Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Effect of thyroxine, insulin and cholesterol on the amount of cyclic adenosine monophosphate in normal and oncornavirus infected cells]

The intracellular concentration of cyclic AMP in normal cells and in cells infected with the Rous sarcoma virus (chicken fibroblasts) was determined by the method of saturation analysis (method of concurrent binding with protein). Thyroxine (2 mcg/ml) increased the level of cyclic AMP both in normal and infected cells, insulin (2 mcg/ml) decreased the concentration of cyclic AMP in infected cells, but cholesterol (5 mcg/ml) exerted no effect on this parameter in infected cells. The data are discussed from the point of view of a possible influence of hormones on the cholesterol accumulation in cells and on the adenylatecyclase activity in cell membranes.     

TSH induces insulin receptors that mediate insulin costimulation of growth in normal human thyroid cells

The mitogenic/goitrogenic effects of thyrotropin (TSH) on human thyrocytes in vitro and in vivo depend on permissive comitogenic effects of insulin-like growth factors (IGFs), which are mimicked in vitro by the low-affinity binding of high supraphysiological concentrations of insulin to IGF-I receptors. Contrary to general assumption, we show here that very low concentrations of insulin, acting through insulin receptors but not IGF-I receptors, can also support the stimulation of DNA synthesis by TSH in primary cultures of normal human thyrocytes. Moreover, TSH through cAMP increases the content of insulin receptors demonstrated by Western blotting and the cells' responsiveness to low insulin concentrations. These observations provide the first in vitro evidence in normal human thyroid cells of a functional interaction between TSH and insulin acting through its own receptor.     

Kinetics of herpes simplex virus photoinhibition by haematoporphyrin in both diploid and heteroploid cells

The kinetics of inhibition of herpes simplex virus type 1 (HSV 1) on both diploid (CEF) and heteroploid cells (HEp2) by light-irradiated haematoporphyrin (HP) was studied. The inactivation of HSV1 by HP was drug-dose dependent and light-irradiation dependent; the viruses grown in heteroploid cells being in all cases more sensitive to inhibition than viruses grown in diploid cells. Cell toxicity by HP was markedly more evident on HEp2 cells than on CEF. The highest viral sensitivity to photodynamic inactivation by HP was found to be between the 4th and the 5th hour after cell infection, when the viral DNA synthesis is at its peak and before it is incorporated into complete virions. Microfluorometric and spectrofluorometric assays revealed that virus infected cells always take up more HP than uninfected cells, and heteroploid cells incorporated more HP than diploid cells. The possibility that an increased uptake of HP and modifications of the cell micro-environment in virus infected cells could account for the viral-inhibiting properties of HP, is discussed.     

Laser effects on cellular adhesion and influence on the interrelation between HeLa S3 and human diploid cells

The interactions between HeLa S3 tumoral cells and human fibroblasts after nitrogen-laser irradiation (337.1 nm) have been studied by using an in vitro cell invasion model. For the quantitative and morphological evaluation of nitrogen-laser radiation action upon tumoral adhesion to the fibroblast monostrate, we used: a) 3H-thymidine labelling of HeLa S3 tumoral cells; b) morphological modifications studies by phase contrast and scanning electron microscopy. The results emphasized the following aspects: 1. In non-irradiated cell cultures we noticed three interaction stages: adhesion, tumoral spreading and displacement with fibroblasts destruction; on the other side, we found a reduced adhesion to non-irradiated human fibroblasts of laser irradiated tumoral cells. 2. Significant percent increasing of non-irradiated tumoral cells adhesion to fibroblast monostrate, irradiated with various laser fluences (e.g. 0.2 kJ/m2--48.1%; 0.8 kJ/m2--63.8% and for 1.6 kJ/m2--79.5%). This phenomenon evidenced the close interrelation between irradiation fluences and tumoral adhesion rates. 3. The importance of numerical ratio between tumoral cells and fibroblasts in tumoral adhesion and invasion processes (e.g. ratio 1:10 tumoral adhesion reached 8.1%; in 1:5--25.9%; in 1:1--59.4% and for 2:1--83.9%). 4. Marked cytotoxic effects for both cell types after exposure to high and very high laser fluences (1.6--6.4 kJ/m2). Our results emphasize near UV-laser irradiation effects upon some of tumoral adhesion and invasion mechanisms and demonstrate the interrelations between cell populations manifesting a different vital potential.     

