Search PubMed⌕ Search

Biomedical subjects

G Giese

Publications and source records attributed to G Giese.

At least 37 records · Page 2Linked to original sources
PubMed1991

Subclones of C6 rat glioma cells differing in intermediate filament protein expression.

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Animals↗
PubMed1990

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells.

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.

Animals↗
PubMed1990

Differentiation and retrodifferentiation of U937 cells: reversible induction and suppression of intermediate filament protein synthesis.

Significant morphological and functional changes were observed when human monoblastoid U937 tumor cells growing in suspension were induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h to differentiate along the monocyte/macrophage pathway. These include adherence of the cells to each other and to the substratum, alterations in cell-surface antigen expression and cessation of autonomous proliferation. In this study, we show by both, hybridization analysis of RNA and immunoblotting that an enhanced expression of the intermediate filament (IF) subunit proteins vimentin, lamin A and lamin C accompanied the TPA-induced differentiation process. After long-term culture of differentiated U937 cells in the absence of TPA (more than 28 days), however, the adherent cells retracted their pseudopodia, detached and started again to proliferate. This "retrodifferentiation" process, not previously described was paralleled by a rapid down-regulation of both, IF mRNA and protein synthesis back to the level of undifferentiated U937 control cells. These data suggest a functional relationship between the expression of vimentin and lamins A and C and the differentiation process taking place in these cells.

Cell Differentiation↗
PubMed1989

Expression of nuclear lamins in mammalian somatic cells lacking cytoplasmic intermediate filament proteins.

Using immunofluorescence and immunoblotting techniques, we have examined the composition of the nuclear lamina in murine plasmacytoma cells, MPC-11, exposed to the phorbol ester TPA as well as in two cell lines devoid of cytoplasmic intermediate filament proteins, the human adrenal cortex carcinoma-derived cells SW-13 and the clone C6-M-D4 derived from the rat glial cell line C6. Our results show that the inhibition of proliferation and the induction of vimentin synthesis observed in TPA-treated MPC-11 populations are not paralleled by changes in the lamin complement of these cells, which contain lamin B but lack lamins A and C. Furthermore, the analysis performed on SW-13 and C6-M-D4 cell lines clearly demonstrates that mammalian somatic cells display considerable variations in lamin expression and indicates that lamin B may be the only lamin species constitutively expressed in mammalian cells.

Adrenal Cortex Neoplasms↗
PubMed1989

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Animals↗
PubMed1989

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60.

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1988

Induction of vimentin synthesis in a murine myeloma cell line by TPA is strongly dependent on the composition of the cell culture medium.

MPC-11 mouse plasmacytoma cells virtually lacking intermediate filament (IF) proteins can be induced to synthesize and accumulate the IF protein vimentin by treatment with the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Like MPC-11 cells, X63-Ag8.6.5.3 mouse myeloma cells (Ag8) proved to be vimentin-negative, as assayed by immunoblotting of whole cellular protein using goat antiserum to vimentin and [125I]protein A. Vimentin synthesis could be elicited by a TPA concentration as low as 10(-9) M in cells grown in HB-102 serum-free medium. Transfer of these cells to medium containing 15% fetal calf cerum (FCS) greatly reduced the ability of these cells to synthesize vimentin upon TPA treatment. After 50 generations of culture in the presence of FCS, induction of vimentin synthesis was barely detectable even at a TPA concentration of 10(-6) M. Addition of FCS to cells grown in serum-free medium partially suppressed vimentin induction by TPA. This suppression seems to be due, at least in part, to nondialyzable, heat-sensitive components of FCS, since the dialyzable fraction even enhanced vimentin induction by TPA. When cells grown in the presence of FCS were transferred back to serum-free medium, their ability to synthesize vimentin in response to TPA treatment was readily restored. The individual components of serum-free medium which proved to support vimentin induction by TPA were insulin and the unsaturated fatty acids oleic acid and linoleic acid. An even stronger TPA response could be elicited by a combination of these components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Induction of vimentin synthesis in mouse myeloma cells MPC-11 by 12-0-tetradecanoylphorbol-13-acetate.

Mouse myeloma cells (MPC-11 cell line) known to lack intermediate filaments were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). Asynchronous cell cultures were screened for vimentin by indirect immunofluorescence microscopy, whole cell lysates derived from such cultures by immunoblotting using goat antiserum to vimentin. The minimum TPA concentration sufficient for the induction of vimentin synthesis was found to be 3 X 10(-9) M; substantially larger amounts of vimentin could be detected after treatment of cells with TPA at a concentration of 3 X 10(-8) M. At each effective TPA concentration tested, the maximum level of vimentin was reached after 18 to 24 h; it was dependent on the TPA concentration. In addition, vimentin synthesis was demonstrated employing two-dimensional polyacrylamide gel electrophoresis in combination with either fluorography or immunoblotting and autoradiography. Vimentin purified from TPA-treated MPC-11 cells as well as a protein species in whole lysates from cells labelled with [35S]methionine after TPA treatment for at least 2 h comigrated with vimentin isolated from Ehrlich ascites tumor cells. The fact that only poly(A) +RNA from TPA-treated MPC-11 cells was able to direct vimentin synthesis in vitro suggests that in MPC-11 cells vimentin production is regulated at the transcriptional level.

Animals↗
PubMed1984

Thermal diminution and augmentation of the retention of transportable rRNA in nuclear envelope-free nuclei.

