Biomedical subjects

N Paweletz

Publications and source records attributed to N Paweletz.

At least 37 records · Page 2Linked to original sources

PubMed1996

An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells.

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.

Chymotrypsin↗

PubMed1996

Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome.

HeLa cells growing in vitro were treated with the peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). Immunofluorescence studies of treated cells revealed the formation of massive perinuclear aggregates rich in ubiquitin and proteasomal antigens, which on the ultrastructural level appeared as perinuclear aggregates of electron-dense material, usually in the vicinity of Golgi cisternae. Histochemical studies disclosed that these cells contained protein-rich perinuclear aggregates detected by amido black staining, while unusual accumulations of lipids, carbohydrates, or nucleic acids were not present. Inhibition of protein synthesis by cycloheximide prevented the formation of aggregates, whereas microtubule disruption by nocodazole induced a dispersion of the aggregates. We hypothesize that aggregates induced by PSI treatment correspond to accumulations of proteasome-substrate complexes in a well-defined region, where the proteolytic processes of the ubiquitin-proteasome pathway seem to be somehow centered. We propose to call this region the proteolysis center.

Blotting, Western↗

PubMed1995

A critical appraisal of synchronization methods applied to achieve maximal enrichment of HeLa cells in specific cell cycle phases.

Synchronization of mammalian cell cultures is a prerequisite for studies of molecular mechanisms of cell cycle control. Many researchers routinely use widely spread tumor cell lines like HeLa for these purposes, and a great variety of synchronization protocols has been described. Generally, they have been developed for monolayer cultures, usually with satisfactory results. However, we found that is not necessarily the case for cells cultivated in suspension. A critical appraisal of different standardized methods for selective enrichment of HeLa cells in suspension in all phases of the cell cycle has been undertaken. Our results reveal that only a few of the applied procedures can really yield high numbers of synchronized cells in G1, S, G2, and M phases, working with suspension cultures.

Cell Cycle↗

PubMed1995

Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes.

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A, -B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.

Animals↗

PubMed1995

Localization of proteasomal antigens during different phases of the cell cycle in HeLa cells.

We localized two different proteasome-associated epitopes with the use of two specific monoclonal antibodies, MCP21 and anti-p25, in interphase and mitotic HeLa cells cultured in vitro. We provide evidence for an association of those antigens with the spindle poles in metaphase and with the midbody region in anaphase and telophase. Triton-nonextractable structures are also labeled in interphase. Moreover, the labeling pattern of interphase cells obtained with the monoclonal antibodies MCP21 and anti-p25 differs considerably, and in the case of MCP21 displays clear cell cycle-specific changes: nuclear labeling is absent or very weak in G1 and S phases of the cell cycle, whereas it is strong in G2 phase. This might reflect the existence of different subpopulations of proteasomes in the cell.

Antibodies, Monoclonal↗

PubMed1994

Nucleolar organization as revealed in cycloheximide treated cells.

The nucleolar organisation has been studied in cycloheximide treated rat kangaroo cells. Within 1 h of treatment most nucleoli become loose and reveal a nucleolonemal network. With longer treatment the granules are scarce and the nucleolonemal network becomes more prominent. The network reveals units comparable to the rDNA transcriptional units in length. The nucleolonemal units consists of tufts of fibrils. When cycloheximide is withdrawn, granules reappear and obliterate the nucleolonemal network.

Animals↗

PubMed1994

Making first contacts between the spindle and the chromosomes in HeLa cells.

To guarantee an ordered bipartition of the genetic material during mitosis, the chromosomes must be incorporated into the mitotic spindle. In HeLa cells, this process starts early in prophase when the nuclear envelope is still nearly complete, but only a few small holes in the double membranes offer access to the chromosomes for individual microtubules growing out from the poles. Inside the nuclear domain, these microtubules make contact with the kinetochore/centromere complexes which can be found in the vicinity of the hole. These complexes seem to be distributed at random during early prophase until early prometaphase. Therefore, the chromosomes become incorporated in a sequential order. No accumulation of the complexes in the nucleus near the centrosomes can be recognized. The individual microtubules attach tangentially to the kinetochores. This contact can already take place before the kinetochore is fully developed.

Centromere↗

PubMed1994

Defined tumor cell-host interactions are necessary for malignant growth.

Analyzing the different steps of malignant growth (primary tumor, metastasizing tumor cells, secondary tumor), one recognizes an intense interaction between normal and malignant cells. Tumor cells not only induce activities of normal cells, which normally are rarely activated, but also they exploit properties of normal cells for their own purposes. The major mechanisms and processes of this "parasitism" are described in more detail and the results are discussed. Tumors cannot grow beyond a certain size without a supply of blood and lymph vessels by the host (angiogenesis). Metastasizing tumor cells cannot leave the vessel (extravasate) in which they are transported without the cooperation of the respective endothelial cells of the host. An appropriate environment formed by the host tissues is essential for the settlement of tumor cells at secondary sites. Historically, these are a few examples that show intense cooperation between host and tumor. More are given in the present contribution.

