Membrane dynamics correlate with formation of signaling clusters during cell spreading

The morphology and duration of contacts between cells and adhesive surfaces play a key role in several biological processes, such as cell migration, cell differentiation, and the immune response. The interaction of receptors on the cell membrane with ligands on the adhesive surface leads to triggering of signaling pathways, which allow cytoskeletal rearrangement, and large-scale deformation of the cell membrane, which allows the cell to spread over the substrate. Despite numerous studies of cell spreading, the nanometer-scale dynamics of the membrane during formation of contacts, spreading, and initiation of signaling are not well understood. Using interference reflection microscopy, we study the kinetics of cell spreading at the micron scale, as well as the topography and fluctuations of the membrane at the nanometer scale during spreading of Jurkat T cells on antibody-coated substrates. We observed two modes of spreading, which were characterized by dramatic differences in membrane dynamics and topography. Formation of signaling clusters was closely related to the movement and morphology of the membrane in contact with the activating surface. Our results suggest that cell membrane morphology may be a critical constraint on signaling at the cell-substrate interface.     

Unscheduled DNA synthesis of rat hepatocytes in monolayer culture

Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimurium. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of ³H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM ³H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM ¹⁴C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity and/or their interactions with variuos cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing ³H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while α-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic and/or genotoxic metabolites from various xenobiotics.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

Immortalized phenotype and the presence of active oncogenes correlate with the capacity of culture cells to induce reactivation of DNA synthesis in macrophage nuclei in heterokaryons

Several types of culture cells with limited life span (rat embryo fibroblasts, rat chondrocytes and mouse premacrophages) were found to be unable to induce the reactivation of DNA synthesis in the nuclei of non-dividing differentiated cells (mouse peritoneal resident macrophages) in heterokaryons. By contrast, malignant HeLa cells have this ability. In heterokaryons formed by fusion of mouse macrophages with HE239 cells (Syrian hamster fibroblasts transformed with a ts mutant of the SV40 virus), DNA synthesis in macrophage nuclei is reactivated only at the permissive temperature (33° C), at which viral T antigen is stable. Immortalization of rat chondrocytes by transfection with p53 gene enables to induce DNA synthesis in macrophage nuclei upon fusion. All the evidence indicates that the function of immortalizing oncogenes is necessary for the resumption of the DNA synthesis in macrophage nuclei in heterokaryons.     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.     

Use of cryopreserved hepatocytes for unscheduled DNA synthesis assays

Hepatocytes were isolated from Fischer rats by perfusion and tested for unscheduled DNA synthesis (UDS) induction or cryopreserved for long-term storage at -196 degrees C. Thawed cells could be recovered at greater than 90% viabilities, and were cultured on fibronectin-coated coverslips. The cells attached and spread, and could be used for UDS assessment. Data were compared for fresh and frozen cells from the same animal. Results obtained for net nuclear grains and dose response were similar for the fresh and frozen cells, in response to the carcinogenic compounds methyl methanesulfonate and 7,12-dimethylbenzanthracene, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265–2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC₅₀ = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor ). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.