Aldose reductase is involved in long-term adaptation of EUE cells to hyperosmotic stress.

Aldose reductase has been shown to be expressed in large amount by human embryonic epithelial cells (EUE) in response to osmotic stress. This conclusion is the result of studies undertaken following the purification to homogeneity of two forms of a 35-kDa protein overexpressed in EUE cells grown in hypertonic saline culture medium as compared to EUE cells grown in isoosmotic medium. Amino-acid composition, molecular weight and partial internal amino-acid sequence showed that the above proteins are two different forms of aldose reductase. These findings were confirmed by the observation that aldose reductase activity increased about 150-fold in adapted cells and returned to basal levels in de-adapted cells.

Hypertonic stress induces c-fos but not c-jun expression in the human embryonal EUE epithelial cell line.

Recent evidence has indicated a role for the two early response genes c-fos and c-jun in transcriptional regulation of genes acting in osmoregulation. On this basis we investigated their expression in response to hypertonic stress in the human embryonal EUE epithelial cell line. EUE cells have proven to be a useful tool for studying long-term in vitro adaptation to hypertonic stress. After culturing EUE cells in hypertonic medium a marked c-fos induction was observed, both at the mRNA and the protein level. Northern analysis of fos-mRNA showed a peak expression at 4 h, followed by a progressive decline till complete extinction at 8 h. Immunofluorescence analysis of FOS protein evidenced a similar, although slightly delayed kinetics of expression. Conversely, neither c-jun nor c-myc up-regulation could be detected. The treatment of EUE cells with cycloheximide led to superinduction of c-fos expression, (with high levels up to 12 h), and to a c-jun expression that was just detectable. Hypertonic stimulation of the transformed cell lines A549, MCF7 and JR induced both c-fos and c-jun only in JR cells. Hypertonic shock was also effective in inducing c-fos expression in fetal human diploid fibroblasts, although the response was earlier and more transient than in EUE cells. These findings indicate that c-fos is a primary response gene in hypertonic stress-activated cells, although the pattern and kinetics of its induction may differ according to the type of cell.

Effects of hypertonic conditions on cytoskeletal and adhesion structures of EUE cells.

The distribution of cytoskeletal structures has been studied by electron and immunofluorescence microscopy in human embryonic epithelial cells (EUE cells) exposed to a hypertonic medium containing 0.274 M NaCl. A first noticeable effect involved an increase of cell size. Microtubules, microfilaments and intermediate filaments were also considerably changed under these experimental conditions. The most marked effect was on intermediate filaments of the keratin type which formed very thick bundles around the nucleus and gave rise to an intracellular cagework which is likely i) to increase mechanical resistance and ii) to avoid cell collapse in conditions of hyperosmolarity. A remarkable increase in complexity of the microfilamentous network was also found: stress fibers became thicker and more densely arranged and vinculin-containing streaks at focal cell-substratum contacts increased in number and size; this indicated improved cellular adhesion. The phenotypic adaptation of EUE cells to conditions of hyperosmolarity is slowly reversible under defined experimental conditions.

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.

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 G0/1 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 G0/1 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.

Liability to chromosome damage in lymphocytes of "cancer family" subjects: a study of spontaneous and induced chromosomal fragility.

Spontaneous chromosomal fragility was detected in seven tumor patients and one healthy member from two families with a high recurrence of cancer. Major chromosome lesions, such as terminal deletions and rearranged chromosomes, were found at levels significantly higher than those reported for control individuals. The prevalence of these aberrations in comparison to minor ones (chromosome gaps and chromatid breaks) in this group of patients seems to indicate that the fragility observed is the end-point of a process of chromosomal instability, which may have already been brought to expression. Study of other parameters of genetic instability in the most unstable karyotypes showed that the chromosome damage observed was neither paralleled by abnormal SCE frequency nor sustained by defective DNA repair mechanisms or expression of inherited or constitutional fragile sites. As all the subjects investigated here had previously been shown to display intraindividual variations in the C-banded region of chromosome 1, it is possible that spontaneous fragility and acquired C-heterochromatin polymorphism may be markers that, combined with chromosomal instability, create genetic predisposition to cancer.

Enzymatic characterization of E.U.E. (embryonal human explants) cells adapted to hypertonic media.

E.U.E. cells (general population) were submitted to biochemical and cytoenzymatic tests to compare the enzymatic profile of E.U.E. cells (controls) with that of E.U.E. adapted to hypertonic medium. The adapted cells are characterized by very high oxoreductase activity (LDH, HBDH, G-6-P DH) and very high alkaline-phosphatase activity. Clones derived from general population were also submitted to biochemical tests to characterize those more strictly related to the enzymatic profile of adapted cells. The profile of clone N. 13 resembles on this respect that of the adapted cells. The high redox activity is a prerequisite supporting energy supply for osmotic work. The increased activity of plasma membrane enzymes of the adapted cells is also demonstrable in cells exposed for short time to salinity.