Animals↗

PubMed1993

Sequence of centromere separation. Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse.

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.

Animals↗

PubMed1993

Failure of sister chromatid separation leads to formation of diplochromosomes in colcemid treated PtK1 cells.

The origin of diplochromosomes has been traced in multinucleate rat kangaroo cells (PtK1) obtained after colcemid treatment. In these cells the diplochromosomes were shown to originate from restitution nuclei, indicating that they were formed due to the omission or failure of sister chromatid separation and not due to endoreduplication. In this context the mechanism of sister chromatid separation has been discussed. The independence of this mitotic event from other associated processes, such as chromosome condensation, nuclear envelope breakdown or spindle formation has been stressed.

Animals↗

PubMed1993

Failure of centromere separation leads to formation of diplochromosomes in next mitosis in okadaic acid treated HeLa cells.

High concentrations of okadaic acid, sufficient to inhibit phosphatase 1 and 2A activities, induces formation of diplochromosomes in HeLa cells. It has been shown that this is due to a failure of sister chromatid separation in earlier mitosis in the presence of okadaic acid in the medium and not due to bypassing of mitosis (endoreduplication). Moreover, it has been demonstrated that the sister chromatid adherence does not depend on any under-replicated chromatin segment shared by the sister chromatids which might happen in okadaic acid induced premature mitosis, but due to the failure of the centromeres to separate at metaphase-anaphase transition. The role of phosphatase 1 in sister chromatid separation has been discussed.

Centromere↗

PubMed1992

Failure of kinetochore development and mitotic spindle formation in okadaic acid-induced premature mitosis in HeLa cells.

The mitotic events associated with okadaic acid (OA)-induced premature chromosome condensation (PCC) in S-phase-blocked HeLa cells were studied at the light microscope, immunofluorescence, and electron microscope level. The development of PCC in these cells has been compared with that in multinucleate cells and also in uninucleate hamster cells induced by caffeine. In OA-induced PCC, the nuclear envelope breaks down and chromosomes condense, but the mitotic spindle and trilaminar kinetochores fail to develop. In S-phase PCC in multinucleate cells, only the mitotic spindle does not develop, whereas in caffeine-induced PCC, all these events are found to be associated. The possible difference in their pathways of induction and, in this connection, the dissociability of the early mitotic events have been discussed.

Caffeine↗

PubMed1992

Okadaic acid inhibits sister chromatid separation in mammalian cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid inhibiting phosphatase 2A activity alone or in addition to phosphatase 1 activity. Phosphatase 2A inhibition alone had no visible effect on mitosis, but inhibition of both phosphatase 1 and 2A produced mitotic abnormalities, including inhibition of anaphase mimicking the effect of colchicine. Recovery experiments in okadaic acid-free medium showed formation of diplochromosomes, indicating a failure of sister chromatid separation in the treated mitotic cells. The universality of the phosphatase 1 requirement in sister chromatid separation is discussed.

Chromatids↗

PubMed1992

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.

Cell Nucleolus↗

PubMed1992

Effects of okadaic acid on mitotic HeLa cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid (OA), known to inhibit phosphatase 1 and 2A activities. The cytological effects on the course of mitosis were studied at the light microscopic, immunoflourescence and electron microscopic levels. At the lowest concentration used (1 nM), OA did not show any effect on mitosis, but at higher concentrations it showed pronounced effects. The mitotic chromosomes became scattered, the mitotic spindle became deranged and the cells failed to enter anaphase. At the electron microscopic level formation of isolated microtubules and regular trilaminar kinetochores were observed. An extensive growth of the endoplasmic reticulum could be noted in these cells. Decondensation of chromatin and nuclear envelope re-formation could be seen only after withdrawal of OA. A high frequency of multinucleate cells could be found after 24 h of recovery. Cells treated with 100 nM OA for 3 hours showed diplochromosomes in over 50% of mitotic cells after 24 h recovery. These were presumably formed due to the failure of sister chromatid separation in the earlier mitosis in the presence of OA. At the electron microscopic level the diplochromosomes showed a quadruplet structure. The role of phosphatase 1 in controlling some late mitotic events, i.e. sister chromatid separation, MPF-inactivation and nuclear envelope re-formation etc., is discussed.

Chromosomes, Human↗

PubMed1992

Deuterium oxide (heavy water) arrests the cell cycle of PtK2 cells during interphase.

