Position Of Individual Cells Research Articles

Curcumin Selectively Induces Apoptosis in Deregulated Cyclin D1-expressed Cells at G2 Phase of Cell Cycle in a p53-dependent Manner

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.

Measurement of biological effects of high-energy carbon ions at low doses using a semi-automated cell detection system

Purpose : To study the effects of low-dose, high-LET irradiation in order to detect possible deviations from a linear-quadratic dose dependence. Materials and methods : Experiments using charged particle irradiation were performed, in particular in the low-dose range from 0 to 2 Gy, and compared with experiments using photon radiation. The survival of V79 cells was studied by means of a semi-automated system, which allowed the detection and relocation of positions of individual cells at regular intervals. It consists of an inverted microscope equipped with a computerized stage control and image processing system. With such a technique, the precision of the survival assay is increased by avoiding the stochastic uncertainties of the conventional dilution assay. Furthermore, by means of daily observations, the system allows a detailed study of the growth kinetics of individual cells up to approximately 1 week after irradiation. Results and conclusion : Up to now, high-LET experiments have been performed using mainly 100 MeV/u carbon ions (LET: 28 keV/ μ m). A significant hypersensitivity at low doses (0.1 Gy) has been detected for these ions. It is the first report of such a pronounced effect for charged particle irradiation. Although hypersensitivity after carbon ion irradiation apparently is in contrast to other data reported for peak pion irradiation at similar LET values, the difference might possibly be due to differences in the track structure of the particles.

Cellular ras and cyclin D1 are required during different cell cycle periods in cycling NIH 3T3 cells.

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

Monoclonal antibodies reactive with human osteogenic cell surface antigens

Monoclonal antibodies (McAbs) against the surface of osteoblastic cells have been used to characterize the osteogenic lineage. In view of the paucity of probes against the surface of normal human osteogenic cells, we sough to generate McAbs which could be used for both in vivo and in vitro studies. We raised a series of McAbs against early osteoblastic cell surface antigens by immunizing mice with human mesencymal stem cells (MSCs) that had been directed into the osteogenic lineage in vitro. After screening against the surface of osteogenic cells at various stages of differentiation in vitro, as well as evaluating in situ reactivity with human fetal limbs, we isolated three hybridoma cell lines referred to as SB-10, SB-20, and SB-21. Immunocytochemical analyses during osteogenic differentiation demonstrate that SB-10 reacts with MSCs and osteoprogenitors, but no longer reacts with cells once alkaline phosphatase (APase) is expressed. Flow cytometry documents that SB-10 is expressed on the surface of all purified, culture-expanded human MSCs, thus providing further evidence that these cells are a homogeneous population. By contrast, SB-20 and SB-21 do not react with the progenitor cells in situ, but bind to a subset of the APase-positive osteoblasts. None of these antibodies stain terminally differentiated osteocytes in sections of developing bone. Furthermore, these McAbs were not observed to react in samples from chick, rat, rabbit, canine, or bovine bone, although selected extraskeletal human tissues were immunostained. In all cell and tissue specimens examined, SB-20 immunostaining is identical to that observed with SB-21. We have used these McAbs to refine our understanding of the discrete cellular transitions that constitute the osteogenic cell lineage. We suggest a refined model for understanding osteoblast differentiation that is based on the proposition that the sequential acquisition and loss of specific cell surface molecules can be used to define positions of individual cells within the osteogenic cell lineage.

Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis.

Morphogenesis is characterized by orchestrated changes in the shape and position of individual cells. Many of these movements are thought to be powered by motor proteins. However, in metazoans, it is often difficult to match specific motors with the movements they drive. The nonmuscle myosin II heavy chain (MHC encoded by zipper is required for cell sheet movements in Drosophila embryos. To determine if myosin II is required for other processes, we examined the phenotypes of strong and weak larval lethal mutations in spaghetti squash (sqh), which encodes the nonmuscle myosin II regulatory light chain (RLC). sqh mutants can be rescued to adulthood by daily induction of a sqh cDNA transgene driven by the hsp70 promoter. By transiently ceasing induction of the cDNA, we depleted RLC at specific times during development. When RLC is transiently depleted in larvae, the resulting adult phenotypes demonstrate that RLC is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When RLC is depleted in adult females, oogenesis is reversibly disrupted. Without RLC induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping). Finally, we show that in sqh mutant tissues, MHC is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail-binding protein p127. This suggests that sqh mutant phenotypes are chiefly caused by sequestration of myosin into inactive aggregates. These results show that myosin II is responsible for a surprisingly diverse array of cell shape changes throughout development.

Spatiotemporal analysis of simultaneous single-unit activity in the dragonfly

Continuous neural spiking records were obtained from the mesothoracic ganglion of the dragonfly. For analysis the 12 s records of all 58 discriminated cells were "tracked" across three continuous behavioral states: pre-flight, flight and post-flight. The recorded spike amplitudes and angles (widths) for each cell were used to construct a simple map of individual cell positions relative to each other within the ganglion. Individual cell activity patterns were then characterized both with respect to neighboring cell locations and patterns of cell spiking observed across three behavioral states. The results indicated that this technique for constructing a "neighboring cell map" effectively reflects the known histological features of the ganglionic cell architecture. The gross firing histories of individual cells were found to correspond to the overall spike patterns of neighboring ganglionic cells as opposed to more distal cells. Such relationships suggest that the physical layout of this ganglionic network may help to determine or bias individual cell firing histories that occur during different behavioral states in the dragonfly.

Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content.

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

The Yeast Cell Cycle: Coordination of Growth and Division Rates

Publisher Summary The fungi, and especially the yeasts, have an important role to play in the development of the biochemistry of the cell cycle. It is clear that the basic processes in eukaryotic cell division are well represented in the yeasts. Thus, an examination of the current status of cell cycle studies in yeast may be useful to researchers interested in mammalian and plant cell-division, as well as those interested in yeast. This review is timely because the rapid advances in the molecular biology of yeasts promise exciting advances in this decade. The patterns of accumulation and synthesis of a number of different cellular components have been studied in yeast. In many ways yeast is an ideal organism for the studies of cell growth and division. This organism progresses through a number of well-defined morphological and genetic steps during cell division that allow a precise delineation of the cell-cycle position of individual cells. Photomicroscopy of growing cells is possible because yeast grows rapidly on solid media. Large quantities of synchronously growing cells can be generated by a variety of methods. Mutants defective in cell division are available for study.

Nucleolar and nuclear RNA synthesis during the cell life cycle in monkey and pig kidney cells in vitro.

The incorporation of 5-(3)H-uridine and 5-(3)H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-(3)H and cytidine-(3)H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G(1) in both the nucleus and the reconstituting nucleolus. Thereafter, through the G(1) and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G(1) or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.