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.

Selective distribution of calretinin in adenocarcinomas of the human colon and adjacent tissues.

The expression of calretinin, a calcium-binding protein, has been studied in a series of 82 human colorectal adenocarcinomas. In 22.5% of the cases, part of the tumor cells were calretinin-positive, whereas the cells of the normal and paratumoral mucosa were always negative. Two types of cells from the tumoral mass reacted positively and selectively with calretinin-antisera: the tumor cells and giant fibroblasts. The neurons of enteric ganglia and reactive mesothelial cells also reacted positively to the same antibody. The results obtained by immunochemistry have been confirmed by Western blot analysis and in situ hybridization for calretinin mRNA. There is a correlation between the expression of calretinin and the degree of differentiation of the tumor. Well-differentiated tumors express calretinin in only 5% of the cases, whereas this percentage is 20% for moderately differentiated tumors and 66.6% for poorly differentiated or undifferentiated tumors. We conclude that calretinin is expressed by most undifferentiated colorectal adenocarcinomas, but only by a limited number of cells in well-differentiated tumors. The degree of its expression coincides also with additional signs of malignancy, such as an increase in the number of metastases in the regional lymph nodes and in other organs.

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.

In vitro detection of calretinin immunoreactivity in chicken embryo dorsal root ganglion neurons: a possible developmental marker.

Calretinin, a cytosolic calcium-binding protein, is widely expressed in mammalian and chicken neurons, including subpopulations of dorsal root ganglion neurons. In chicken embryo spinal ganglion cells, calretinin first appears on the 9th day of incubation. To determine whether the expression of this protein is maintained in vitro and depends on the developmental stage, dissociated dorsal root ganglion cultures from chick embryos at E6 and E10 (before or after establishing connections) were processed for calretinin immunohistochemistry. Cultured neurons from E6 embryos never showed calretinin immunoreactivity at any culture duration, whereas neurons from E10 embryos displayed strong immunostaining immediately after attaching to the culture dish. Quantitative evaluation revealed that the percentage of calretinin-positive neurons increased until day 3 in culture and afterwards declined in parallel the total cell number. These results indicate that primary sensory neurons express calretinin in vitro similarly as in vivo and the gene expression depends from the establishment of connections with peripheral targets.

Expression of calretinin in human mesothelioma cell lines and cell cycle analysis by flow cytometry.

Human mesothelioma cell lines were studied concerning the expression of the calcium-binding protein calretinin (CR), and the relation of the DNA index to their cell cycle. The results obtained for cell lines with different morphological characteristics, were compared to those from human mesothelial cells transfected with SV40 to escape senescence. Immunocytochemical expression of calretinin (confirmed by immunoblot) was observed in all mesothelioma cell lines but not in the control cells. It is suggested that calretinin is active in the first steps of carcinogenesis in all human mesotheliomas and during several stages in the evolution towards malignancy.

Microcinematographic and electron microscopic study of the death of human tumor cells induced by histiocytic cells.

The cells from a malignant fibrous histiocytoma were enzymatically isolated and cultured in vitro. The cultures were observed by microcinematography and with an electron microscope after fixation and embedding. The interactions between histiocytic and tumor cells resulted in tumor cell death. The microcinematographic study revealed that filiform projections of the histiocytic cell protrude toward the tumor cell surface making contacts for varying periods of time. The tumor cell then contracts and almost simultaneously some granules (lysosomes?) appear in the tumor cell cytoplasm. These granules eventually fuse with each other thus increasing in volume. The resulting granulation heads for the periphery of the tumor cell at a site previously touch by a projection of a histiocytic cell; at this site the membrane bursts and the tumor cell dies leaking amorphous cytoplasmic material. Before bursting the tumor cell does not show any noticeable morphological changes.

Heterogeneity of expression of the calcium-binding protein calretinin in human colonic cancer cell lines.

