Neidhard Paweletz

Tumor-related angiogenesis

In recent years, tumor-related angiogenesis has become an important field of research in oncology. It could be stated that growth of solid tumors is completely dependent on neovascularization to provide the tumor with all required nutrients. Special compounds (tumor angiogenesis factor[s]) are released by tumor cells into the environment to stimulate different types of normal cells to become active for the tumor. In particular, endothelial cells of neighboring capillaries are induced to react. They disintegrate their own basal lamina, detach from their neighbors, enter the extracellular matrix, and migrate toward the tumor mass. Cell divisions occur within such sprouts, thereby increasing the number of migrating endothelial cells. Strands of such cells are formed, and inter- and intracellular lumina develop. Loops of these hollow strands anastomose to form a network of new vessels which become connected with the blood circulation. The tumor mass thus becomes vascularized and can continue to grow. The prevention of neoangiogenesis has an enormous impact on cancer treatment by inhibiting the growth of the tumor. In this review, all important aspects of tumor-related angiogenesis are presented.

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice.

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.

Proteasome activator (PA28) subunits, α, β and γ (Ki antigen) in NT2 neuronal precursor cells and HeLa S3 cells

The catalytic activity of the 208 proteasome can be modulated by endogenous proteins. A proteasome activator protein termed PA28 or 118 regulator, composed of two homologous subunits (α and β) and a separate but related protein termed Ki antigen or PA28γ have been characterized. To explore the functional relationship of these proteins, NT2 clone Dl human neuronal precursor cells, as well as HeLa S3 cells were labeled by immunofluorescence and immunoelectron microscopy with three different antisera directed against peptides derived from their sequences. It was found that both PA28α and PA28β antisera label the cytoplasm and the nucleoli. In contrast, the PA28γ antiserum labels the nucleus but not the nucleoli while in the cytoplasm it labels two different classes of structures identified as microtubular-like extensions and inclusion bodies that are most likely autophagosomes. The latter do not contain proteasome δ subunit antigen. The microtubular-like structures colocalize with β-tubulin, are dispersed by nocodazole and are not affected by brefeldin A treatment. PA28α and PA28β are co-localized in the cell whereas PA28γ has a different distribution. PA28γ complexed with the proteasome may serve a function other than or in addition to activation and may also have a proteasome-independent function.

Transmission electron microscopic studies on the mitotic cycle of nucleolar proteins impregnated with silver

Hela cells were impregnated with silver according to Paweletz et al. (1967). In cells in mitosis not only the nucleolar organizer regions (NORs) are strongly impregnated but also part of the nucleolar material, which accumulates in and around the chromosomes. The treatment with adenosine, which in interphase cells spreads the nucleolar material within the nucleus, also distributes the argentophilic material in and around the chromosomes. During the reconstruction phase this material reassembles around the NORs to form parts of the new nucleolus. The silver impregnation technique clearly demonstrates that two main components are responsible for the argentophily of the nucleolus. This is in agreement with the results obtained by Lischwe et al. (1979).

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.

Cytological identification and characterization of the nuclear matrix

Abstract The presence of a matrix system in the nucleus has been cytologically demonstrated in intact untreated and α-amanitin-treated HeLa and rat kangaroo cells. The matrix is Feulgen negative and is stained very faintly with toluidine blue, whereas it can be brightly stained with haemalum or light green. It is highly susceptible to pepsin digestion but resistant to RNase. Ultrastructurally the matrix consists of 50–60 A thick fibrils. Some granules, which may be ribonuclear protein granules are also found associated with the fibrils. The matrix is essentially a network of acidic protein.

MEMBRANES IN THE MITOTIC APPARATUS OF MAMMALIAN CELLS

HeLa cells in mitosis are fixed by KMnO4 to obtain a good preservation of membranes. Inside and around the mitotic apparatus a vesicular, a tubular and a cisternal type of membranes are demonstrated. Small spherical or ellipsoidal vesicles are distributed at random in the spindle area. Zones around the poles and in the midbody of telophase cells are almost free from vesicles. Elements of the tubular ER are often aggregated in parallel arrays at the periphery of the spindle region, thus resembling dictyosomes. During telophase tubular ER is most prominent at the cleavage furrow. Layers of concentrically arranged flat cisternae surround the spindle region during the middle stages of mitosis. Radiating from the poles such membranes form large asters, thereby interrupting the concentric arrangement. This pattern might act as a barrier against larger cytoplasmic organelles or as a cisternal system to bring about an oriented transport of molecules from or to the poles. Fragments of the two large indentations of the nuclear envelops in prophase are aggregated in the spindle region and later on contribute to form new envelopes around the daughter nuclei.

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.

Membranes and microtubules of the mitotic apparatus of mammalian cells

HeLa cells in mitosis are fixed with glutaraldehyde followed by fixation in a saturated aqueous solution of KMnO4. In this combination microtubules are preserved and membranes stand out more clearly than after conventional fixation. The microtubular bundles between poles and chromosomes are accompanied by elongated cisternae of the ER, vesicles are found all over the spindle area, within the bundles of microtubules and in the neighborhood of kinetochores. In different regions of the mitotic apparatus microtubules are in close connection with membranes. Some vesicles are in direct contact with elongated cisternae, indicating a close relationship between the two different membranous systems. The role of vesicles as easily transportable “containers” is discussed.

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.