Treatments with saline solutions and DNase I have different effects on DNA content and distribution in human and in mouse chromosomes.

The aim of this work was to re-examine, on a quantitative basis, the relationship between banding pattern after Giemsa staining and the amount (and distribution) of DNA along the length of the chromatid arms. To do this, we investigated by cytofluorometric methods the occurrence of possibly different extraction of chromosome DNA after some alternative G-banding procedure, i.e. treatment of chromosomes with saline solutions, or DNasi I digestion in situ. The G-banding procedure entailing trypsin pretreatment is known to be difficult to standardize; in the present investigation, it was also found that trypsin induced a massive, although quantitatively variable, extraction of DNA from fixed metaphase chromosomes. G-banding-like patterns may be obtained, by treating chromosomes preparations with saline solutions. Both PBS and Tris-HCl treatment for the times considered induced a G-banding-like pattern after Giemsa staining, regardless of the age of chromosome preparations; no banding was observed after staining DNA with PI, nor extraction of DNA was found to occur. DNase I digestion initially induced a G-banding in both human and mouse chromosomes after Giemsa staining, with concomitant extraction of DNA (but without apparent G-banding-like pattern after PI staining); after 30 min digestion, a C-banding-like pattern was observed after both Giemsa and PI staining. Exposure to PBS or Tris-HCl buffer at room temperature may therefore be recommended as a G-banding inducing treatment, since it allows the classification of single chromosomes after Giemsa staining, without determining significant displacement of genomic DNA, which can be submitted to further analysis in situ.

[Monosomy 9p. Clinical and cytogenetic aspects]. / La monosomia 9p. Aspetti clinici e citogenetici.

The Authors describe a new case of monosomy 9 p in a newborn, confirmed by bands technique. The parents had a normal karyotypes and this alteration was defined as deletion 9 p "de novo" arisen. The main morphological anomalies are described: these anomalies reproduce that reported by others and, for many aspects, remember Down syndrome. It is very important that the staff of the Neonatological Departments has a good knowledge of this syndrome since for the abnormal objective features, the diagnosis is possible already from the birth.

[Partial trisomy 9q: description of a new case]. / Trisomia parziale 9q: descrizione di un nuovo caso.

The authors describe one case of partial 9q trisomy they observed. The malformations they observed are correspondent to the very little amount of existing documented cases. And just because we have only a few observations, we thought useful publishing this case, to better define the clinical features among the alterations of chromosome 9 (trisomy 9 p and 9q). Head, neck, bones, heart and urogenital apparatus seen to be the most frequently involved in the phenotypic expression of the 9q trisomy.

Cytologic and genetic features of mammalian cultured cells adapted to hypertonic medium.

The response to changes in the osmotic pressure and the capability to adapt to raised tonicity of culture medium, obtained by increasing the NaCl molarity (0.137M), varies among different Mammalian cell lines. Cells cultured in permissive hypertonic medium become resistant to more drastic hypertonic solutions, which are incompatible with cell survival, in the absence of any pretreatment. When cells grown in hypertonic medium are fed again with normal medium, they do not maintain their greater resistance acquired by adaptation; this points to a reversible physiological process rather than to a stable regulatory change at a genetic level. Cells of the EUE line (human embryonic epithelium), grown in medium containing a doubled NaCl concentration (0.274M), show a growth rate slower than cells cultured in a isotonic medium, whereas the plating efficiency is apparently unaffected. The chromosome number distribution in EUE cells cultured in hypertonic medium shows a significant increase in the frequency of cells with a double chromosomal set (20% in treated cells versus 1.5% in control cells). The induction of polyploidy in hypertonic media can be a useful tool in experiments of genetic analysis based on chromosome segregation via tetraploidy. Furthermore the osmotic adaptation of cells in vitro can provide a useful selective system for the isolation of cellular hybrids.

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.

Response to DNA-damaging agents in cultured cells from patients with X-linked duchenne muscular dystrophy phenotype: male DMD, female DMD, possible carriers.

Using blood cultures the response to gamma (γ) radiation was examined in a male DMD and his mother, in a female DMD and her mother and in a normal control. In a series of experiments chromosome aberrations were determined after 3 separate γ-irradiation dose levels: 0, 150, 300 rads. The DMD patients showed a response to ionising radiations different from control, in fact the percentage of aberrations was lower than the control. In this preliminary study a slight difference between normal and possible carriers was also found.

[Parameters of a cell population synchronized for radiosensitivity studies (author's transl)]. / Parametri di una populazione cellulare, sincronizzata per lo studio della radiosensibilità.

Human cultured cells (EUE) were synchronized by the method of the mitotic harvest and the degree of synchronization, during the first duplication interval, determined by various tests which include growth curves, mitotic index cell-size distributions. Values of synchronization index initially greater than 90% and desynchronization rate of about 2%/hour were evaluated. The survival after 1.75 Gy of 31 MeV protons irradiation shows a sensitive period in the late G1 followed by an increase in radioresistance to a maximum in S.

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.