seen to serve as backbones for the DNA attachment. Artificial redistribution occurring during fixation with glutaraldehyde suggests that the salt-resistant filaments are not stably cross-bridged into a three-dimensional network. The existence of reversible cross-bridges in vivo cannot be excluded, however. From the available data it is inferred that the filaments represent a decondensed from of the chromosome scaffolds and play a basic role in the organization of the genome throughout the nuclear cycle.