We searched for the presence of calretinin (CR) in 12 colonic cancer cell lines using immunohistochemistry, Western blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). We were able to demonstrate that calretinin is expressed in the cell lines HT-29, WiDr, LoVo, LS180, CO112, CO115. SW480, SW620, COLO205 and SK-CO-1, while no detectable amounts were found in the cell lines SW1116 and Caco-2. In general, rapidly proliferating cell lines expressed calretinin, whereas the protein was absent from cell lines with a low multiplication rate. A possible role for calretinin in maintaining the proliferative cycle of tumor cells is discussed.

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.

[Histochemical study of nucleic acids and proteins by cytophotometry and histoautoradiography in fibroblasts cultivated in hyperoxia (author's transl)]. / Etude histochimique des acides nucléiques et des protéines par cytophotométrie et histoautoradiographie sur des fibroblastes cultivés en hyperoxie

Cells cultivated in 80% O2 were shown, by cytophotometric and histoautoradiographic methods, to undergo alterations concerning DNA, RNA and total proteins synthesis. These changes appear almost at the same time for all the above-named biological metabolites. Proliferative cell cycle undergoes changes before the blocking of the cells first in post-synthetic (G2) and then in pre-synthetic period (G1). It was also seen that protein synthetic rate of the cells is proportional to their DNA content, in other words tetraploid cells synthesize a double protein amount in comparison to diploid cells. Nucleolus labelling after Uridine-3H incorporation disappear the last under the effect of hyperoxia. Finally it was observed that DNA values (measured with scanning microscope) of prometaphase are higher than the mean values of other mitotic phases.

[The mucous secretion anisotropy of the sublingual gland of the guinea-pig by means of topo-optical histochemical reaction (author's transl)]. / Anisotropie du mucus de la glande sous-linguale du cobaye étudiée au moyen des réactions histochimiques topo-optiques

The authors study the mucous secretion of sublingual gland of the guinea-pig by means of topo-optical histochemical reactions with polarized light. They show a "paracristalline" organization of glucidic and protidic portion of these macromolecules. The results are discussed and compared with those of the literature.

In vitro responses of proliferating and non-proliferating cells to high doses of 17 beta-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.

[Analysis of the structure and distribution of chromatin in interphase nuclei of different cell types from a pleural effusion of tumoral origin in man. Cytophotometric and electron microscopic studies]. / Analyse de la structure et de la distribution de la chromatine des noyaux en interphase des différents types cellulaires se trouvant dans un épanchement pleural humain d'origine tumorale. Etude par cytophotométrie et microscopie électronique.

The authors have studied the distribution and the structure of the chromatin in the different cell types from a human pleural effusion of tumoral origin (mammary adenocarcinoma). The results show that in the entire nuclei the distribution and the structure of the chromatin cannot be considered as characteristic morphological features of the cells of the same cellular type. Thus making a clear distinction among differentiating cells and cells in DNA synthesis or in G2 period, only based on these properties of the chromatin, is very debatable. On the other hand, the distribution and the structure of the chromatin permit to characterize the different cell types in G1 period. The authors discuss the possible differential cytological diagnosis of the smears from human effusions of tumoral origin.

Comparative studies of calretinin expression by WiDr cell line in vivo in xenografts in nude mice and in vitro.

WiDr cells from a human colon adenocarcinoma cultivated in vitro express the calcium binding protein calretinin. The immunoreactivity is present in some interphasic cells and decreases after seven days in culture together with the augmentation of the cell number. Calretinin expression is maintained in the undifferentiated cells of the tumoral mass developed in nude mice and in recultivated isolated tumour cells from the xenograft. From the experiments here described, the protein expression is quantitatively influenced in vitro by the addition of drugs, such as colchicine and taxol, which intervene in cytoskeleton organisation. The percentage of the calretinin immunoreactive cells increases after the addition of colchicine to the medium while the immunoblot analysis shows a higher calretinin content in the cells treated with taxol.

Morphological aspects of lymphocyte-macrophage interactions in human tumoral effusions "in vivo" and "in vitro".

