A. M. Fuhrman Conti

Relative biological effectiveness for protons of energies up to 31 MeV.

The relative biological effectiveness (RBE) was determined for proton beams of 31, 12, and 8 MeV produced at the Milano University Cyclotron. Survival curves for human cells grown in monolayer at different proton energies and for γ rays from60 Co were determined. The minimum clone size to be chosen for definition of true survivors was examined. RBE values of 1.0 ± 0.1, 1.4 ± 0.2, and 1.5 ± 0.2, respectively, were found and compared with the results of other experiments in this energy range.

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de‐adaptation of EUE cells to a hypertonic medium

Abstract. EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle‐related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/l range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the Go/1 range of DNA contents: this suggests that a G0 block occurs under long‐term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.

Effects of low-power 632 nm radiation (HeNe laser) on a human cell line: Influence on adenylnucleotides and cytoskeletal structures

HeNe (632 nm) irradiation (5, 15 and 30 min) of an embryonal human cell line (EUE) was used to study the short-term effects on energy charge and the rapid, energy-dependent, remodelling processes of cytoskeletal and adhesion structures. The adenosine triphosphate (ATP) concentration, tested by luminometric and high performance liquid chromatography (HPLC) procedures, is constant after 15 and 30 min of HeNe treatment; the lower phosphorylated nucleotides, i.e. adenosine diphosphate (ADP) and adenosine monophosphate (AMP), change after 30 min in opposite directions: the ADP concentration decreases by 39% whilst that of AMP increases about sixfold. The adenylate energy charge (AEC) decreases by 21.7% in treated EUE cells (AEC = 0.65) in comparison with untreated EUE cells (AEC = 0.83). In HeNe-treated cells, the remodelling of cytoskeletal and adhesion molecules becomes evident after 15 min of treatment. The following events are important: (1) modification of stress fibre assembly and increase in vinculin-containing adhesion plaques; (2) assembly and bundling of intermediate filaments; (3) increase in laminin and L-cell adhesion molecules (L-CAM) expression. The lowered energy charge in irradiated cells is related to the increase in AMP production at the expense of ADP. ATP is dynamically constant despite its requirement in short-time remodelling processes of the cytoskeletal network which are enhanced in irradiated cells.

Multinucleate Cells and Micronucleus Formation in Cultured Human Cells Exposed to 12 MeV Protons and γ-rays

Cultured human cells of the EUE line were exposed to different doses of 12 MeV protons, plated and allowed to grow for 8 days; colonies were then scored for the presences of multinucleate cells and micronuclei. The frequency of both effects is an increasing function of the dose; the evaluated exponents of the dose-response equation (e = bDn) are n = 1.0 %/- 0.1 for multinucleate cells and n = 1.6 +/- 0.1 for micronuclei. By comparison with the results obtained with gamma irradiations, r.b.e. values were obtained for both effects. The correlation between the logarithm of the surviving fraction and the yield of the studied effects has been proved to be statiscally significant.

Chromosome aberrations induced by protons up to 31 MeV in cultured human cells

SummaryChromosome aberrations were induced in cultured human cells by proton beams of 31, 12, and 8 MeV. The frequencies of isocromatid breaks and dicentrics have been analysed as a function of proton energy and dose. Both effects are largely dependent on proton energy; isochromatid breaks increase linearly with the dose, whereas dicentrics show a definite parabolic behaviour. The experimental data were fitted to the analytic formY = KDn andY = αD +βD2 and the best fitted values of the parameters are reported and discussed. The values of RBE for the isochromatid breaks are in the ratio 1.7: 1.3: 1 for 8, 12, and 31 MeV respectively.In the case of the dicentrics the RBE values are dose-dependent function of the typeCD−n. The three distributions of dicentrics among the cells do not fit a Poisson distribution.

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.

Cytogenetic analysis and muscle differentiation in a girl with severe muscular dystrophy.

SummaryThe uncommon case is described of a girl severely affected with Duchenne muscular dystrophy. Cytogenetic analysis revealed no numerical or structural abnormalities of the X-chromosome in any of the cells examined (leucocytes and myoblasts). No abnormality in morphology, growth pattern or differentiation was observed in the dystrophic muscle cultures as compared with control cultures.

NUCLEAR PHOSPHOLIPIDS DURING THE ADAPTATION OF HUMAN EUE CELLS TO HYPERTONIC STRESS

The phospholipid component of interphase nuclei was analysed in EUE cells (an established cell line from embryonic human epithelium) grown in an isotonic culture medium and during the adaptation process to a hypertonic medium, using a highly specific ultracytochemical procedure, viz. labelling with the phospholipase A2 gold-complex. Within the nucleus, the phospholipids were localized in domains involved in different steps of the synthesis and processing of the RNA. These localizations did not vary at the two key steps of the adaptation process to hypertonic medium: short-term treatment (6 h) representing critical shock condition, and long-term growth (5 days) representing the adapted cells under survival conditions. On the contrary, deep changes of the labelling intensity of phospholipids at these sites occurred at the different times of hypertonic treatment and followed the same course as those observed in the ultramorphological patterns of transcription: the chromatin condensation, as evaluated by image analysis, the permanent nucleolar components, the interchromatin and the perichromatin granules. These data endorse the hypothesis that nuclear phospholipids could be involved in different steps of the transcriptional activity. They are indicative of the deep changes occurring in the EUE cells submitted to hypertonic stress.

Characterization of glycoconjugates in an embryonic human epithelial line and changes consequent to adaptation to a hyperosmotic medium

SummaryCell surface and cytoplasmic glycoconjugates were characterized in embryonic human explant cells (a transformed heteroploid line) cultured in iso-osmotic medium (0.137m NaCl) and in hyperosmotic medium (0.274m NaCl) for 10 days in order to study the changes induced in these compounds by hyperosmoticity. Cytochemical and ultracytochemical staining selective for glycoconjugates was carried out. The following results were obtained: (1) glycoproteins, glycosaminoglycans and glycolipids are present on the cell surface and in the cytoplasm of the explant cells; (2) lectin histochemistry combined with glycosidase digestion demonstrated the presence of the disaccharides fucose-N-acetylglucosamine and sialic acid-β-galactose as terminal sequences; (3) histophotometric evaluation of lectin labelling showed a noticeable decrease in histochemical reactivity of adapted cells; (4) plasma membrane cell coat decreased in adapted cells, which was emphasized by ultracytochemical reactions and a rearrangement of glycolipids in the cytoplasm.

Induction of 8-azaguanine Resistant Mutants in Human Cultured Cells Exposed to 31 MeV Protons

We report results on the induction of 8-azaguanine (8-AG)-resistant mutants in cultured human cells (EUE) exposed to 31 MeV protons. The spontaneous frequency of mutants was 5.6 +/- 0.7 x 10(-6) per viable cell. Gamma rays were taken as reference radiation. Expression times giving the highest frequency of mutants after 31 MeV protons and gamma irradiation were found to be about 10 days for both radiations. The dose-response relationship for mutant induction by protons, as determined at the optimal expression time, was compared to that obtained after gamma rays. The relative biological effectiveness (RBE) is 2.4 +/- 0.5, this value being higher than the RBE value determined for cell survival.