Deuterium oxide (D2O, heavy water) exerts an antiproliferative effect on a variety of cells in vitro and on some organisms. This effect is mainly ascribed to a tubulin-mediated antimitotic action. We evaluated the morphology, the mitotic activity, and the dynamics of the cell cycle of PtK2 cells grown in vitro in the presence of 75% D2O for up to eight weeks by microspectrophotometric DNA measurements as well as flow cytometric analysis and a determination of mitotic indices. Substitution of heavy water for water in the culture medium initially increased the mitotic index by a (pro-) metaphase block but after 2 to 3 days of incubation no mitotic figures were seen. Analysis of cells grown for 6 days in medium containing 75% D2O revealed accumulation of cells in S/G2-phase. Extended treatment stabilized the high level of cells in this specific phase, when compared to normal growing cells. Cells grown for 1 to 6 weeks in the presence of D2O remained non-proliferating, nevertheless, they were able to divide again after recovery in non-deuterated medium. The time needed for resumption of the mitotic activity was proportional to the duration of deuterium oxide exposure. Cells incubated for 8 weeks in 75% D2O did not recommence mitotic activity. Light and electron microscopic examination revealed characteristic morphological changes of size and ciliation in PtK2 cells subjected to prolonged deuteration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Derangement of microtubule arrays in interphase and mitotic PtK2 cells treated with deuterium oxide (heavy water).

The extent and pattern of the rearrangements of microtubule arrays in interphase and mitotic PtK2 cells treated with deuterium oxide (2H2O) were evaluated using light, immunofluorescence and electron microscopy. Combined labelling with anti-tubulin antibodies and staining with a DNA-specific fluorochrome revealed that 2H2O influences the reassembly of the cytoplasmic microtubule complex (CMTC) of interphase cells after depolymerization of microtubules (MTs) with nocodazole. In cells entering mitosis in the presence of 75% 2H2O the conversion of the CMTC into the mitotic spindle was affected, resulting in a retardation of the prophase/prometaphase transition. (Pro)metaphase cells did not assemble a regular mitotic spindle and the metaphase/anaphase transition was blocked. Immunofluorescence and ultrastructural studies suggest that separation of centrosomes, nucleation of MTs around centrosomes, organization of MTs into the mitotic spindle, as well as the ultrastructure and positioning of the mitotic poles, are affected in deuterated PtK2 cells. In comparison with control cells, a significantly higher proportion of multipolar divisions was found after stimulation of proliferation in the presence of 25-50% 2H2O or during recovery after a long-term exposure to 75% 2H2O. On the basis of these results we discuss the mechanism of the antimitotic action of deuterium oxide and suggest that, apart from perturbation of MT polymerization, it could also encompass disturbances in MT reorganization, most probably by impairment of the microtubule-organizing centres (MTOC).

Animals↗

PubMed1991

First steps of tumor-related angiogenesis.

We present morphological data of the early steps of tumor-induced angiogenesis and show the distribution of the three main components of the basal lamina (BL), laminin, collagen IV, and fibronectin during these early processes. Tumor cells of a line of BSp73 AS a nonmetastasizing tumor isolated from a pancreatic adenocarcinoma were injected subcutaneously into the back of BDX rats. Two days after tumor inoculation, the BL of the dilated mother vessels around the whole circumference of the vessel has either disappeared, become fragmented, or developed several successive layers. By immunoelectron microscopy, we demonstrate that the fragmented and multilayered BL is strongly stained for laminin and collagen IV but less strongly for fibronectin. Around the surface of the dilated mother vessels which are free of any detectable BL material (by electron microscopy standards), we can see accumulation of all three components in the connective tissue. Simultaneously with the alteration of the BL, the proliferation of the endothelial cells (EC) and the pericytes and the migration of the EC from the wall of the mother vessel have started. EC migration begins in two different ways. Either one EC migrates from the wall of the mother vessel into the surrounding connective tissue, or two or more EC form nearly parallel processes toward the connective tissue. The tips of these processes are connected by intracellular junctions. Around the cellular protrusions of these cells material of the BL deposited into the nearby connective tissue can be observed neither by conventional nor by immunoelectron microscopy. During the outgrowth and migration, the EC remain in contact via junctions with the EC of the original vessel. When migration during which the EC retain their polarization continues, a slit-like lumen forms immediately between the migrating EC. This lumen always remains in direct connection with the lumen of the mother vessel. It is sealed at its border by intercellular junctions. Such junctional complexes can develop a length (in sections) of several hundred micrometers. A BL detectable in the electron microscope can neither be found around the tip of the migrating EC nor around young capillaries not yet surrounded by pericytes. By immunoelectron microscopy, however, only the cellular protrusions at the tip of migrating EC are free of deposited material of the BL. The basal surface of longer (new) capillaries is covered by a continuous layer of amorphous material.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