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.     

Effect of A-nor steroids and oestradiol on progesterone production by human luteal cells

Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase. Progesterone production was assessed after short-term incubation of luteal cell suspensions. Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0.01 i.u./ml, and the response was 5 times higher than the basal production. Oestradiol (1-100 microM) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production. The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 microM. The ED50 values were approximately 3 microM, 75 microM and 100 microM for oestradiol, AF-45 and anordrin respectively. Anordrin showed no significant effects on basal progesterone production. In addition, oestradiol markedly inhibited the activity of 3 beta-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low. The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.     

Effect of antioxidants on the proliferation of human diploid cells at various phases of the culture growth

Some antioxidants (2-ethyl-6-methyl-3-hydroxypyridine chlorhydrate and some of its derivatives and 4-methyl-2,6-di-tert-butylphenol) have been shown to stimulate proliferation of young and old diploid cell at all phases of culture growth (lag and stationary phases). It is supposed that the mechanism of this effect may depend on stimulation of dreaming cell to division.     

The interaction of Mengo virus ribonucleic acid with L, HeLa and human diploid cells: a comparative study

A study, designed to define the optimal conditions for infecting L, HeLa and human diploid cells with Mengo RNA, showed that the three cell types differ with respect to (a) the concentration of sucrose (in PBS) that produces maximum stimulation of infectious center (I.C.) formation (0.6 M, 1.2 M and 1.0 M for L, HeLa and human diploid cells respectively) and (b) the optimal combination of sucrose and dimethyl-sulfoxide (DMSO). With all three cell types, diethylaminoethyldextran (DEAE-D) was found to produce maximum stimulation of I.C. formation when present, in PBS, at a concentration of 100 μg/ml. Direct comparisons of I.C. formation in the three cell types using the sucrose, sucrose-DMSO and PBS-DEAE-D media optimal for each revealed that (a) the best medium for titration in L and HeLa cells is PBS containing 100 μg DEAE-D/ml, (b) DMSO produces a 4–8 fold increase in I. C. formation in L cells, but is only marginally effective in HeLa cells, and (c) the optimal medium for titration in human diploid cells is sucrose-DMSO. The results of studies of the “uptake” of ³H-Mengo RNA showed that there is no correlation between the amount of RNA that becomes cell associated and the number of I.C.'s that are produced in the cell population.     

Fabrication and growth mechanism of ZnO nanostructures and their cytotoxic effect on human brain tumor U87, cervical cancer HeLa,and normal HEK cells

ZnO nanostructures of diverse shape were grown via a solution process with different precursors and conditions. Morphological investigation of the nanostructures was carried out using field emission scanning electron microscopy and transmission microscopy observations and revealed that the nanostructures exhibit a wurtzite phase with an ideal lattice fringe distance of approximately 0.52 nm. The powder crystallinity was examined via X-ray diffraction spectroscopy. Screening results from anticancer studies of the effects on human brain tumor U87, cervical cancer HeLa, and normal HEK cells of ZnO nanostructures of diverse shape were obtained and indicate promising activity that varies with changes in the structure and the size of the particles. Treatment-induced cell death [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and survival assay], growth inhibition, cytogenetic damage (formation of micronuclei), and apoptosis were studied as parameters for the cellular response. Treatment with nanostructures enhanced growth inhibition and cell death in a concentration-dependent manner in both U87 and HeLa cell lines. At higher concentrations (above 15.6 μg/ml) the cytotoxic effects of the nanoparticles were highly synergistic and mainly mediated through apoptosis, implying the possible interactions of lesions caused by the agents. The enhanced cell death due to nanoparticles was accompanied by a significant increase (2–3 fold at 31.25 μg/ml) in the formation of micronuclei in U87 cells. The increase in the formation of micronuclei observed after treatment indicates that these structures may interfere with the rejoining of DNA strand breaks. Among all the nanostructures, nanoparticles and sheets exhibited potent activity against both HeLa and U87 cells. However, despite potent in vitro activity, all nanostructures exhibited diminished cytotoxicity against normal human HEK cells at all effective concentrations.