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1.
Biochem Pharmacol ; 52(9): 1339-46, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8937443

ABSTRACT

Using the DNase I inhibition assay, fluorimetric measurements, and immunoblot analysis, we studied quantitatively changes in the actin polymerization dynamics in primary cultures of normal and malignant human lymphocytes, normal human endometrial cells, and in various leukemic and endometrial adenocarcinoma cell lines. The G/total-actin ratio of malignant cells was found to be 1.37 to 1.81-fold higher compared to normal cells, indicating that malignant cells express reduced amounts of polymerized actin. The above findings were corroborated by fluorescence measurements of the amounts of rhodamine-phalloidin-labeled F-actin in normal and neoplastic cells, which showed significantly lower F-actin content in malignant cell preparations. Moreover, the total actin content, as quantitated by the DNase I inhibition assay and by immunoblot analysis, was found to be significantly decreased in the primary cultures of malignant human lymphocytes and endometrial cells when compared to the total actin levels in corresponding normal cells. Proliferation and viability measurements of normal and neoplastic cells in culture, treated equally with cytochalasin B (CB), revealed an increased susceptibility of malignant cells to this anticytoskeletal agent. This was not due to increased CB incorporation in neoplastic cells, as indicated by 3H-CB uptake experiments. In addition, fluorescence microscopy, in the presence of graded concentrations of CB, showed destabilization of microfilaments in the poorly differentiated endometrial adenocarcinoma HEC-50 cells, compared to the well-differentiated Ishikawa cells. In conclusion, all investigated malignant cells are characterized by: (a) higher G/total-actin ratio; (b) decreased F- and total-actin content; and (c) lower resistance to CB treatment. These quantitatively determined parameters may represent potential biochemical indicators reflecting malignant transformation. Moreover, it seems worthwhile to explore whether or not the differential sensitivity of malignant cells to anticytoskeletal drugs may provide a valuable approach to the manipulation of malignant cells.


Subject(s)
Actins/metabolism , Cytochalasin B/pharmacology , Polymers/metabolism , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytochalasin B/administration & dosage , Dose-Response Relationship, Drug , Endometrial Neoplasms/metabolism , Endometrium/metabolism , Female , Humans , Leukemia/metabolism , Lymphocytes/metabolism , Microscopy, Fluorescence , Tumor Cells, Cultured
3.
Coron Artery Dis ; 5(4): 359-64, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8044347

ABSTRACT

BACKGROUND: A lipid-lowering diet has been shown to lower total cholesterol but also high-density-lipoprotein (HDL) cholesterol. The effect of the first-step lipid-lowering diet (as suggested by the European Atherosclerosis Society) on HDL levels was studied in 129 Greek patients aged 52.7 +/- 9.8 years, of whom 78 were men and 51 women of similar ages. METHODS: Total, HDL, and low-density-lipoprotein (LDL) cholesterol, and the total: HDL cholesterol and triglyceride ratio were assessed before and 3 months after the diet. RESULTS: Overall, total cholesterol decreased by 12% (P < 0.001), LDL by 15% (P < 0.001), HDL by 3% (NS), triglycerides by 12% (P < 0.01), and total: HDL cholesterol ratio by 11% (P < 0.001). A difference was found in the response to diet according to baseline HDL levels: in patients with HDL of 39 mg/dl or higher (group A), HDL decreased by 10% and the total: HDL cholesterol ratio by 3%, whereas in those with HDL lower than 39 mg/dl (group B) HDL increased by 17% and the total: HDL cholesterol ratio decreased by 22%. The difference between the groups was statistically significant (P < 0.001) for these two values as well as for triglycerides, but not for total cholesterol and LDL. No difference in the responses between men and women was found. CONCLUSION: This differential response to diet should be taken into account when planning treatment. Patients with baseline HDL levels higher than 39 mg/dl should probably be considered for early treatment not only by diet but by lipid-lowering-HDL-raising drugs as well.


Subject(s)
Cholesterol, HDL/analysis , Diet, Atherogenic , Hyperlipidemias/diet therapy , Adult , Female , Humans , Hyperlipidemias/blood , Male , Middle Aged , Triglycerides/analysis
4.
Biochem Cell Biol ; 70(9): 779-86, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1482554

ABSTRACT

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cytoskeletal Proteins/analysis , Heat-Shock Proteins/analysis , Adenocarcinoma , Cytoskeletal Proteins/antagonists & inhibitors , Detergents , Endometrial Neoplasms , Female , Humans , Immunoblotting , Microscopy, Fluorescence , Neoplasm Proteins/analysis , Octoxynol , Polyethylene Glycols , Solubility , Tumor Cells, Cultured
5.
Cell Biochem Funct ; 9(1): 23-8, 1991 Jan.
Article in English | MEDLINE | ID: mdl-2065433

ABSTRACT

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.


Subject(s)
Captan/pharmacology , Microtubules/drug effects , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cell Line , L Cells , Macromolecular Substances , Mice , Microscopy, Electron , Microscopy, Fluorescence , Microtubules/metabolism , Nephelometry and Turbidimetry , Tubulin/metabolism
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