Role of topoisomerase II in the structural and functional evolution of mitogen-stimulated lymphocyte nuclei.

To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 microM. DNA synthesis, however, was already significantly inhibited at 0.05 microM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [3H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 microM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo II beta isoform is involved in the very early phases of lymphocyte activation, with function of the Topo II alpha isoform, which is more sensitive to VM26, being required for progression through S phase.

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.

Taxol protects the microtubules of concanavalin A-activated lymphocytes from disassembly by methylmercury, but DNA synthesis is still inhibited.

We have shown previously that there is a good correlation between the degree of microtubule disassembly by methylmercury (MeHg) and the extent of inhibition of DNA replication in Concanavalin A (Con A)-stimulated mouse splenic lymphocytes. The purpose of this study was to determine if these two events are causally related and to examine the effects of MeHg-induced microtubule disassembly on earlier events of the stimulation process. We show that early steps constituting the activation pathway, such as the Con A-induced increase in Ca2+ influx and the expression of interleukin 2 receptor, are not inhibited by concentrations of MeHg that disassemble microtubules. RNA synthesis is not affected by short-term (3 h) treatment with MeHg, but longer treatment (24 h) inhibits RNA synthesis. In contrast, DNA synthesis is effectively inhibited by a 3-h treatment with MeHg. In lymphocytes treated with taxol, microtubules are not disassembled by MeHg; however, the inhibition of RNA and DNA synthesis persists. We conclude that the inhibition of nucleic acid synthesis by MeHg is not causally related to MeHg-induced microtubule disassembly.

Effects of methyl mercury on the microtubule system of mouse lymphocytes.

The effects of in vivo and in vitro methyl mercury (MeHg) treatments on the microtubule system of murine splenic lymphocytes were examined by immunofluorescence microscopy. In vitro exposures to 1 to 10 microM MeHg resulted in time- and concentration-dependent microtubule disassembly. Lymphocytes isolated from mice receiving a single 10 mg/kg injection displayed microtubule damage when examined 2 and 5 days post-treatment. The capacity of in vivo and in vitro treated lymphocytes to respond to the mitogen concanavalin A (Con A) was generally inhibited by MeHg. There was a good correlation between the degree of microtubule disassembly and the inhibition of mitogen responsiveness. In vivo and in vitro treatments that resulted in extensive microtubule damage suppressed the ConA response and blocked lymphocytes early in the stimulation sequence. In vitro MeHg treatment late in mitogenesis caused a rapid, concentration-dependent inhibition of [3H]thymidine incorporation. These results suggest that damage to the microtubule system can serve as an indicator of MeHg toxicity and may underlie the toxicant's effects on lymphocyte functions.

Monoclonal antibodies against nuclear matrix detect nuclear antigens in mammalian, insect and plant cells: an immunofluorescence study.

We have applied monoclonal antibodies generated against nuclear matrix in an immunofluorescence study of a variety of plant and animal species. Antibodies P1 and I1 detected antigens in all species examined, including higher and lower plants. Antibodies PI1 and PI2 stained only animal cells, and showed some tissue and/or species-specific variability in staining pattern. The presence of similar nuclear matrix components in such diverse species suggests that nuclear order may be maintained by similar mechanisms in all eukaryotes.

Localization of nuclear antigens during preparation of nuclear matrices in situ.

Nuclear matrix structure closely resembles the organization of nonchromatin components of nuclei in situ. However, reports on the extent to which nuclear components are reorganized during matrix isolation have produced conflicting results, and the reality of an in situ nuclear matrix is still in question. We have prepared nuclear matrices by processing cells still attached to the growth substrate through the extraction steps, thus avoiding mechanical disruption due to homogenization and centrifugation. Furthermore, the extensive residual cytoskeleton seems to keep the residual nuclei "stretched out" so that they retain many features of intact nuclei. Indirect immunofluorescence staining was used to compare the distribution of nuclear antigens in intact nuclei with their organization in nuclear matrices, as well as at each stage of nuclear matrix preparation. We have applied monoclonal antibodies P1, I1, PI1, and PI2, which had been generated against isolated matrices, as well as autoimmune sera detecting lamins, perichromin, and centromere antigens. Chromatin and RNA extraction was monitored with Hoechst 33258, ethidium bromide, and antihistone. The lamins, PI1, and, to a great extent, PI2 and centromere antigens were little affected by the extraction. The data suggest furthermore that PI1 is a fundamental nuclear matrix component and may serve in integrating peripheral and internal nuclear functions. P1 and perichromin were extensively redistributed after chromatin extraction, supporting a role for these antigens in spatial ordering of chromatin. I1 was progressively extracted at each stage of nuclear matrix preparation and was artifactually associated with matrices which had not been digested with RNase. This study demonstrates unequivocally that the organization of many nuclear matrix components in final preparations reflects their organization in situ. It does indicate, however, that some components retained in matrices are extensively redistributed during nuclear matrix preparation and that their role in nuclear organization must be evaluated in consequence.

Effects of taxol on microtubule organization in mouse splenic lymphocytes and on response to mitogenic stimulation.

We have used immunofluorescence staining with antibodies to tubulin and electron microscopy to examine the effects of microtubule assembly-promoting drug taxol on the organization of microtubules in unstimulated and in mitogen stimulated mouse splenic lymphocytes. A 4-h exposure to 10 microM taxol of unstimulated or stimulated cells results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules which extend from the centrosome. Concomitantly, the centrosome is displaced from the normal position near the nucleus to a position near the plasma membrane. The Golgi apparatus is not disrupted by this treatment and also appears to be displaced in association with the centrosome. We then examined the capacity of lymphocytes with taxol-reorganized microtubules to respond to stimulation by the mitogen concanavalin A. The taxol-induced reorganization of microtubules has no effect on the increase in cell size (blastogenesis), the changes in structure of the nucleus and cytoplasm, or on the DNA replication that occurs in response to mitogen. The stimulated cells, however, do not proliferate and appear to accumulate in mitosis with the appearance of multiple asters. We conclude that all of the major events of mitogenic stimulation up to the first mitosis can occur in the presence of a highly reorganized microtubule system. Our results suggest that taxol will be a useful drug in determining those lymphocyte functions that are dependent on the presence of normal microtubule organization.

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.

Measurement of proteolysis in natural waters.

Microbiological proteolysis in Lake Champlain water was measured in situ and in vitro by the spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder. Water samples sterilized by microfiltration were never proteolytic. In situ proteolysis was found to be very dependent upon water temperature (1 to 23 degrees C). No measurable activity was observed below 4 degrees C. The in vitro proteolysis rate at 20 degrees C was found to be 2.3 times the rate at 15 degrees C and 6 times the rate at 10 degrees C. Water taken from beneath the ice-covered lake throughout the winter and tested in the laboratory at 20 degrees C was found to show an increasing proteolytic potential during the winter months. The highest activity was obtained as the ice broke up in early spring. Microbiological proteolysis in water from Burlington Harbor was often four times that found in center lake water. In most experiments proteolysis was inhibited completely by 2 mug of Cu and inhibited 67% by 0.75 mug/ml. Proteolysis was markedly stimulated by 20 to 40 mug of Casitone or Casamino Acids per ml. The predominant bacteria growing in the proteolysis flasks were species of Pseudomonas and Flavobacterium. Pure cultures of Pseudomonas required traces of Casitone, Casamino Acids, or yeast extract for proteolysis of hide powder azure, whereas those of Flavobacterium did not. The requirement could not be met by a mixture of 21 amino acids and eight vitamins.