We have examined the effect of temperature on the rRNA transport from nuclei isolated from Tetrahymena after removal of both nuclear membranes and pore complexes by 1% Triton X-100. These nuclei export rRNA as precursor ribosomal ribonucleoprotein particles at both 28 degrees C and 8 degrees C which are qualitatively the same in terms of rRNA pattern, sedimentation coefficients and buoyant densities. At 8 degrees C, however, significantly fewer ribosomal ribonucleoprotein particles can be maximally exported than at 28 degrees C, though nuclei contain enough potentially transportable particles. These are increasingly released with increasing temperatures. Under conditions non-permissive for export, temperature elevation decreases the number of the potentially transportable ribosomal ribonucleoprotein particles in nuclei. Our data show: transportable ribosomal ribonucleoprotein particles inside nuclei are not 'free', but rather are subject to a complex temperature-sensitive retention: this retention is gradually diminished under export conditions and augmented under non-permissive export conditions with increasing temperatures. These retention mechanisms operate at an intranuclear level preceding the ribosomal ribonucleoprotein passage through the nuclear envelope pore complexes, i.e., the nuclear envelope regulates neither the number of potentially transportable ribosomal ribonucleoprotein particles in nuclei nor the number of those particles which can be maximally exported from nuclei at a given temperature. We suggest that these retention mechanisms involve temperature-sensitive domains of the nuclear matrix.

Animals↗
PubMed1984

Thermal down-regulation of exportable rRNA in nuclei.

The export of rRNP particles from nuclei isolated from Tetrahymena was investigated after preincubating the nuclei at different temperatures under nonpermissive export-conditions. We observed a new phenomenon: Temperature elevation from the sublethal cells' growth temperature, 8 degrees C, to the optimal temperature, 28 degrees C, lead to a gradual down-regulation in the maximal proportion of rRNP particles subsequently exported from nuclei at 28 degrees C. This thermal down-regulation is apparently not due to qualitative changes in the exported rRNP particles, a derangement in the gross nuclear organization, a degradation and/or nicking of the nuclear rRNA, a gross decomposition of the major nuclear proteins, a random cross-linking of nuclear components by disulfide bonds, or an elution of nuclear factors possibly required for rRNP export. Moreover, there is a corresponding thermal down-regulation in nuclear envelope-free nuclei. Our data indicate that nuclei possess a mechanism that regulates the number of potentially exportable rRNP particles at a level preceding the rRNP passage through the nuclear envelope.

Animals↗
PubMed1983

In vitro ribosomal ribonucleoprotein transport. Temperature-induced "graded unlocking" of nuclei.

We examine the effect of temperature on the export of ribosomal precursor particles from nuclei isolated from Tetrahymena. A new phenomenon is observed. Temperature does affect not only the export rate, but also the maximal portion of particles exported. At 8 degrees C, for example, the export kinetics reveals a significantly lower saturation plateau which does not equilibrate with the higher plateau at 28 degrees C even after 3 h. This nonequilibration is not due to (i) a different physical quality of the exported particles, (ii) a degradation of the nuclear rRNA, (iii) a backward import of exported particles into nuclei, (iv) an irreversible inactivation of potentially transportable nuclear ribosomal ribonucleoprotein (rRNP) particles, or (v) a thermodynamic equilibrium between transportable rRNP particles associated with nuclei and those exported from nuclei. We conclude, therefore, that potentially transportable rRNP particles are somehow "locked" in nuclei at low temperature and temperature raising induces a "graded unlocking."

Animals↗
PubMed1983

In vitro nuclear transport of ribosomal ribonucleoprotein: temperature affects quantity but not quality of exported particles.

The in vitro export of ribosomal ribonucleoprotein (rRNP) from Tetrahymena nuclei was investigated at the optimal growth temperature of 28 degrees C and at the nonlethal temperature of 8 degrees C. At both temperatures, nuclei exported ribosomal precursor particles that revealed the same physical qualities of size, appearance in negative-staining electron microscopy, sedimentation coefficient, buoyant density, and rRNA pattern. Surprisingly, fewer rRNP particles were exported at 8 than at 28 degrees C, as was revealed by a lower saturation plateau in the export kinetics from nuclei prelabeled with [3H]uridine. Upon a temperature increase from 8 to 28 degrees C, additional rRNP particles were exported. We conclude that nuclei export only a defined portion of rRNP particles at a given temperature, although enough potentially transportable rRNP particles are present in nuclei. Obviously, the reactivity of at least one of the reactants involved directly or indirectly in rRNP export changes with temperature.

Biological Transport↗
PubMed1980

In vitro ribosomal ribonucleoprotein transport upon nuclear expansion.

The interdependence of nuclear rRNA release and nuclear size is investigated in macronuclei isolated from Tetrahymena. Nuclei are induced to contract and to expand, without any structural disintegration of the nuclear envelope, by final Ca2+/Mg2+ (3:2) concentrations of 5 and 1.5 mM, respectively. Upon expansion, the average volume of nuclei increases from 600 +/- 42 to 811 +/- 76 micron3. Concomitantly, nuclei begin to release RNA following saturation kinetics. This RNA release stops immediately upon nuclear contraction. Similar to the in vivo situation, only advanced rRNA processing products are released in the form of ribosomal precursor particles, as identified in detail by polyacrylamide gel electrophoresis and rate zonal and isopycnic density gradient centrifugation. Three particle ty9es are released having average buoyant densities of 1.495, 1.470, and 1.532 g/cm3, exhibiting average sedimentation coefficients of 62, 62, and 35 S, and containing the immediate precursor to the 25S rRNA, 26S rRNA, and 17S rRNA, respectively. Tje rRNP release if ATP independent and noncoincident with the release of endogenous nuclear Pi, though it is Be2+ sensitive. Our data are compatible with the views that nuclear expansion is the prerequisite rather than the cause for the rRNP release and that nuclear pore complex associated ATPases play only, if at all, a minor role in nucleocytoplasmic exchange of rRNP.

Animals↗