V. Gotzos

The Calcium Binding Protein Calretinin is a Selective Marker for Malignant Pleural Mesotheliomas of the Epithelial Type

In a series of 23 cases of mesothelioma of either the epithelial, sarcomatoid or the mixed type, the expression of three calcium-binding proteins (calretinin, parvalbumin and calbindin-D28k) was studied using immunohistochemical techniques on paraffin sections. The results show that calretinin is expressed in mesotheliomas of the epithelial type (papillary, adenomatous or solid) and by the epithelial component of the mixed tumours. The immunohistochemical reaction is specific and reproducible. The tissues of the pulmonary parenchyma and of the pleura are negative for calretinin except for the rare fibroblasts and some skeletal muscle fibres situated in the interstices of, or near the epithelial tumour mass. The sarcomatoid mesotheliomas and the sarcomatoid component of the mixed tumours do not express calretinin. Parvalbumin and calbindin-D28k are expressed neither in mesotheliomas nor in normal lung tissue. Primary adenocarcinomas of the lung are negative for all three calcium binding proteins cited. Thus, calretinin seems to represent a selective marker for mesotheliomas of the epithelial type and allows their differentiation from metastases of lung adenocarcinomas.

Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells

In some neurological diseases, injury to neurones reflects an over-stimulation of their receptors for excitatory amino acids. This response may disturb the Ca(2+)-homeostasis and lead to a pronounced and sustained increase in the intracellular concentration of this ion. On the basis of data derived from correlative studies, calcium-binding proteins have been postulated to play a protective role in these pathologies. We tested, directly, the capacity of the three calcium-binding proteins calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) to buffer [Ca(2+)], and to protect cells against excitotoxic death. We used P19 murine embryonic carcinoma cells, which can be specifically induced (by retinoic acid) to transform into nerve-like ones. The differentiated cells express functional glutamate-receptors and are susceptible to excitotoxic shock. Undifferentiated P19-cells were stably transfected with the cDNA for CR, CB or PV, induced to differentiate, and then exposed to NMDA, a glutamate-receptor agonist. The survival rates of clones expressing CR, CB or PV were compared with those of untransfected P19-cells using the lactate-dehydrogenase assay. CR- and CB-expressing cells were protected from death during the first 2 h of exposure to NMDA. This protection was, however, transient, and did not suffice to rescue P19-cells after prolonged stimulation. Two of the three PV-transfected clones raised were vulnerable to NMDA-induced excitotoxicity; the third, which expressed the lowest level of PV, was protected to a similar degree as that found for the CR- and CB-transfected clones. Our results indicate that in the P19-cell model, CR and CB can help to delay the onset of cell death after excitotoxic stimulation.

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.

Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation

Calretinin is a Ca(2+)-binding protein of the EF-hand family which is expressed in colon adenocarcinomas and colon-derived tumor cell lines (e.g. WiDr), but is absent from normal human enterocytes. Its function has not as yet been elucidated, but some lines of evidence lead us to postulate its involvement in cell proliferation in these cells. In order to test whether calretinin is correlated with an undifferentiated, proliferating, or with a differentiated, state of cells, its expression was studied in the human colon adenocarcinoma clonal cell line HT29-18, which can be caused to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (glucose starvation differentiation). Treatment of HT29-18 cells with galactose led to a drop in the calretinin mRNA level and in protein expression as evidenced by immunocytochemical staining and Western blot analysis of cytosolic cell extracts. These results suggest that calretinin is present in HT29-18 cancer cells, mostly in those which are in the undifferentiated state. The possibility that calretinin is involved in maintaining the cells in an undifferentiated (cancerous) state is discussed.

Action de différents mélanges gazeux sur l'artère mésentérique de l'embryon de poulet explantée in vitro

The effect of various gas mixtures on the mesenteric artery of chicken embryo explanted in vitro has been studied. The expiants were cultivated in normal air, with addition of 3% of CO2, and in hypo- and hyperoxic atmosphere. The best survival of explanted arteries is obtained with atmospheric air. The hypoxic gas mixture (2% O2) has the most toxic effect on the expiants. The hyperoxic conditions show also a toxic effect, but in a lesser degree than hypoxic conditions.

Culture in vitro de cellules de liquide peritoneal humain

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5–6 months). Three cellular types are described with different morphological features, namely the cellular

In vitro responses of proliferating and non-proliferating cells to high doses of 17β-estradiol: Cytochemical study

The authors studied the effect of 17 beta-estradiol on the proliferation in vitro of chick embryo fibroblasts and human macrophages by microdensitometry, cytofluorometry, and autoradiography. For fibroblasts 17 beta-estradiol shortens the duration of the preparing period to mitosis, particularly of the synthetic phase (S) and has no effect on the duration of mitosis. For macrophages, which have temporarily lost their mitotic capacity, 17 beta-estradiol cannot induce mitotic division.

Peculiar Ultrastructural Features in the Cytoplasm of Cells from Human Effusions Associated with Malignant Disease

A peculiar structure revealed by the electron microscope in a few cells from two human pleural fluids, showing the morphologic features of metastasizing cancer cells in effusions, is described. It has the appearance of a rod-shaped pentalaminary structure approximately 25-35 nm thick formed by an outer double membrane, surrounding a central, more dense axis showing transverse striations at about 10-nm intervals. The double membrane often exhibits a terminal expansion connected with the endoplasmic reticulum and it is sometimes associated with microfilaments. These structures could be a variant of or represent a step in the formation of confronting cisternae, but the periodic striations they show in the more dense lamella give them some resemblance to a Langerhans cell granule. To date, such striations have not been seen in confronting cisternae.

Étude histochimique des acides nucléiques et des protéines par cytophotométrie et histoautoradiographie sur des fibroblastes cultivés en hyperoxie

Resume Au cours de ce travail, il a ete demontre par des methodes cytophotometriques et histoautoradio-graphiques que les cellules cultivees en presence de 80 % d’O 2 subissent des alterations de la synthese d’ADN, d’ARN et des proteines totales. Ces alterations interviennent presque simultanement pour tous les metabolites biologiques susnommes. Le cycle cellulaire proliferatif subit des modifications avant que la vie cellulaire soit irreversiblement alteree. Il a ete montre que les cellules se bloquent d’abord en post-synthese (G 2 ) et ensuite en presynthese (G 1 ). On a aussi constate que l’activite de synthese protidique des cellules est proportionnelle a leur contenu en ADN, c’est-a-dire que les cellules tetraploides en ADN synthetisent une quantite double de proteines par rapport aux cellules diploides. Le marquage du nucleole apres incorporation d’Uridine- 3 H est le dernier a disparaitre au niveau cellulaire sous l’action de l’hyperoxie. Enfin on a observe que la prometaphase et la metaphase vues de face donnent des valeurs en ADN (mesures au microscope a balayage) superieures a la moyenne de celles des autres phases mitotiques.

Etude cytofluorométrique et histoautoradiographique de la synthèse d'ADN et de protéines sur des cellules mésothéliales humaines

The study of nuclear DNA synthesis of human mesothelial cells showed that cells do not divide by mitosis, even if an important percentage of them have a tetraploid amount of DNA. Concerning the cell cycle of mesothelial cells, the authors conclude that some of them are blocked in postsynthesis.