"In vivo" and "in vitro" morphological analysis of associations of cells ("rosettes") involved in immune response in human tumoral effusions revealed the existence of cell interactions either by simple membrane apposition between the cell projections or by gap-like junctions between two adjacent cells; endocytotic phenomena were also observed. The giant fibroblastic cells seen "in vitro" ("myofibronoblasts") reacting positively to anti-human macrophage Mabs, might be the cells presenting antigen to lymphocytes.

[Cells affected by apoptosis in effusions of tumor origin in man]. / Cellules affectées par l'apoptose dans des épanchements d'origine tumorale chez l'homme.

The cells from 3 human effusions of tumoral origin (adenocarcinoma) were observed by light and transmission electron microscopy. The presence and the type of cells undergoing apoptosis were studied. Among the different cell types present in these effusions, only some lymphocytes, macrophages and eosinophils (cells concerned in cell-mediated tumor immunity) are affected by the morphological changes of apoptosis. We did not find, as other authors did, tumor cells or other cell types (mesothelial cells, histiocytes, other granulocytes) showing apoptic alterations. The authors conclude that this peculiar morphological type of cell death affects the effector cells of the cell-mediated tumor immunity being at the end of their life-span.

[In vitro culture of human peritoneal fluid cells]. / Culture in vitro de cellules de liquide péritonéal 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 shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.

[Mitotic anomalies observed in fibroblasts of chicken embryo cultured in vitro with 80% oxygen]. / Anomalies mitotiques observées dans des fibroblastes d'embryon de poulet cultivés in vitro avec 80% d'oxygène

Fibroblasts cultivated in vitro were observed during mitosis. Already after 24 h of culture in 80% oxygen, some anomalies affect mitosis (atypical mitosis): abnormalities of the usual mitosis stages, chromosomal aberrations. Mitosis is interrupted at metaphase during the first and second day of culture in hyperoxia and at prophase (prometaphase) during the third day. The new mode of nuclear division is discussed.

[Clonogenic culture of identified cells of lung tumors and correlation with certain histopathologic aspects of the tumor mass]. / Croissance clonogène des cellules provenant de tumeurs pulmonaires et sa corrélation avec certains aspects histopathologiques de la masse tumorale.

Clonogenic cultures from 27 lung cancers were realised. For 68% of cases a clonogenic growth was observed, but no direct correlation could be done with the differentiation of the tumor or with the presence of ganglion metastases. EGF in medium increased the number of colonies obtained in 60% of cases.

Observation of living cells using the atomic force microscope.

We used an atomic force microscope (AFM) to image samples immersed in a fluid in order to study the dynamic behavior of the membranes of living cells. AFM images of cultured cells immersed in a buffer were obtained without any preliminary preparation. We observed surface changes and displacements which suggest that the cells were still alive during the measurements. Some membrane details imaged with the AFM have also been observed using a scanning electron microscope and their dynamic behavior has been confirmed by microcinematography. We believe that the AFM will offer new insights into the exploration of dynamic changes affecting cell membranes.

A quantitative microdensitometric and autoradiographic study of the effect of 4'-demethyl-epipodophyllotoxin-beta-D-thenylidene glucoside (VM-26) on the cell cycle of cultured fibroblasts.

The effect of 4'-demethyl-epipodophyllotoxin-beta-D-thenylidene glucoside (VM-26) , a semi-synthetic derivative of podophyllotoxin, on the cell cycle was studied with chick embryo fibroblasts cultivated in vitro. DNA, RNA and protein content, as well as NADH-diaphorase activity were determined by quantitative microdensitometry and cytofluorometry. The incorporation of [3H]thymidine and [3H]leucine into DNA and proteins were analysed by autoradiography. These metabolic data correlated with morphological observation showed that VM-26 blocks the cell cycle at different moments of its kinetics depending on both the dose and the time exposure. NADH-diaphorase activity is the first to be affected, then biochemical changes (involving the metabolism of RNA and proteins) and morphological alterations (especially of mitochondria) follow. This suggests that VM-26 may act primarily upon the mechanism of respiration of the cell.