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G Giese

Publications and source records attributed to G Giese.

13 recordsLinked to original sources
PubMed1992

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).

Animals↗
PubMed1991

Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of c-fos and c-jun (AP-1).

Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation and expression of the c-jun and c-fos early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in c-jun and c-fos mRNA levels in U-937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA-induced increases in lamin A, lamin C, and vimentin transcripts. Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the c-jun and c-fos genes in myeloid leukemia cells are regulated by protein kinase C (PKC). The finding that dexamethasone has no effect on TPA-induced activation of PKC suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run-on assays demonstrate that: (1) induction of c-jun and c-fos expression by TPA is regulated by transcriptional mechanisms, (2) TPA-induced expression of c-jun and c-fos does not require protein synthesis, and (3) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted c-jun promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene. Increases in CAT activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region (-97 to -20) of the promoter that contains the AP-1 binding site. This induction of CAT activity was sensitive to dexamethasone. These findings suggest that dexamethasone down-regulates TPA-induced transcription of the c-jun gene during monocytic differentiation by inhibiting activation of the AP-1 site.

Actins↗
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↗
PubMed1978

Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane-bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein-framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.

Animals↗